Histidine-rich peptides are endosomal escape agents used in nanomedicine for drug delivery. The cellular endosomal uptake pathway is known as the rate-limiting barrier for bioactive molecules. Endosomal escape is known as the bottleneck in intracellular delivery. During drug delivery, many bioactive molecules get trapped in the endosomal compartment. Therefore effective strategies are needed to enhance endosomal escape and cytosolic bioavailability of therapeutic molecules.

Most uptake routes converge into endocytic vesicles. Both vesicle and cargo may be enzymatically degraded when late endosomes transform into lysosomes. The histidine's imidazole group has a pK of ~6 which gets protonated under acid conditions in the endosome—the positive charge recruits chloride ions (Cl^-). The result is osmotic swelling and cracking of the endosome (according to the "proton sponge" model). The result is the cytoplasmic release of nanoparticles and the escape from lysosomal enzymes.

Histidine-rich peptides can be incorporated into polymers, liposomes, and proteins, including virus-like particles. However, poly-histidines are neutrally charged at neutral pH, minimizing nonspecific binding to serum proteins and inactivation of the particle.

Table 1:  Examples of Histidine-rich peptides used in nanomedicine as endosomolytic agents (Adapted from Ferrer-Miralles et al. 2011).

Abbreviations: aas, amino acids; pDNA, plasmid DNA; PEI, poly (ethylene imine); Lau, lauryl; Pal, palmytoil; chit. 4 gen, chitosan 4 generation; CMPLH, carboxymethyl poly (L- histidine); PbAE, poly (b-amino ester); STR, staeroyl; FGF-2, fibroblast growth factor-2.

Reference

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Peptide-conjugated antisense oligonucleotides enable exon-skipping therapeutics.

Antisense Oligonucleotides (ASOs or AONs) are small single stranded chemically modified nucleic acids designed for targeting specific gene transcripts. The small size of the ASO is crucial for cell delivery.

Chemical modification of the ASO affects stability, solubility, toxicity, affinity, and resistance to degradation. The conjugation of peptides to oligonucleotides allows the generation of peptide-conjugated antisense oligonucleotides. Modifying the backbone of ASOs increases their stability and affinity to target sequences. Peptide-conjugated antisense oligonucleotides potentially enable the systemic correction of the Duchenne muscular dystrophy phenotype. Also, the conjugation of ASOs to arginine-rich cell-penetrating peptides improves their cell transduction properties.

ASOs targeting exon 53 of dystrophin pre-mRNA on the Duchenne muscular dystrophy (DMD) gene alter pre-mRNA splicing by removing specific exons in the dystrophin mRNA during the splicing process. The ASO hides the exon splicing sites from the splicing machinery and restores the reading frame of the transcript. Examples are the FDA-approved phosphorodiamidate morpholino ASOs viltolarsen and golodirsen.

Duchenne muscular dystrophy is a severe muscle degenerative X-linked allelic disorder resulting in the absence of functional dystrophin. The human gene called dystrophin (DMD) spans 24 kb of genomic DNA with its 79 exons. It encodes dystrophin, a 427 kDa protein localized on the cytoplasmic side of the sarcolemma of skeletal and cardiac muscle fibers and the cortical/cerebellar synapses.

Utilizing polyclonal antibodies directed against fusion proteins that contained two distinct regions of the mDMD cDNA, Hoffman et al., in 1987, identified the protein product of human Duchenne muscular locus (DMD) and its mouse homolog (mDMD). A mutation disrupting the reading frame in the dystrophin gene results in the absence of the functioning protein. Non-randomly distributed deletions involving one or more exons are the most common mutations.

Exon skipping is a novel therapeutic approach to correct mutations in DMD patients and restore the proper expression of dystrophin. During the last decade, researchers investigated exon skipping ASOs as tools for the correction of missplicing diseases. ASOs can correct splicing defects through exon skipping.

ASOs targeting exon 53 of dystrophin pre-mRNA on the Duchenne muscular dystrophy (DMD) gene alter pre-mRNA splicing by removing specific exons in the dystrophin mRNA during the splicing process. The ASOS hides the exon spicing sites from the splicing machinery and restores the reading frame of the transcript. Examples are the FDA-approved phosphorodiamidate morpholino ASOs viltolarsen and golodirsen.

Figure 1: Extracellular membrane and cytoplasmic components of the dystrophin-glycoprotein complex (DGC), cardiac and skeletal muscle membrane specialization (Wiki Commons).

The DGC is a multimolecular complex linking the extracellular matrix to the cytoskeleton. The DGC is essential for the maintenance of normal cardiac and skeletal muscle. The DGC is a mechanosignaling complex that has mechanical and nonmechanical membrane-stabilizing functions. Destabilization of the DGC leads to membrane fragility and loss of membrane integrity. The result is a degeneration of skeletal muscle and cardiomyocytes.The cytoskeletal protein dystrophin links cytoplasmic γ-actin to the transmembrane components of the DGC. Dystrophin also binds to β-dystroglycan, and dystroglycan binds to the extracellular matrix protein laminin-α2. The sarcoglycan complex is composed of multiple subunits.

Yin et al., in 2008, reported the systemic correction of dystrophin expression in the muscle cells of adult dystrophic mdx mice. A single dose of a peptide-conjugated morpholino phosphoroamidate antisense oligonucleotide was sufficient to restore dystrophin protein expression. The treatment resulted in the improvement of muscle function in the mice. Since this work, antisense oligonucleotides have become the basis of exon skipping therapeutic approaches to correct defects in splicing events and repair the disrupted open reading frame.

However, the therapeutical use of unmodified ASOs is quite limited because they are subject to degradation by endonucleases and exonucleases. Therefore, in recent years researcher scientists have tested multiple chemical modifications of the phosphoribose backbone to improve the stability, efficacy, and pharmacokinetics of ASOs. To allow for high-affinity interaction with target mRNA during splicing, ASOs should not support ribonuclease H activity. Several studies tested ASOs containing 2’-O-methyl (2’-OMe) and 2’-O-methoxyethyl (2’-MOE) with a phosphorothioate backbone (PS) in cells. Further investigations revealed that ASOs containing bridged nucleic acids (BNAs, LNAs), peptide nucleic acids (PNAs), and phosphorodiamidate morpholinos (Mos) are also potent ASOs in cells. However, the non-ionic nature of the phosphorodiamidate linkage minimizes nuclear uptake. Adding charged cell-penetrating peptides to ASOs improves their uptake by cells.
 

Reference

Karen A. Lapidos, Rahul Kakkar, and Elizabeth M. McNally; The Dystrophin Glycoprotein Complex. Signaling Strength and Integrity for the Sarcolemma. Karen A. Lapidos, Rahul Kakkar, and Elizabeth M. McNally;  Originally published30 Apr 004 https://doi.org/10.1161/01.RES.0000126574.61061.25 Circulation Research. 2004; 94:1023–1031.

HaiFang Yin, Hong M. Moulton, Yiqi Seow, Corinne Boyd, Jordan Boutilier, Patrick Iverson, Matthew J.A. Wood, Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function, Human Molecular Genetics, Volume 17, Issue 24, 15 December 2008, Pages 3909–3918, https://doi.org/10.1093/hmg/ddn293

DMD dystrophin [ gene ]

Golodirsen [ drugbank]

Viltolarsen [ drugbank ]

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 The anticancer drug Taxol has been used extensively to treat breast or ovarian cancer (also lung cancer, esophageal cancer, head and neck cancer, pancreatic cancer).  Taxols' anticancer property derives from its ability to interfere with 'dynamic instability', the mechanism through which microtubule polymers are maintained in vivo, thus interfering with chromosome segregation during the mitosis.  Taxol was originally isolated from the bark of pacific yew plants; however, the processing of the bark (provides immunity) leads to the death of the plants (38,000 trees yield 25 kilogram Taxol annually).  Yet the demand for Taxol continues to rise drastically (~250 kg annually as of 2012) for treating cancer and other diseases.

 This had led to the semisynthesis of Taxol from 10-deacetylbaccatin isolated from the needles of European yew (Taxus baccata), which leaves the plants viable.  Though the total organic synthesis (40 steps) of Taxol was achieved chemically (Nicolaou et al., 1994), it did not reach commercial application due to a low yield.  More recently, Bristol-Meyer Squibb Pharmaceutical has been relying on a plant cell line (Taxus) to harvest Taxol (by purifying Taxol secreted into growth medium via chromatography and crystallization).  For this, Phyton Biotech Inc. (Germany), which operates the largest GMP plant cell fermentation facility, supplied the Taxol-containing medium.

 For clinical application, Taxol (due to its poor solubility in aqueous media) is dissolved in Cremophor EL (polyoxyethylated castor oil in 1 : 1 mixture with dehydrated ethanol), which causes significant side effects (i.e. hypotension, brochospasm, hypersensitivity) (Surapaneni, et al., 2012).  To avoid using organic solvents for dissolution, 'Abraxane' was developed, which consists of Taxol encased in a nanoparticle comprised of albumin (Wilson et al., 2012).  The intravenously administered Abraxane exits blood vessels via binding to albumin receptor present on endothelial cells, followed by its putative binding to SPARC (Secreted Protein Acidic Rich in Cysteine) overexpressed in various solid tumors.  However, to prevent clogging of capillaries, other types of experimental nanoparticles are being developed using ingredients such as PLGA [poly(lactic-co-glycolic acid)] (Surapaneni, et al., 2012).
     
                                                                                       

The latest innovation concerns the attempt to administer Taxol orally.  Normally, intestinal accumulation of toxic products is prevented by p-glycoprotein (a transporter expressed by the intestinal cells), which pumps them out of the cells.  Unfortunately, p-glycoprotein also prevents the uptake of cytotoxic drugs through effluxing, causing multi-drug resistance.  To allow intestinal absorption, patients with metastatic breast cancer were treated with Encequidar, an inhibitor of p-glycoprotein, along with Taxol in a Phase 3 clinical trial.  Though the study yielded comparable results as intravenously administered Taxol, it led to lower white blood cell counts (neutropenia), increasing the risk of infection (https://www.abstractsonline.com/pp8/#!/7946/presentation/2050 ).  Also, the question remained as to whether the patients alone could follow complex dosing schedule.

 Increasingly, oligonucleotide based drugs (siRNA, gapmer, miRNA, aptamer) are being recognized as a viable therapeutic modality.  The hurdles once thought insurmountable are slowly being chipped away through chemical modification of oligonucleotides (i.e. nucleobase, internucleotide linkage, sugar moieties) as well as improvements in delivery vector (ex. GlcNAc, cholesterol, PEGylation, peptide) (O'Driscoll et al., 2019).  One of the key barriers for oral delivery of oligonucleotide therapeutics is the presence of gastroinstestinal nucleases (ex. DNase), which has prompted encasing of the oligonucleotides in oil drops such as SEDDS (self-microemulsifying drug delivery system) or nanoparticles like the liposomes to avoid degradation (Hauptstein et al., 2015).  The second barrier is the mucus layer (glycoproteins crosslinked by disulfide bonds; 400 micrometer thick; mesh size 10-200 nanometer; dynamic) making it nearly impossible for bulky plasmid DNA (for gene therapy) to penetrate.  To infiltrate, drug delivery vectors that are mucoinert and less than 100 nanometers may need to be administered along with mucolytic agents (ex. enzymes, sulfhydryl compounds).  Intriguingly, viruses with "slippery" exterior due to a high density of of positive and negative charges are known to permeate through mucus efficiently.  The third barrier is electrostatic repulsion of oligonucleotides (negatively charged) by the anionic charge associated with brush border microvilli (contains charged residues) of the epithelial cells lining the gastrointestinal tract.

Despite the challenge, nearly 20 works have been published regarding the oral delivery of oligonucleotide based drugs (antisense, siRNA) or plasmid DNA (O'Driscoll et al., 2019).    Among their targets include the mRNAs encoding Map4kf4, proinfoammatory cytokines, tumor necrosis factor alpha, VEGF (vascular endothelial growth factor) and survivin.  To suppress systemic inflammation, the investigators at the University of Massachusetts (USA) reported the successful oral delivery of oligonucleotides encased in hollow β1,3-D-glucan particles (purified from baker's yeast)  to suppress TNF and Map4kf4 expression in macrophages found in spleen, liver and lung of mice (Aouadi et al., 2009).  Of relevance to cancer, double gene silencers (shRNA downregulating survivin mRNA; siRNA targeting VEGF mRNA) enclosed in a nanoparticle composed of GTC (galactose modified trimethyl chitosan-cysteine) were delivered orally, which suppressed tumor growth in a mouse model (Han et al., 2014).   Another group utilized glycol chitosan-taurocholic acid conjugate to protect gold-siRNA (inhibit Akt2) conjugate from gastrointestinal degradation, which inhibited colorectal liver metastasis in a murine model after oral delivery (Kang et al., 2017).

 The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/covid-19.aspx

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/tew/fda-approves-3rd-sirna-therapeutic-oxlumo-to-treat-the-metabolic-disease-primary-hyperoxaluria-type-1-and-the-structural-significance-of-2-nucleotide-3-overhang.aspx

 References

Aouadi M, Tesz GJ, et al. Orally delivered siRNA targeting macrophage Map4k4 suppresses systemic inflammation.  Nature.  458:1180-4 (2009).  PMID: 19407801

Han L, Tang C, et al. Oral delivery of shRNA and siRNA via multifunctional polymeric nanoparticles for synergistic cancer therapy.  Biomaterials. 35:4589-600 (2014).  PMID: 24613049

Hauptstein S, Prüfert F, et al.  Self-nanoemulsifying drug delivery systems as novel approach for pDNA drug delivery.  Int J Pharm.  487:25-31 (2015).  PMID: 25839413

Kang SH, Revuri V, et al. Oral siRNA Delivery to Treat Colorectal Liver Metastases.  ACS Nano.  11:10417-10429 (2017).  PMID: 28902489

Nicolaou KC, Yang Z, et al.  Total synthesis of taxol. Nature.  367:630-4 (1994).  PMID: 7906395

O'Driscoll CM, Bernkop-Schnürch A, et al.   Oral delivery of non-viral nucleic acid-based therapeutics - do we have the guts for this?  Eur J Pharm Sci.  133:190-204 (2019).  PMID: 30946964

Surapaneni MS, Das SK, et al. Designing Paclitaxel drug delivery systems aimed at improved patient outcomes: current status and challenges.  ISRN Pharmacol.  2012:623139 (2012).  PMID: 22934190

Wilson SA, Roberts SC.  Recent advances towards development and commercialization of plant cell culture processes for the synthesis of biomolecules. Plant Biotechnol J.  10:249-68 (2012).   PMID: 22059985

Following the identification of the coronavirus that causes respiratory diseases in Wuhan (China) in late 2019, multiple variants arose globally.  The most notable among them are Alpha (B.1.1.7; United Kingdom), Beta (B.1.351; South Africa), Gamma (P.1; Brazil), and Delta (B.1.617.2; India).  The continued emergence of variants has fueled multiple 'waves of outbreaks' experienced since then (Tao et al., 2021).

A high number of mutations found in the genome of Omicron (B.1.1.529) coronavirus has necessitated further classification into the sublineages BA.1​/B.1.1.529.1, BA.2​/B.1.1.529.2, and BA.3​/B.1.1.529.3.  Among the sixty mutations identified are 50 nonsynonymous (alters amino acid), 8 synonymous (amino acid remains unchanged), and 2 non-coding types, which impact various viral genes (Nsp3, Nsp4, Nsp5, Nsp6, Nsp12, Nsp14, membrane protein, nucleocapsid protein, envelope protein).  A significant fraction of these mutations (3 deletions, 1 insertion; 30 amino acid-changing) occurred in the spike protein, with 15 in the receptor-binding domain alone, raising the speculation whether vaccination may have imposed selection pressure on the virus to evolve (Chen et al., 2021).  Further, 6 mutations (A1892T, P314L, I189V, K38R, V57V, T492I) affect RNA-dependent RNA polymerase (RdRP) (Bansal et al., 2021).

Subsequently, it was found that the novel mutations in Omicron may abrogate the humoral immunity acquired either naturally (via exposure to the prior COVID-19 strains) or through vaccination.  Humoral immunity relies on the antibodies secreted by B cells to neutralize incoming viruses, thus playing a key role in blocking infectivity.  One report utilizing a topology-based artificial intelligence model predicted that Omicron may be more infectious than Delta coronavirus (Chen et al., 2021).

 Another report determined that the efficacy of mRNA vaccine prepared by Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) diminished considerably (as well as other vaccines targeting the spike protein) against Omicron (Cameroni et al., 2021).   The potential benefit of lessening the impact through booster (for mRNA vaccine) was described (Garcia-Beltran et al., 2021).   

Similarly, the activity of most antibody-based drugs targeting the spike protein was lost against Omicron though a handful of them retained activity.  The efficacy of antibody drugs targeting a region outside the receptor-binding motif was less impacted (Sotrovimab) (Cameroni et al., 2021).

Interestingly, the decline in humoral response was lesser (5 times) for those who have been previously infected with COVID-19 (plus vaccinated), suggesting that the antibody-based immunity targeting multiple antigenic sites (in addition to spike protein) may provide greater protection (Cameroni et al., 2021).

Cellular immunity is based on T cells and involves the activation of cytotoxic T cells (CTL), activation of macrophages and natural killer cells, and secretion of cytokines in response to antigen.  A critical component of cellular immunity is cytotoxic T lymphocyte recognizing specific antigens.  Previously, the ability of mRNA vaccine (ex. mRNA-1273 by Moderna) to induce CD4+ T helper cells and CD8+ CTLs was documented in vaccinated individuals (Mateus et al., 2021). 

To determine if the T cell-based immunity was compromised, the investigators at the Johns Hopkins School of Medicine (USA) examined the Omicron sequence encoding viral epitopes recognized by CD8+ T-cells (Redd et al., 2021).  They showed that only 1 of 50 Omicron-associated mutations occurred in the epitope (GVYFASTEK) recognized by CD8+ T-cells.  Further, the epitope (recognized by HLA type HLA*A03:01 and HLA*A11:01) represents a "low-prevalent" target.  The study involved 30 COVID-19 infected individuals, accounting for 52 unique COVID-19 epitopes.  The retaining of T cell-based immunity may account for lesser severity and hospitalization observed despite the greater transmissibility of Omicron.  Its impact on cancer patients remains to be determined.

References

Bansal K, Kumar S.  Mutational cascade of SARS-CoV-2 leading to evolution and emergence of omicron variant.  bioRxiv (2021). Preprint.  doi: https://doi.org/10.1101/2021.12.06.471389

Cameroni E, Saliba C, et al.  Broadly neutralizing antibodies overcome SARS-CoV-2 Omicron antigenic shift.  bioRxiv. 2021 Dec 14:2021.12.12.472269.  (2021) Preprint.  PMID: 34931194

Chen J, Wei GW et al. Omicron (B.1.1.529): Infectivity, vaccine breakthrough, and antibody resistance.  ArXiv.  Dec 1:arXiv:2112.01318v1 (2021).  Preprint.  PMID: 34873578

Garcia-Beltran WF, St Denis KJ, et al.   mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 Omicron variant.   medRxiv. Dec 14:2021.12.14.21267755 (2021).  Preprint.  PMID: 34931201

Mateus J, Dan JM, et al.  Low-dose mRNA-1273 COVID-19 vaccine generates durable memory enhanced by cross-reactive T cells.  Science.  374(6566):eabj9853  (2021).  PMID: 34519540

Redd AD, Nardin A, et al. Minimal cross-over between mutations associated with Omicron variant of SARS-CoV-2 and CD8+ T cell epitopes identified in COVID-19 convalescent individuals.  bioRxiv. Dec 9:2021.12.06.471446. (2021).  Preprint.  PMID: 34909772

Tao K, Shafer RW, et al. The biological and clinical significance of emerging SARS-CoV-2 variants.  Nat Rev Genet. (12):757-773 (2021).  PMID: 34535792

Specific RT-qPCR assays enable rapid identification of newly emerging SARS-COV-2 variants such as the Omicron (B.1.1.529) virus variant of concern. The assays target characteristic mutations in the nsp6 (Orf1a), spike, and nucleocapsid genes. Also, multiplexing PCR may be a reliable assay to identify B.1.1.529 in suspected samples.

A list of mutations for a variety of variants can be reviewed at here! 

The Omicron variant (SARS-CoV-2 B.1.1.529) first emerged in November 2011 in South Africa and Botswana.

Travel-related cases were already identified in Hong Kong, Belgium, and Israel at the end of November.

As of 01-11-2022, Omicron has now been discovered in all seven continents. However, since Omicron variants with and without deletions in the spike protein and others are co-circulating, the correct diagnose using PCR tests is quite complicated.

GISAID is tracking Omicron and other variants of SARS-CoV-2 and reported 271,805 Omicron genome sequences on 01-11-2022.  Oran et al. recently reported four RT-qPCR assays that allow the rapid identification of the newly emerging SARS-CoV-2 Omicron variant of concern. With its 30 mutations in the spike protein alone, this variant of concern (VOC) can potentially increase transmissibility and reduce vaccine effectiveness. Therefore, assays that allow the specific detection of this variant are needed. RT-qPCR assays can be implemented and performed in any molecular diagnostic laboratory without requiring specific brand instruments or unique reagents.

Primer and Probes for Specific Detection of the Omicron Virus

Note: TaqMan® or FRET probes are usually labeled at the 5′-end with the reporter molecule 6-carboxyfluorescein (FAM) and with the quencher, Black Hole Quencher 1 (BHQ-1) at the 3′-end. Alternatively, probes can also be labeled at the 5′-end with the reporter molecule 6- carboxyfluorescein (FAM) and with a double quencher ZEN™ Internal Quencher positioned between the ninth (9th) and tenth (10th) nucleotide base in the oligonucleotide sequence and Iowa Black® FQ (3IABkFQ) located at the 3’-end.

In addition, a ROX probe reaction mixture may be used as well.


Reference

Corman VM, Landt O, Kaiser M, Molenkamp R, Meijer A, Chu DK, Bleicker T, Brünink S, Schneider J, Schmidt ML, Mulders DG, Haagmans BL, van der Veer B, van den Brink S, Wijsman L, Goderski G, Romette JL, Ellis J, Zambon M, Peiris M, Goossens H, Reusken C, Koopmans MP, Drosten C. 2020. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill 25:2000045. [PMC]

Oran Erster, Adi Beth-Din, Hadar Asraf, Virginia Levy, Areej Kabat, Batya Mannasse, Roberto Azar, Ohad Shifman, Shirley Lazar, Michal Mandelboim, Shay Fleishon, Ella Mendelson, Neta S Zuckerman; SPECIFIC DETECTION OF SARS-COV-2 B.1.1.529 (OMICRON) VARIANT BY FOUR RT-qPCR DIFFERENTIAL ASSAYS; medRxiv 2021.12.07.21267293;  [ medrxiv ]

US CDC Omicron Update


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Bio-Synthesis provides a full spectrum of high quality custom oligonucleotide modification services including back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
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Multidrug-resistant gram-negative bacteria are a serious and growing risk to public health. Gram-negative bacteria can cause antibiotic-resistant infections, often leading to death. Resistance to the antibiotic amikacin and other aminoglycosides in clinical settings is often due to the enzymatic acetylation of the antimicrobial molecule. In antibiotic-resistant Gram-negative bacteria, the enzyme 6’-N-acetyltransferase type Ib [AAC(6’)-Ib] mediates resistance to amikacin. This enzyme catalyzes the transfer of an acetyl group from acetyl coenzyme A to the 6’ position of the antibiotic molecule using acetyl-CoA as a donor substrate.

Magallon et al. recently observed that adding Zn²⁺ to in-vitro enzymatic reactions obliterated acetylation of the acceptor antibiotic. When added to amikacin-containing culture medium in complex with ionophores such as zinc pyrithione, it prevents the growth of resistant strains.

The anti-fungal drug zinc pyrithione is widely used in medical shampoos to treat dandruff and seborrheic dermatitis. Zinc pyrithione is a solid coordination complex of zinc with a molecular weight of 317.7 g/mol and a logP = 0.88. The bioactive monomeric molecule is the anti-fungal agent.

Magallon's research group in Tolmaky's lab found that a zinc pyrithione based inhibitor helps fight bacterial antibiotic resistance. More recently, in 2022, the researchers showed that water-soluble pyrithione compounds allow the design of inhibitors to fight bacterial resistance to the aminoglycoside amikacin.

Inhibiting the acetylation reaction will again render the antibiotic active against Gram-negative bacteria. To enable further use of amikacin against antibiotic resistant infections, inhibitors of the inactivating reaction can be combined with the antibiotic. Magda et al. previously derivatized pyrithione with the amphoteric group di(ethylene glycol)methyl ether at position 5 to increase its water solubility.

Figure 1: Chemical structures of zinc pyrithione.

Amikacin is a 4,6-linked aminoglycoside modified at position N1 of the 2-deoxystreptamine ring by the (l)-α-hydroxy-γ-aminobutyric amide (L-HABA) group. Amikacin binds specifically to the A site like its parent compound, kanamycin. The introduction of the L-HABA group on ring II of the aminoglycoside increases the affinity to the bacterial A site. Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site where they induce misreading of the genetic code.

Figure 2: Structure of the bacterial ribosomal decoding site containing the bacterial ribosomal A site in complex with amikacin [Kondo et al. 2006. PDB ID 4P20].

One prominent mechanism of antibiotic resistance is the enzymatic modification of the active compound. The change prevents its binding to the cellular target. One common mechanism is N-acetylation at the 6’-position. The aminoglycoside 6’-N-acetyltransferase (AAC(6')) catalyzes the reaction. This enzyme's two functional classes have been described: AAC(6′)-I, which confers resistance to amikacin but not to gentamicin, and AAC(6′)-II, with reciprocal selectivity. Both enzyme classes also acetylate kanamycin, tobramycin, neomycin, netilmicin, and sisomicin. Some isoforms of the AAC(6')-Ib subclass also modify amikacin and gentamicin and potentially some fluoroqinolones.

Figure 3: Structural model of aminoglycoside acetyltransferase AAC(6’)-Ib in complex with coenzyme A and kanamycin [Maurice et al. 2008. PDB ID 2QIR].  

In 2013 Tada et al. observed that bacteria that acquire the aac(6′)-Iaj gene gain resistance to all aminoglycosides tested except to gentamicin. Thin-layer chromatography revealed that AAC(6′)-Iaj acetylated all tested aminoglycosides except gentamicin. The study found that AAC(6′)-Iaj is a functional acetyltransferase that modifies the amino groups at the 6′ positions of aminoglycosides and contributes to aminoglycoside resistance of P. aeruginosa NCGM1588, including arbekacin.

Kanamycin binds to four nucleotides of 16S rRNA and a single amino acid of protein S12 which interferes with the decoding site in the vicinity of nucleotide 1400 in 16S rRNA of the 30S subunit. This region interacts with the wobble base in the anticodon of tRNA leading to interference with the initiation complex resulting in misreading of mRNA so that incorrect amino acids are inserted into the polypeptide. The result is the synthesis of nonfunctional or toxic peptides and the breakup of polysomes into nonfunctional monosomes.

Synthesis of water soluble zinc pyrithione (according to Magda et al. 2008).

[1]   Bromination of 2-Bromo-5-methyl-pyridine under efflux for 8 hours.

[2]   Followed by PEGylation of 2-Bromo-5-bromomethylpyridine.

[3] Oxidation of 2-Bromo-5-CH₂OPEG-pyridine.

[4]  Sodium sulfide reaction with 2-Bromo-5-CH₂PEG-pyridine-N-Oxide.

[5]  Preparation of zinc complex of 2-mercapto-5-CH₂PEG-pyridine-N-Oxide..

Time-kill assays performed by Magallon et al. showed that the addition of amikacin to bacterial cell cultures resistant to amikacin together with the water-soluble ionophore  zinc pyrithione inhibited bacterial growth.


Reference

Amikacin

Kanamycin

Kondo J, François B, Russell RJ, Murray JB, Westhof E. Crystal structure of the bacterial ribosomal decoding site complexed with amikacin containing the gamma-amino-alpha-hydroxybutyryl (haba) group. Biochimie. 2006 Aug;88(8):1027-31. [PubMed]

Magallon J, Vu P, Reeves C, Kwan S, Phan K, Oakley-Havens CL, Rocha K, Jimenez V, Ramirez MS, Tolmasky ME. Amikacin potentiator activity of zinc complexed to a pyrithione derivative with enhanced solubility. Sci Rep. 2022 Jan 7;12(1):285. [PMC]

Magda D, Lecane P, Wang Z, Hu W, Thiemann P, Ma X, Dranchak PK, Wang X, Lynch V, Wei W, Csokai V, Hacia JG, Sessler JL. Synthesis and anticancer properties of water-soluble zinc ionophores. Cancer Res. 2008 Jul 1;68(13):5318-25. [Cancer Research]

Mangion SE, Holmes AM, Roberts MS. Targeted Delivery of Zinc Pyrithione to Skin Epithelia. Int J Mol Sci. 2021 Sep 8;22(18):9730. [PMC]

Maurice F, Broutin I, Podglajen I, Benas P, Collatz E, Dardel F. Enzyme structural plasticity and the emergence of broad-spectrum antibiotic resistance. EMBO Rep. 2008 Apr;9(4):344-9. doi: 10.1038/embor.2008.9. [PMC]

Reeves CM, Magallon J, Rocha K, Tran T, Phan K, Vu P, Yi Y, Oakley-Havens CL, Cedano J, Jimenez V, Ramirez MS, Tolmasky ME. Aminoglycoside 6'-N-acetyltransferase Type Ib [AAC(6')-Ib]-Mediated Aminoglycoside Resistance: Phenotypic Conversion to Susceptibility by Silver Ions. Antibiotics (Basel). 2020 Dec 31;10(1):29. [PMC]

Tada T, Miyoshi-Akiyama T, Shimada K, Shimojima M, Kirikae T. novel 6'-n-aminoglycoside acetyltransferase AAC(6')-Iaj from a clinical isolate of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2013 Jan;57(1):96-100. doi: 10.1128/AAC.01105-12. [PMC]

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Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.

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The conversion of adenosines to inosines by ADARs is known as RNA editing.

RNA is the blueprint for protein production in cells. Adenosine deaminases acting on RNA (ADAR) present in cells can catalyze single-base changes in RNAs. Adding a guide RNA to ADAR allows RNA editing of complementary strands. Compared to DNA editing, RNA editing is reversible since cells constantly metabolize new RNA copies. A transcript’s sequence is altered during RNA-editing by insertion, deletion, or modification of nucleotides. The result is a change in information content from the original encoded genomic transcript.

Adenosine deaminases acting on RNA (ADARs) enzymes convert adenosine to inosine in duplex RNA via a deamination reaction of adenosine. Inosine functions similarly to guanosine in many cellular processes including during splicing, translation, and reverse transcription by base pairing with cytidine.

Adenosine-to-inosine editing can form alternative splice variants, alter microRNA processing and targeting, change codon sequences and suppress the activation of the innate immune system by endogenous double-stranded RNAs.

Dysregulated RNA editing is linked to neurological disorders such as epilepsy, seizures, and Amyotrophic Lateral Sclerosis (ALS) involving ADAR2. Apparently, mutations in the ADAR1 gene also cause the autoimmune disease Aicardi-Goutieres Syndrome and Dyschromatosis Symmetrica Hereditaria.

Adenosine-to-inosine (A-to-I) editing enables the treatment of guanosine-to-adenosine (G-to-A) mutations. A-to-I editing involves the hydrolytic deamination of adenosine to an inosine base. The protein family of RNA-specific deaminases called “adenosine deaminases acting on RNA (ADARs) mediate this reaction by acting on different types of RNA. However, editing events in coding regions of mRNAs are of particular interest since every A-to-I change is read as an A-to-G change during translation. Utilizing this reaction allows the recoding of RNA sequences to correct genetic mutations within mRNAs. One major challenge is re-directing ADAR’s activity towards A’s that are not naturally edited.

Discovery of RNA editing

In 1986, Benne et al. reported that the mitochondrial cytochrome oxidase (cox) subunit II gene from trypanosomes contains a frameshift at amino acid 170. Since no second version of the coxII gene was detected, the research group concluded that the extra nucleotides are added during or after transcription of the frameshift gene by an RNA-editing process.

Demonstration of the possibility of RNA editing

In 1995, Woolf et al. pointed out that treating genetic diseases caused by specific base substitutions is possible by rationally designed RNA editing of mutated RNA sequences. To demonstrate that therapeutic RNA editing is possible, the research group used a synthetic complementary RNA oligonucleotide to direct the correction of a premature stop codon mutation in dystrophin RNA.

The researchers employed complementary RNA oligonucleotides in conjugation with cellular double-stranded RNA adenosine deaminase (dsRAD). Directed RNA editing involves hybridization of the complementary RNA oligonucleotide to a premature stop codon followed by treatment with nuclear extracts containing the cellular enzyme double-stranded RNA adenosine deaminase. This experiment resulted in a dramatic increase in the expression of a downstream luciferase coding region. The analysis of the cDNA sequence revealed the deamination of the adenosine in the UAG stop codon to inosine by double-stranded RNA adenosine deaminase. As a proof of principle, the injection of oligonucleotide-mRNA hybrids into Xenopus embryos also increased luciferase expression.

.Figure 2: (Left) Adenosine Deaminase Acting on dsRNA mutant E488Q in complex with a dsRNA from the human GLI1 gene sequence. (Right) Reaction mechanism of ADAR2 based deamination.

ADARs contain two main structural motifs. The first motif contains a double-stranded RNA binding Domain (dsRBD). The second motif contains a Deaminase Domain (DD) carrying out the catalytic activity. ADARs use a flipping mechanism to move the adenosine out of the A-form RNA helix into the enzyme’s catalytic pocket where the A-to-I conversion occurs.

Exon-skipping antisense oligonucleotide strategy

Kim et al. recently used an exon-skipping antisense oligonucleotide (ASO) strategy in cultured human bronchial cells expressing the mutation to achieve gene-specific NMD evasion in the hope to cure the disorder. A mixture of two antisense oligonucleotides (ASOs) was utilized that promotes the skipping of exon 23 of the CFTR-W1282X mRNA resulting in NMD resistance thereby preserving the reading frame. The hope is to develop a therapeutic approach allowing the treatment of CF in affected people.

Cystic fibrosis (CF) is an inherited disorder of the lungs, digestive system, and other organs of the human body. Cystic fibrosis affects the cells producing mucus, sweat and digestive juices. In people with CF, a gene defect causes these secretions to become sticky and thick. These thick secretions plug up tubes, ducts and passageways, especially in lungs and pancreas. Presently, there is no treatment available for CF caused by the CFTR-W1282X mutation located on CFTR exon 23. Nonsense-mediated messenger RNA (mRNA) decay (NMD) degrades the CFTR-W1282X mRNA. The result is a low level of functional CFTR protein.

Reference

Bhakta S, Tsukahara T. Artificial RNA Editing with ADAR for Gene Therapy. Curr Gene Ther. 2020;20(1):44-54. [PubMed]

Benne R, Van den Burg J, Brakenhoff JP, Sloof P, Van Boom JH, Tromp MC. Major transcript of the frameshifted coxII gene from trypanosome mitochondria contains four nucleotides that are not encoded in the DNA. Cell. 1986 Sep 12;46(6):819-26. [PubMed]

Bhakta Sonali and Tsukahara Toshifumi *, Artificial RNA Editing with ADAR for Gene Therapy, Current Gene Therapy 2020; 20(1) . [Eurekaselect]

Cystic Fibrosis

Kim, Y.J., et al., “Exon-skipping antisense oligonucleotides for cystic fibrosis therapy”, PNAS, January 18, 2022. [PNAS]

Thuy-Boun AS, Thomas JM, Grajo HL, Palumbo CM, Park S, Nguyen LT, Fisher AJ, Beal PA. Asymmetric dimerization of adenosine deaminase acting on RNA facilitates substrate recognition. Nucleic Acids Res. 2020 Aug 20;48(14):7958-7972. [PMC]

Woolf TM, Chase JM, Stinchcomb DT. Toward the therapeutic editing of mutated RNA sequences. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8298-302. [PMC]

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Nevertheless, the resultant antibody-drug conjugates continued to exhibit side effects.  In the case of Enhertu, side effects include nausea, diarrhea, anemia, decreased clotting, etc.  Kadcyla is associated with thrombocytopenia (low platelet count), hepatotoxicity, heart damage, neuropathy, etc.  Several of these symptoms also occurred following the Lumoxiti treatment--i.e. in addition to the swelling of limbs, hypotension, breathing difficulty, abnormal red blood cell destruction, etc.

 To explain the off-target effects of antibody-drug conjugates, potential uptake of monoclonal antibodies lacking terminal galactose on the Fc domain by mannose receptors expressed in various immune cell types, leading to their depletion, has been suggested (Gorovits et al., 2012).   Further, the binding of antibodies to reticuloendothelial system may cause toxicity to the liver, bone marrow, and spleen.   Other limitations of using antibodies include failure to reach the brain through blood -brain barrier, poor penetration of solid tumors due to high molecular weight (~160 kD), and premature release of drugs while in circulation.  The difficulty of their preparation along with the exorbitant cost for large-scale production represents other disadvantages (Le Joncour et al., 2017).

 To address these shortcomings, peptides are increasingly being tapped for targeted drug delivery.  Peptides (as short as 7 to 12 mer) can adopt myriad conformations by altering their sequences.  Previously, these properties have been exploited to isolate 'homing peptides' (Joliot et al.., 2004).    Due to their minuscule molecular weight (1 to 2 kD), peptides allow deeper penetration into tissues--as has been demonstrated for solid tumors (Hong et al., 2000).  Additionally, peptide-conjugated drugs exhibit excellent tolerability, lesser immunogenicity, are easier to produce in large quantities, simpler purification scheme, etc.  Plus, developing peptide-drug conjugate using previously FDA-approved drugs is far less costly than discovering novel drugs, and the conjugation process is generally less complex and rapid (Vrettos et al., 2018).  

                         

During the last several decades, significant efforts have been made by the biopharmaceutical industries to improve their application.  First, to isolate 'homing peptides', bacteriophage-based random peptide-display technology has been used extensively.  The externally displayed random peptides as part of the coat protein of M13 bacteriophages served as the main 'work horse' to isolate peptides targeting various tumors or normal tissues (Brown, 2010).  Others peptides have been derived from naturally occurring sources like the neurotransmitters (ex. octreotide) or matrix proteins (ex. RGD).  These include peptides targeting cancer cells directly (ex. LyP-1, HN-1, TGN peptide), tumor-associated vasculature (ex. RGD, NGR), tumor-associated macrophages, tumor lymphatics, etc. (Arap et al., 1998; Gray et al., 2014).  For other types of disorders, peptides that target various normal tissues (ex. immune cells, cardiac cells (ex. CTP peptide), muscle cells, kidney cells, liver cells) have been isolated (Gray et al., 2014).  The majority of previously isolated peptides bind to cell surface molecules albeit a minority retains the potential to internalize.

 Second, peptides have been engineered to improve their pharmacokinetic (traffic) or pharmacodynamic (mechanism) properties.  To increase binding affinity, peptides may be cyclized via 'stapling' (to lock their secondary structures into desired conformations).  To increase half-life, the N- or C-terminus may be chemically modified (to avoid degradation by exoproteases).   Further, specific amino acids may be replaced with D-configuration (potentially alternate residues) or unnatural amino acids (Le Joncour et al., 2018).  To avoid renal clearance, higher molecular weight entities such as PEG, fatty acid, branched amylopectin, polysialic acid, hydroxyethyl starch, etc. may be linked.  To improve oral bioavailability, it may be coated with the acid-stable coating (ex. citric acid) in the stomach, which breaks apart when it reaches higher pH of the intestine (Cooper et al., 2020).  Nevertheless, some of these modifications may run the risk of attenuating/losing the binding properties.

 Third, to conjugate peptides to drugs, both the cleavable and non-cleavable types of the linker are available (Hoppenz et al., 2020).  The cleavable types include disulfide (S-S) linkers that become cleaved upon reduction by glutathione intracellularly, pH-sensitive linkers (ex. hydrazine, acetals, imines) that are hydrolyzed at acidic pH in endosomes, etc.  Cleavable linkers also include peptides that are cleaved by matrix-metalloproteinases (MMPs) extracellularly and others (ex. Valine-Alanine or Valine-Citrulline) that are cleaved by cathepsin B inside the endosome (albeit the latter becomes cleaved in the mouse plasma) (Cooper et al., 2021).  Commonly used linkers containing 'enzyme hydrolyzable unit' may consist of carboxylic ester or an amide bond (Vrettos et al., 2018).  The 'self-immolative' (self-destructive) linker PABC (para-amino benzyl alcohol) can be used to connect peptide with drug as it could be cleaved at the enzyme hydrolyzable unit to release the peptide, and then undergo 1,6-elimination to release the unmodified drug.  Alternatively, aryl sulfate linkers that undergo 1,6-elimination to release unmodified drugs have been utilized.  The non-cleavable type, which is more stable in circulation, may be used when the peptide is intended to undergo degradation upon internalization, releasing the drug-linker complex into the cytosol.

 Recently, U. S. FDA issued the first approval of the peptide-drug conjugate to treat gastroenteropancreatic neuroendocrine tumors.  Somatostatin is a neurotransmitter and there are 5 subtypes of somatostatin receptors (SSTR); among them, SSTR2 and SSTR5 are highly expressed in neuroendocrine tumors.  Neuroendocrine tumors are derived from neuroendocrine cells and may occur in various tissue types.  Somatostatin analogs have been used as probes to image tumors in the 1980s-1990s.  Octreotide labeled with radioisotope ¹¹¹In (gamma emitter; Octreoscan, Mallinckrodt) was useful for tumor imaging (albeit less effective in therapy), and was FDA approved for imaging in 1994 (ex. for PET scan with positron emitting isotope or SPECT with gamma ray emitter).  Subsequently, the FDA approved (in 2018) LUTATHERA (⁷⁷Lu-DOTA-octreotate; Novartis), representing ⁷⁷Lu (beta emitter) conjugated to Octreotate using the bi-functional chelating agent DOTA, was able to achieve longer progression-free survival with a higher therapeutic response rate in advanced midgut neuroendocrine tumors (Hennrich et al., 2019).

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies. It provides custom conjugation of small molecules such as chemical drugs, metabolites and labeled compounds with synthetic or natural polymers (enzymes, peptide, protein, oligonucleotide, antibody, dendrimer, nanoparticle, etc).  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  It has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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References

Arap W, Pasqualini, R, et al.  Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model. Science 279: 377-380 (1998).  PMID: 9430587

Brown KC. Peptidic tumor targeting agents: the road from phage display peptide selections to clinical applications.  Curr Pharm Des.   16:1040-54 (2010).  PMID: 20030617

Cooper BM, Iegre J, et al. Peptides as a platform for targeted therapeutics for cancer: peptide-drug conjugates (PDCs).     Chem Soc Rev.  50:1480-1494 (2021).  PMID: 33346298

Gorovits B, Krinos-Fiorotti C.  Proposed mechanism of off-target toxicity for antibody-drug conjugates driven by mannose receptor uptake.  Cancer Immunol Immunother.  62:217-23 (2013).  PMID: 23223907

Gray BP, Brown KC. Combinatorial peptide libraries: mining for cell-binding peptides.  Chem Rev.  114:1020-81 (2014).  PMID: 24299061

Hennrich U, Kopka K. Lutathera®: The First FDA- and EMA-Approved Radiopharmaceutical for Peptide Receptor Radionuclide Therapy.  Pharmaceuticals (Basel).  12:114 (2019).  PMID: 31362406

Hong FD, Clayman GL.  Isolation of a peptide for targeted drug delivery into human head and neck solid tumors.  Cancer Res.  60:6551-6 (2000).  PMID: 11118031

Hoppenz P, Els-Heindl S, et al.  Peptide-Drug Conjugates and Their Targets in Advanced Cancer Therapies.  Front Chem. 8: 571 (2020).  PMCID: PMC7359416

Joliot A, Prochiantz A.  Transduction peptides: from technology to physiology.  Nat Cell Biol.  6:189-96 (2004).  PMID: 15039791

Le Joncour V, Laakkonen P.  Seek & Destroy, use of targeting peptides for cancer detection and drug delivery.  Bioorg Med Chem. 26:2797-2806 (2018).  PMID: 28893601

Vrettos EI, Mező G, et al. On the design principles of peptide-drug conjugates for targeted drug delivery to the malignant tumor site.  Beilstein J Org Chem. 14:930-954 (2018).  PMID: 29765474

Synthetic and natural RNA molecules can contain sequence motifs that stimulate the immune system. In addition to mediating RNA interference (RNAi), silencing RNAs (siRNAs) can induce the innate immune system as well.

Pattern recognition receptors (PRRs) recognize and respond to RNA sequence motifs uniquely. In general, when designing regular siRNAs, the presence of immune-stimulating sequence motifs is not desired. Therefore, three sequence motifs, "UGUGU," "GUCCUUCAA," and "AUCGAU(N)nGGGG," need to be included in an immune-motif avoidance list. Also, U-rich sequences or sequences with biased nucleotide content, such as (G + U) >> (C + A) or with A + U or G + U rich motifs, also need to be avoided.

However, some clinical applications of RNA interference (RNAi) may benefit from siRNAs' activation of the immune system. In particular, the design of small interfering RNAs (siRNAs) with antitumor and antiviral activities.

Blood immune cells express a receptor set that helps detect pathogens and help mount an appropriate immune response. For example, Toll-like receptors (TLRs) detect DNA, RNA, or bacterial and fungal components together with modified endogenous molecules such as oxidized low-density lipoprotein (LDL) or fibrillar amyloid-β peptides. siRNAs can recruit immune receptors specialized in RNA detection, for example, TLR3 and TLR7. Double-stranded RNA can induce immune activity associated with producing a range of cytokines. Usually, this is perceived as an unwanted nonspecific effect of in vivo siRNA when administered. The use of chemically modified nucleic acids in siRNA can prevent this response, for example, using 2’-O-methyl ribonucleotides or bridged nucleic acids (BNAs).

Design of immuno-stimulatory siRNAs

Incorporating specific mismatches in the siRNA duplex's passenger strand allows the creation of an immune-stimulatory motif. The introduction of such a mismatch between bases 9 and 12 from the 5’-end of the passenger strand increases RNAi potency. However, the introduction of such a mismatch strongly depends on the siRNA sequence selected.

According to Gantier:

[1]  A uridine bulge replacing bases 9 to 12 in the passenger strand of the siRNA activates TLR8.

[2]  The fusion of a CpG DNA moiety containing phosphorothioate bonds to the 5’-end of the guide strand also increases the immune response.

[3]  Adding a triphosphate group to both strands' 5'- ends activates RIG-I.

Triphosphate moieties can be added chemically or via in-vitro transcription.

In 2015, Xu et al. identified a natural viral RNA motif in Sendai virus RNA that optimizes sensing of viral RNA by RIG-I. This RNA motif named DVG70-114 is essential for the potent immunostimulatory activity of 5′-triphosphate-containing Send virus (SeV) iDVGs. According to Xu et al., DVG70-114 enhances viral sensing by the host cell independently of the long stretches of complementary RNA flanking the iDVGs. The motif retains its stimulatory potential when transferred to otherwise inert viral RNA. In vitro analysis showed that DVG70-114 augments the binding of RIG-I to viral RNA and promotes enhanced RIG-I polymerization. The study defined a new natural viral PAMP enhancer motif that promotes viral recognition by Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) and confers potent immunostimulatory activity to viral RNA.

Table 1: RNA sequence motifs that stimulate immune responses

The cytosine-phosphate-guanine class C (CpG-C) immunostimulatory sequence oligodeoxynucleotides (ISS-ODNs) also activate human B cells and dendritic cells (DCs).  These properties suggest a potential use as an adjuvant to enhance vaccine efficacy. 

Reference

Lopez C.; Method and composition of stimulating immune response using potent immunostimulatory RNA motifs: PubChem [Internet]. Bethesda (MD): National Library of Medicine (US), National Center for Biotechnology Information; 2004. PubChem Patent Summary for US-10624964-B2. [US Patent]

Gantier, M.P.; Strategies for designing and validating immunostimulatory siRNAs. In Debra J. Taxman (ed.); siRNA design: Method and Protocols. MMB 942. Chapter 10 pp 179. Humana Press 2013.

Forsbach A, Nemorin JG, Montino C, Müller C, Samulowitz U, Vicari AP, Jurk M, Mutwiri GK, Krieg AM, Lipford GB, Vollmer J. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J Immunol. 2008 Mar 15;180(6):3729-38. doi: 10.4049/jimmunol.180.6.3729. PMID: 18322178. [Jimmunol]

Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol. 2002;20:709-60. [Annual Reviews]

Zhongji Meng and Mengji Lu; RNA Interference-Induced Innate Immunity, Off-Target Effect, or Immune Adjuvant? Front. Immunol., 23 March 2017 [Frontiersin]

Netea, M.G., Domínguez-Andrés, J., Barreiro, L.B. et al. Defining trained immunity and its role in health and disease. Nat Rev Immunol 20, 375–388 (2020). [nature]

Xu J, Mercado-López X, Grier JT, Kim WK, Chun LF, Irvine EB, Del Toro Duany Y, Kell A, Hur S, Gale M Jr, Raj A, López CB. Identification of a Natural Viral RNA Motif That Optimizes Sensing of Viral RNA by RIG-I. mBio. 2015 Oct 6;6(5):e01265-15.  [PMC]

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An effective drug delivery system enables the release of the active ingredient where it is most needed to achieve the desired therapeutic effect. For cellular therapeutics to work well, the precise delivery of drugs is essential. Off-target effects of drugs are a significant hurdle in designing efficient therapies. Drug delivery technologies allowing the accurate targeting of disease-causing molecules or pathways hold the potential to minimize off-target effects during systematic drug administration.

For example, drugs administrated into blood vessels need to penetrate various biological barriers before reaching target sites. When administered, drugs encounter several obstacles, including enzymes, such as proteases and nuclease, renal filtration, sequestering by the mononuclear phagocyte system, or the endothelial barrier. Hence, delivery systems are needed to target specific tissues and cell compartments. Tissue- and cell compartment-specific drug delivery has the potential to avoid unnecessary toxicities resulting in the improvement of quality of life and patient well-being.

Peptide delivery into the nucleus

Loregian et al. in 1999 reported an intracellular peptide delivery system for targeting specific cellular compartments. The research group designed a chimeric protein (EtxB-Pol = EtxB-peptide) consisting of the nontoxic B subunit of Escherichia coli heat-labile enterotoxin.

The researh group fused EtxB to a 27-mer peptide derived from the DNA polymerase of herpes simplex virus 1. The study revealed that the peptide dissociates the interaction complex between an accessory factor, UL42, encoded by the virus, and the DNA synthesis machinery. The chimeric protein entered cells through the acidic endosomal compartments where the Pol peptide is cleaved from the protein before translocating into the nucleus. After translocation, the antiviral peptide is localized in the nucleus. As a result, the nontoxic receptor-binding component of E. coli enterotoxin (EtxB) mediates the intracellular delivery to access the nucleus. The selected peptide corresponds to the C-terminal 27 amino acids of HSV-1 DNA polymerase (POL). This polymerase is involved in the interaction with UL42. The free peptide or EtxB alone does not affect virally infected cells; however, the EtxB-Pol fusion protein selectively inhibits HSV-1 replication.

The delivery system described by Loregian et al. enables the delivery of peptides into the nucleus allowing the disruption of specific protein-protein interactions.

The B subunits of cholera toxin (CtxB) and Escherichia coli heat-labile enterotoxin (EtxB) are potent systemic and mucosal adjuvants. These adjuvants bind to their ganglioside receptor GM1 present in lipid rafts. After binding, the proteins undergo rapid cross-aggregation and internalization. This property of EtxB has led to its use as a carrier molecule for the intracellular delivery of exogenous peptides or antigens.

The heat-labile enterotoxin (EtxB) is a carrier molecule for the intracellular delivery of exogenous peptides or antigens.

Examples are the delivery of peptide sequences derived from ribonucleotide reductase and DNA polymerase of herpes simplex virus type 1 to intracellular compartments. The protein complex remained functionally intact. 

This delivery system also allowed the delivery of peptides corresponding to known major histocompatibility complex (MHC) class I-restricted epitopes from the ovalbumin and influenza nucleoprotein to the MHC class I antigen processing and presentation pathway in murine dendritic cells.

Reference

Adepu S, Ramakrishna S. Controlled Drug Delivery Systems: Current Status and Future Directions. Molecules. 2021 Sep 29;26(19):5905. [PMC]

de Haan, L., A. R. Hearn, A. J. Rivett, and T. R. Hirst. 2002. Enhanced delivery of exogenous peptides into the class I antigen processing and presentation pathway. Infect. Immun.70:3249-3258. [PMC]

Intranuclear Delivery of an Antiviral Peptide Mediated by the B Subunit of Escherichia coli Heat-Labile Enterotoxin

Lencer, W. I., T. R. Hirst, and R. K. Holmes. 1999. Membrane traffic and the cellular uptake of cholera toxin. Biochim. Biophys. Acta1450:177-190. [ScienceDirect]

Loregian A, Papini E, Satin B, Marsden HS, Hirst TR, Palù G. Intranuclear delivery of an antiviral peptide mediated by the B subunit of Escherichia coli heat-labile enterotoxin. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):5221-6. doi: 10.1073/pnas.96.9.5221. [PMC]

Marcello, A., A. Loregian, A. Cross, H. Marsden, T. R. Hirst, and G. Palu. 1994. Specific inhibition of herpes virus replication by receptor-mediated entry of an antiviral peptide linked to Escherichia coli enterotoxin B subunit. Proc. Natl. Acad. Sci. USA91:8994-8998. [PNAS]

Matković-Calogović D, Loregian A, D'Acunto MR, Battistutta R, Tossi A, Palù G, Zanotti G. Crystal structure of the B subunit of Escherichia coli heat-labile enterotoxin carrying peptides with anti-herpes simplex virus type 1 activity. J Biol Chem. 1999 Mar 26;274(13):8764-9. PMID: 10085117. [PDB ID 1LTR.] 

Ong KW, Wilson AD, Hirst TR, Morgan AJ. The B subunit of Escherichia coli heat-labile enterotoxin enhances CD8+ cytotoxic-T-lymphocyte killing of Epstein-Barr virus-infected cell lines. J Virol. 2003 Apr;77(7):4298-305. doi: 10.1128/jvi.77.7.4298-4305.2003. [PMC]

Williams, N. A., T. R. Hirst, and T. O. Nashar. 1999. Immune modulation by the cholera-like enterotoxins: from adjuvant to therapeutic. Immunol. Today20:95-101. [PubMed]

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Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.

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Oligonucleotide-conjugated antibodies are valuable tools for protein diagnostics and therapeutics. Oligonucleotide-conjugated antibodies increase the sensitivity of protein assays tremendously and allow multiplexing approaches. Oligonucleotide-conjugated antibodies also allow therapeutic cell targeting with therapeutic antibody-drug conjugates (ADCs). Other applications for oligonucleotide-conjugated antibodies are immune-PCR, hybridization chain reactions technics, enzyme activity studies, sequential fluorescence hybridization methods, and applications combining the conjugates with next-generation sequencing (NGS).

Since site-specific conjugation strategies based on protein tags or unnatural amino acids require recombinant cloning steps, non-site-specific conjugation methods allow the construction of large libraries of oligonucleotide-conjugated antibodies.

Recently, Wiener et al. combined a non-site-directed antibody conjugation method using copper-free click chemistry with ion-exchange chromatography to obtain purified single and double oligonucleotide-conjugated antibodies.

The research group utilized the copper-free click chemistry reaction to obtain single, double, and multiple oligonucleotide-conjugated antibodies. The cross-linking of dibenzocyclooctyne (DBCO) molecules to the antibodies' amino side chains using a hydroxysuccinimide (NHS) reaction allowed functionalization of the antibodies. Employing 3-azidopropionic acid sulfo-NHS ester (3AA-NHS) allowed the functionalization of amine-modified oligonucleotides. In this approach, first, the antibodies are reacted with DBCO-NHS ester. Mixing azide-modified oligonucleotides to DBCO-antibodies generated the conjugates. Oligonucleotides used in this study carried 5’-amino modifier C6 dT and were double HPLC-purified.

DBCO Copper-Free Click Reaction

Step 1: Activate and functionalize the antibody with DBCO.

The research group recommended removing unreacted reagents via dialysis, spin-column-based gel-filtration desalting chromatography, spin column-based ultrafiltration, and cut-off membranes with a 3 or 7 kDA molecular weight cut-off.

Step 2: Activate and functionalize the oligonucleotide with an azide group.

The research group recommended removing unreacted reagents via dialysis, spin-column-based gel-filtration desalting chromatography, spin column-based ultrafiltration, and cut-off membranes with a 3 or 7 kDA molecular weight cut-off.

Step 3: Mix the two activated biomolecules to form the conjugate.

Step 4: Remove excess of azide or DBCO activated biomolecule with a scavenger molecule, via dialysis or purification. Analyze the resulting conjugates via ion exchange chromatography, SDS-electrophoresis or matrix assisted time-of-flight mass spectrometry (MALDI-TOF-MS).

A dialysis step allowed the removal of excess reagents. Ion exchange chromatography enabled the separation of antibody-oligonucleotides conjugates and guided the selection of reaction conditions to improve yields.

The researchers reported that this conjugation workflow allows the conjugation of multiple antibodies in various quantities with quantities as low as 25 ng and a yield of 5 ng.

For quantitative protein detection via amplification with antibody-oligonucleotide conjugates, the purity and the number of conjugated oligonucleotides need to be precisely controlled.

Reference

Wiener J, Kokotek D, Rosowski S, Lickert H, Meier M. Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics. Sci Rep. 2020 Jan 29;10(1):1457. [PMC]

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Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to antibodies, fatty acids and lipids, cholesterol, tocopherol, peptides, other proteins, as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.

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Most recurrent cancers are detected within 5 years of initial diagnosis.  However, recurrent cancer has been documented even after 50 years, ex. renal cell carcinoma (kidney cancer) (Walter et al., 1960).  For breast cancer, 'local recurrence' refers to cancers that have returned to the same organ after the treatment.  'Regional recurrence' refers to cancers that have relapsed at a nearby location (i.e. near neck, armpit, collar bone) containing lymph nodes (as tumor cells may spread via lymphatic vessels).  'Distal recurrence' refers to cancers that have metastasized to distinct organs (ex. bone, lung, liver, brain).

 An initial insight into the existence of "dormant" cancer cells came from transplantation studies.  Certain recipients of organ transplants subsequently developed tumors (~160 out of 1,000,000 organ transplant cases).  Subsequently, it was found that the dormant tumor cells were inadvertently transferred when the transplanted organs came from donors with a history of malignancy (Friberg et al. 2015).  Using the radio-graphically determined volumes of primary and secondary cancers of the same tumor at different time intervals, the growth rate of malignant tumors was calculated (in ~2,000 patients).  The results suggested that >75% of human malignant tumors (ex. breast cancer) may have already metastasized at the time of primary tumor diagnosis (Friberg et al. 2015; Pedersen et al., 2022).  The persistence of dormant cells is supported by the ability to detect circulating tumor cells (CTCs) even decades after the treatment (of primary tumor) (Meng et al., 2004).


                    
                         

Approximately ~30% (ranges from 10 to 40%) of those diagnosed with breast cancer are expected to develop metastasis, with ~90% succumbing to recurrent cancer eventually (Pedersen et al., 2021).  Among the currently used chemotherapeutics to treat metastatic breast cancer are antimicrotubule drugs (ex Taxol) or topoisomerase inhibitors.  However, the use of either drug is associated with severe side effects.  To lessen toxicity, several drugs have been conjugated to tumor-targeting antibodies for selective delivery.

 Several antibody-drug conjugates (ADC) have been approved by FDA for cancer treatment.   Trastuzumab emtansine (T-DM1 by Genentech/Roche) consists of maytansine attached to Her2-recognizing monoclonal antibody.  Sacituzumab govitecan (Trodelvy by Immunomedics) represents SN-38 (inhibits topoisomerase I) linked to monoclonal antibody targeting Trop-2 (tumor-associated calcium signal transducer 2).  Trastuzumab deruxtecan (Enhertu by Daichi Sankyo/AstraZeneca) is comprised of deruxtecan (topoisomerase I inhibitor) conjugated to anti-Her2 antibody (Herceptin).  Despite being guided by antibodies, side effects remain a critical issue for these ADCs.

 As a result, there has been an increasing pharmaceutical attempt to develop peptide-drug conjugates.  A recent report by the investigators at the Yale University School of Medicine (USA) describes the delivery of topoisomerase inhibitors using a peptide (Gayle et al., 2021).  Exatecan, an analogue of camptothecin, is an inhibitor of the topoisomerase I enzyme.  In cancer cells, the preferred processing of glucose via 'anaerobic glycolysis' than via citric acid cycle, i.e. Warburg effect (Nobel prize, 1931), leads to the production of lactic acid (due to conversion of pyruvate by lactate dehydrogenase).  The accumulation of lactic acids confers an acidic milieu to the tumor cell surface (pH 6.0-6.5) and tumor cell microenvironment (pH 6.7-7.1). The pHLIP ('pH-Low Insertion Peptide': AEQNPIYWARYADWLFTTPLLLLDLALLVDADEGT) peptide adopts an alpha helix conformation at low pH for 'directional' insertion across the cell membrane to translocate a C-terminally (the inserting end) conjugated drug into the cytosol (Wyatt et al., 2018).  Exatecan was conjugated to pHLIP (called 'CBX-12') using a glutathione-sensitive (S-S) cleavable linker.  Intraperitoneally (abdominal cavity) injected CBX-12 localized to tumors selectively in a mouse xenograft model of Her2-overexpressing breast cancer.

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies. It provides custom conjugation of small molecules such as chemical drugs, metabolites and labeled compounds with synthetic or natural polymers (enzymes, peptide, protein, oligonucleotide, antibody, dendrimer, nanoparticle, etc).  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  It has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

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References

Friberg S, Nyström A. Cancer Metastases: Early Dissemination and Late Recurrences.  Cancer Growth Metastasis. 8:43-9 (2015).  PMID: 26640389

Gayle S, Aiello R, et al. Tumor-selective, antigen-independent delivery of a pH sensitive peptide-topoisomerase inhibitor conjugate suppresses tumor growth without systemic toxicity.  NAR Cancer.  3:zcab021 (2021).  PMID: 34316708

Meng S, Tripathy D, et al. Circulating tumor cells in patients with breast cancer dormancy.  Clin Cancer Res. 10:8152-62 (2004).  PMID: 15623589

Pedersen RN, Esen BÖ, et al. The Incidence of Breast Cancer Recurrence 10-32 Years After Primary Diagnosis.  J Natl Cancer Inst. 114:391-399 (2022).  PMID: 34747484

Walter CW, Gillespie DR. Metastatic hypernephroma of fifty years' duration.  Minn Med.  43: 123-5 (1960).  PMID: 13782941

Wyatt LC, Moshnikova A, et al. Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors.  Proc Natl Acad Sci U S A. 115:E2811-E2818 (2018).  PMID: 29507241

Shuttle peptides are peptides or peptide conjugates designed to allow the transport or delivery of therapeutic payloads, such as drugs, oligonucleotides, or proteins, through cell or tissue barriers, for example, the blood-brain barrier, to their targets in cells, for instance, to targets in the nervous system.

The delivery of drugs into the brain is a significant challenge. The blood-brain barrier (BBB) prevents most drugs from reaching targets in the brain. Molecular transport vehicles or shuttles based on peptides offer new avenues of crossing the BBB. Well-designed shuttle peptides are of lower cost, have reduced immunogenicity, and are biological and chemical very versatile. Improved drugs for the central nervous system (CNS) can potentially enhance the well-being of many people suffering from neurological diseases such as brain cancer.

The BBB is a complex of tight junctions of the brain’s capillary endothelial cells. Several transport systems are part of this cell complex. For energy, the BBB supplies creatine to the brain, transported by the creatine transporter localized at the brain capillary endothelial cells.

Brain capillary endothelial cells express serotonin and norepinephrine transporters, organic anion transporter 3 (OAT3), and the amino acid transporter ASCT2.

Oller-Salvia et al., in 2016, reported a list of shuttle peptides that cross the BBB (Table 1). 

Table 1: Features of some BBB shuttle peptides

Reference

Jiang T, Olson ES, Nguyen QT, Roy M, Jennings PA, Tsien RY. Tumor imaging by means of proteolytic activation of cell-penetrating peptides. Proc Natl Acad Sci U S A. 2004 Dec 21;101(51):17867-72. [PMC]

Ohtsuki S. New aspects of the blood-brain barrier transporters; its physiological roles in the central nervous system. Biol Pharm Bull. 2004 Oct;27(10):1489-96. [pdf]

Oller-Salvia, Benjamí and Sánchez-Navarro, Macarena and Giralt, Ernest and Teixidó, Meritxell. Blood–brain barrier shuttle peptides: an emerging paradigm for brain delivery, Chem. Soc. Rev., 2016, 45, 17, 4690-4707. The Royal Society of Chemistry. [RSC]

J. Spengler, J. C. Jiménez, K. Burger, E. Giralt and F. Albericio; Abbreviated nomenclature for cyclic and branched homo- and hetero-detic peptides. J. Pept. Res., 2005, 65 , 550-555. [PubMed]

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According to the human genome project, there are 30,000 human genes. Approximately 3,000 are disease-modifying genes, and around 3,000 are druggable genes. These numbers indicate that only 2 to 5 % of the human genome are targets for small molecules (approximately 600–1,500), which presents an opportunity to use peptides or large molecules to treat human diseases.

Peptides have a high potential for targeting and cellular delivery of next-generation therapeutic drugs. However, a significant limitation is their proteolytic susceptibility in biological environments. The chemical enhancement of these peptide types confers resistance to peptides selected for targeted cell delivery. Strategies for chemical enhancement of peptides to increase resistance to proteolysis include:

• Protecting N- and C-terminal ends.

• Backbone modifications.

• Incorporating amino acids with non-natural side chains.

• Conjugation.

The enantio and retro-enantio isomerization approach enhances peptide efficiency when applied to peptides for drug delivery into the brain. However, the cyclization approach also increases peptide transport capacity because of the cyclic peptides' increased protease resistance and affinity.

Historically monoclonal antibodies are the basis of many successful targeting therapeutics. However, peptides have emerged as molecular delivery vehicles with increased tissue selectivity for nano- and biotherapeutics other than antibodies.

Like antibodies, peptides can possess a high affinity and selectivity to biological targets. Peptides are synthetically accessible, easy to modify, and generally have low immunogenicity. The biochemical properties of specific peptides help nano- and biotherapeutics to cross the cell membrane to achieve their intracellular activity.

A variety of chemical modifications allow the enhancement of peptides with increased resistance to proteases. Also, combining more than one modification strategy allows the design and synthesis of "peptidomimetics." Peptidomimetics are small protein-like peptide chains designed to mimic a peptide or a small part of a protein, such as the part of a protein interacting with another protein or DNA or RNA. (Peptidomimetic). Often D-amino acids are utilized for the design of peptidomimetics.

Typical strategies utilized are N- and C-terminal protection, backbone modifications, side-chain substitution, cyclization, and conjugation approaches.

Table 1: A selection of targeting and cell-penetrating peptides
 

Cyclic peptide nomenclature according to Spengler et al. 2005* Trifunctional chemical scaffold. (Source Lucana et al. 2021).

Reference

Hopkins AL, Groom CR. Opinion: the druggable genome. Nat Rev Drug Discov. 2002;1(9):727–30. [PubMed]

Lucana MC, Arruga Y, Petrachi E, Roig A, Lucchi R, Oller-Salvia B. Protease-Resistant Peptides for Targeting and Intracellular Delivery of Therapeutics. Pharmaceutics. 2021 Dec 2;13(12):2065. [PMC] 

Pelay-Gimeno M., Glas A., Koch O., Grossmann T.N. Structure-Based Design of Inhibitors of Protein-Protein Interactions: Mimicking Peptide Binding Epitopes. Angewandte. 2015;54:8896–8927.

J. Spengler, J. C. Jiménez, K. Burger, E. Giralt and F. Albericio; Abbreviated nomenclature for cyclic and branched homo- and hetero-detic peptides. J. Pept. Res., 2005, 65 , 550-555.  [PubMed]

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Chemical conjugation and cross-linking reactions depend on the reactivities of functional groups present in biomolecules and the specificities of conjugation and cross-linking reagents used. In most cases, the selected reaction conditions must preserve the biomolecules' three-dimensional structures to be conjugated or cross-linked. Hence, chemical reagents and reactions need to be chosen that do not inactivate the biological functions of the target molecule. For example, when selecting an amino acid side chain in a protein for conjugation, the amino acid must be located on the protein's surface to be available for the reaction. All proteins are composed of amino acids. The common twenty amino acids have side chains of different sizes, shapes, charges, and chemical reactivity. In the case of proteins, hydrophobic amino acids are generally found in the molecule's interior, forming the hydrophobic core. Amino acids with ionizable side changes are usually located at the protein's surface. Besides amino acids, glycoproteins also contain carbohydrates that provide functional sides for chemical modifications and cross-linking.

Figure 1: Reactive groups present in biomolecules useful for bioconjugation reaction. The amino, the carboxyl, the sulfhydryl, the thioether, the imidazolyl, and the guanidyl group are the six most reactive functional groups. The phenolic group and the indolyl group are the least reactive functional groups.

Figure 2: Common amino acid functional groups in peptides and proteins targeted for bioconjugation.

Most of the modifying reactions utilizing these functional groups are nucleophilic reactions. The incoming attacking nucleophile displaces the leaving group. The rate of the biomolecular nucleophilic substitution reaction, the SN2 mechanism, is dependent on at least two factors:

(i) the ability of the leaving group to leave and

(ii) the nucleophilicity of the attacking group.

The greater the nucleophilicity of the incoming group, the faster the product will be formed. The easier the leaving group can come off, the faster the reaction is. The SN2 mechanism of a nucleophilic substitution reaction is illustrated in Figure 3.

Figure 3: Nucleophilic substitution reaction, the SN2 mechanism. An electron deficient center is attacked by the nucleophile (Nu:) displacing the leaving group (X).

A nucleophile is any molecular species with an unshared pair of electrons, neutral or negatively charged, basically any Lewis base.

The following characteristics govern the nucleophilicity of a species:

[1] A negative nucleophile is always more potent than its conjugate acid. Therefore ArO^- is more potent than ArOH,
     and OH^- is more powerful than H₂O.

[2] Nucleophilicity is roughly proportional to the basicity. The approximate order of nucleophilicity is: 

NH₂^- > RO^-> OH^->ArO^->RNH₂> NH₃> H₂O.

[3] Nucleophilicity increases as one goes down the same column in the periodic table. Hence sulfur is a more powerful
      nucleophile than its oxygen analogs.

However, these rules do not always apply. Edwards and Pearson formulated the following order of nucleophilicity:

RS^-> ArS^-> I^-> CN^-> OH^-> N₃^-> Br^->ArO^->Cl^->pyridine >AcO^-> H₂O.

This scheme suggests that the sulfhydryl group of cysteines is the most potent nucleophile in a protein.

Effect of pH

The pH of the medium used for conjugation affects the rate of many nucleophilic reactions because protonation decreases the nucleophilicity of the molecule to be conjugated. The relationship between protonation and the pH is dependent on the pKa of the nucleophile. Table 2 lists the pKa of reactive groups. Since the pKa is a function of temperature, ionic strength, and the micro-environment of ionizable groups, the listed values are only an approximation.

The Henderson-Hesselbalch equation calculates the ratio of protonated to deprotonated species.

pH = pKa + log{[A-]/[AH]}

Based on the equation, the following general rules hold:

[1]  At one pH unit below the pKa, the ionizable species is 91% protonated.

[2]  At two pH units below the pKa, the ionizable species is 99% protonated.

[3]  At one pH unit above the pKa, the ionizable species is 91% deprotonated.

[4]  At two pH units above the pKa, the ionizable species is 99% protonated.

[5]  When the pH is the same as the pKa, 50% protonation occurs.

At a fixed pH, the most reactive group is usually the one with the lowest pKa.

At a neutral pH, the amino groups are protonated and therefore unreactive.

Carboxyl and imidazolyl groups are unprotonated and reactive at a neutral pH. 

At pH 5, the imidazolyl group will be over 90% protonated but the carboxyl group will remain in the ionic form. 

Other nucleophiles, such as the sulfhydryl group, will react at a higher pH. 

Adjusting the pH according to the low pKas allows for controlling the chemical reaction.

Table 1: Chemical modification reactions of amino acid side chains.

.

 Table 2: pKa of reactive groups in biomolecules

Reduction of Disulfide Bonds

In proteins, disulfide bonds cross-link two portions of a protein where cysteines are located. The oxidized form of sulfur is relatively unreactive but can be activated by the reduction of the sulfhydryl group. Thiol-containing reagents such as dithiothreitol, dithioerythritol, 2-mercaptoethanol, and 2-mercaptoethylamine are useful reducing agents. 

Figure 4: Reduction of disulfide bonds. The reaction involves a disulfide interchange reaction with a free thiol.

The reduction of disulfide bonds is a general reaction used in biochemistry, for example, to prepare immunoglobulin fragments needed for a variety of conjugation reactions.

Interconversion of functional Groups

The introduction of new functional groups by modifying existing functional groups in a biomolecule, such as an amino acid, a peptide, or a protein, allows for a more diverse application of conjugating reactions. For example, it is desirable to convert one functional group into another when preparing immunotoxins.

Conversion of Amines to Carboxylic Acids

The reaction of amino groups with dicarboxylic acid anhydrides will convert these to carboxylic acids. Succinic and malic anhydrides are the two most used dicarboxylic acid anhydrides.

Figure 5: Conversion of amino groups to carboxylic acids. (A) Reaction with malic anhydride. (B) Reaction with succinic anhydride.

Reaction products of maleylation are stable at neutral pH but hydrolyze at acidic pHs. Hence, succinic anhydride is the reagent of choice for chemical conjugation or cross-linking. Succinic anhydride also reacts with tyrosyl, histidyl, cysteinyl, seryl, and threonyl side-chins. However, tyrosyl and histidyl derivatives are reversible. Ester and thioester derivatives are also susceptible to hydroxylamine cleavage at pH 10.

Conjugation of functional groups to amino groups

Several approaches are available to conjugate functional groups or biomolecules to reactive amino groups. Succinic anhydrides, isothiocyanates, and aldehydes allow the linking of functional groups to reactive amino groups.

Figure 6: Chemistry of conjugation reactions of reactive amino groups utilizing succinic anhydrides, isothiocyanates, and aldehydes. General chemical structures of the reaction products are shown.

Conversion of amino to sulfhydryl groups

Several approaches are available to link free thiols to amino groups.

(1)  Thiolation with N-acetylhomocysteine thiolactone

Nucleophilic attack on the carbonyl group of thiolactones opens the ring, releasing the free thiol group. However, the reaction with amino groups is slow, except at pH 10 to 11. Silver ions catalyze the reaction around pH 7.

Figure 7: Thiolation with N-acetylhomeocysteine thiolactone.

(2)    Thiolation with S-acetylmercaptosuccinic anhydride

In this reaction, the amino group first reacts with the thiol-blocking reagent, followed by treatment with hydroxylamine.

Figure 8: Reaction with S-acetylmercaptosuccinic anhydride.

(3)     Thiolation with thiol-containing imidoesters

Several imidoesters allow thiolation of amino groups. Imidoesters react readily with amino groups at pH 7 to 10.

Figure 9: Thiolation with methyl-3-mercaptopropionimidate.

Figure 10: Thiolation with methyl-4-mercaptobutyrimidate.

Figure 11: Cyclization reaction of methyl-4-mercaptobutyrimidate to form iminothiolane also called Traut’s reagent followed by its thiolation reaction.

(4)  Thiolation with SATA

The succinic anhydride N-succinimidyl S-acetylthioacetate (SATA) allows the addition of sulfhydryl groups to proteins and other amine-carrying molecules such as oligonucleotides. The reaction products can be stored for a long time. Treatment with hydroxylamine exposes the labile sulfhydryl group needed for bioconjugation. The chemical reactions are illustrated in figure.

Figure 12: Addition of SATA to amino groups and conversion to free sulfhydryl groups. SATA allows the addition of protected sulfhydryl groups to reactive amines useful for long-time storage. Sulfhydryl groups present in biomolecules, such as oligonucleotides, peptides, and proteins, enable chemical modification of these molecules, for example, for bioconjugation approaches.

Reference

Campodónico PR, Tapia RA, Suárez-Rozas C. How the Nature of an Alpha-Nucleophile Determines a Brønsted Type-Plot and Its Reaction Pathways. An Experimental Study. Front Chem. 2022 Feb 2;9:740161. [PMC]

Edwards J. O., Pearson R. G. (1962). The Factors Determining Nucleophilic Reactivities. J. Am. Chem. Soc. 84, 16–24. [CrossRef]

Hermanson; Bioconjugate Techniques. 3^rd edition. Academic Press. 2013. (b)

March’s Advanced Organic Chemistry. 6^th edition. 2007, 425-431.; (5) Wong S.S.; Chemistry of protein conjugation and cross-linking. CRC Press. 1993. (a).

Weak acids and bases

 

Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.

Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.

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Peptide linkers or drug-peptide linkers enable the development of next-generation antibody-drug conjugates (ADCs) with enhanced plasma stability and function. In addition, linker peptides also allow the design and development of fusion proteins, protein conjugates, and oligonucleotide conjugates, enabling new therapeutics development. A thorough understanding of peptide linker structures, conformations, and functions will significantly facilitate the construction of stable and bioactive recombinant fusion proteins and similar biomolecules for drug delivery applications.

Flexible peptides can link proteins, protein domains, cyclic peptides, oligonucleotides, or proteins and oligonucleotides. Peptide linker sequences that naturally occur in protein complexes stabilize protein structures to allow these to function optimally.

Bifunctional fusion proteins:

1.  Extend plasma half-life by decreasing access to proteases.

2.  Decrease renal filtration.

3.  Alter intracellular routing via receptor-mediated recycling enables absorption across epithelial bilayers by binding
     to transcytosis receptors.

4.  Allow targeting over-expressing in vivo sites or uniquely expressing specific receptors or antigens.

Recent advancements in biotechnology allow the design and synthesis of fusion proteins for novel protein therapeutics that can improve the performance of current protein drugs.

Natural occurring peptide linkers

Natural linker peptides adopt a variety of conformations in secondary structure. Functional proteins contain these peptides sequences as helices, β-strands, coils, bends, and turns. The average length of peptide linkers in natural multi-domain proteins is approximately 6.5 residues according to Argos, P. and 10.0 ± 5.8 residues according to George and Heringa. George and Heringa grouped most natural linkers into helical and non-helical. The α-helical conformation is a rigid and stable structure containing intra-segment hydrogen bonds and a closely packed backbone. Linkers with α-helices can serve as rigid spacers reducing protein domain interactions. Non-helical linkers tend to be rich in prolines. Prolines tend to increase the stiffness of the linker moiety. However, the peptide linker's length, composition, hydrophobicity, and secondary structure are critical for proteins to function correctly (Argos, P. 1990; George & Heringa, G.R. 2022).

The average hydrophobicity of peptide linkers decreases with the increase in length. Longer linkers are more hydrophilic and more exposed to the aqueous solvent than shorter linkers. Natural multi-domain proteins contain proline-rich sequences as interdomain linkers. One example is the linker between the lipoyl and E3 binding domain in pyruvate dehydrogenase, GAAPAAAPAKQEAAAPAPAAKAEAPAAAPAAKA. Another example is the proline 9 (PPPPPPPPP) linker between the central and the C-terminal domains in cysteine proteinase.

The proline P9 linker is also found in the human butyrylcholinesterase tetramer. The C-terminal tryptophan amphiphilic tetramerization (WAT) helices from each subunit mediate tetramerization to assemble the complex. The protein complex is assembled around a central lamellipodia-derived oligopeptide with a proline 12 peptide attachment domain (PRAD) sequence. The proline peptide adopts a polyproline II helical conformation and runs antiparallel. A homologous proline linker is present in the Transformation/transcription domain-associated protein of the human SAGA coactivator complex (TRRAP, core, splicing module) [PDB ID 7KTS] and in Leiomodin-2 in the actin-leiomodin-2 complex [PDB ID 4RWT]. However, the linker peptide is flexible in all structures and, therefore, not observed in the solved x-ray structures.

Figure 1: Structure of the fully glycosylated human butyrylcholinesterase tetramer (PDB ID 6I2T; Leung et al. 2018). The proline peptide is shown in red.

Linkers containing small, polar amino acids such as threonine or serine and glycine are favored because they provide good flexibility due to their small sizes. Furthermore, these linker types may help maintain the stability of the linker structure in aqueous solvents through the formation of hydrogen bonds with water (Whitley et al. 2018; Mottram JC et al. 1989).

As an essential part of recombinant fusion proteins, linkers allow the construction of stable, bioactive fusion proteins. The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamide acetyltransferase subunits (E2p) of the Escherichia coli pyruvate dehydrogenase complex are connected by linker peptide sequences. These linkers are characteristically rich in alanine and proline residues (Turner et al. 1993; Chen et al. 2013). Table 1 shows a list of flexible, rigid, and cleavable linkers utilized in fusion proteins.

Reference

Argos P. An investigation of oligopeptides linking domains in protein tertiary structures and possible candidates for general gene fusion. J Mol Biol. 1990;211:943–958.

Chen X, Bai Y, Zaro J, Shen WC. Design of an in vivo cleavable disulfide linker in recombinant fusion proteins. Biotechniques. 2010;49:513–518.

Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013 Oct;65(10):1357-69. [PMC]

Franco Dosio, Paola Brusa and Luigi Cattel; Immunotoxins and Anticancer Drug Conjugate Assemblies: The Role of the Linkage between Components. Toxins 2011, 3, 848-883. [PMC]

Ducry L, Stump B.; Antibody-drug conjugates: linking cytotoxic payloads to monoclonal antibodies. Bioconjug Chem. 2010 Jan;21(1):5-13.

Feng Tiana, Yingchun Lua, Anthony Manibusana, Aaron Sellersa, Hon Trana, Ying Suna, Trung Phuonga, Richard Barnetta, Brad Hehlia, Frank Songa, Michael J. DeGuzmanb, Semsi Ensarib, Jason K. Pinkstaffc, Lorraine M. Sullivanc, Sandra L. Birocc, Ho Chod, Peter G. Schultze, John DiJoseph, Maureen Dougher, Dangshe Ma, Russell Dushing, Mauricio Lealh, Lioudmila Tchistiakovai, Eric Feyfanti, Hans-Peter Gerber, and Puja Sapra; A general approach to site-specific antibody drug conjugates. (2014) PNAS 111, 5, 1766-1771.

Francisco, J,A, Charles G. Cerveny, Damon L. Meyer, Bruce J. Mixan, Kerry Klussman, Dana F. Chace, Starr X. Rejniak, Kristine A. Gordon, Ron DeBlanc, Brian E. Toki, Che-Leung Law, Svetlana O. Doronina, Clay B. Siegall, Peter D. Senter, and Alan F. Wahl; cAC10-vcMMAE, an anti-CD30–monomethyl auristatin E conjugate with potent and selective antitumor activity. August 15, 2003; Blood: 102 (4).

George R, & Heringa J. An analysis of protein domain linkers: their classification and role in protein folding. Protein Eng. 2002;15:871–879.

SEAN L KITSON, DEREK J QUINN, THOMAS S MOODY, DAVID SPEED, WILLIAM WATTERS, DAVID ROZZELL; Antibody-Drug Conjugates (ADCs) – Biotherapeutic bullets. Chemistry Today - vol. 31(4) July/August 2013.

Leung MR, van Bezouwen LS, Schopfer LM, Sussman JL, Silman I, Lockridge O, Zeev-Ben-Mordehai T. Cryo-EM structure of the native butyrylcholinesterase tetramer reveals a dimer of dimers stabilized by a superhelical assembly. Proc Natl Acad Sci U S A. 2018 Dec 26;115(52):13270-13275. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6310839/

Lewis Phillips GD, Li G, Dugger DL, Crocker LM, Parsons KL, Mai E, Blättler WA, Lambert JM, Chari RV, Lutz RJ, Wong WL, Jacobson FS, Koeppen H, Schwall RH, Kenkare-Mitra SR, Spencer SD, Sliwkowski MX; Targeting HER2-positive breast cancer with trastuzumab-DM1, an antibody-cytotoxic drug conjugate. Cancer Res. 2008 Nov 15;68(22):9280-90. 

McCombs JR, Owen SC. Antibody drug conjugates: design and selection of linker, payload and conjugation chemistry. AAPS J. 2015 Mar;17(2):339-51. Epub 2015 Jan 22. PMID: 25604608; PMCID: PMC4365093. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4365093/

Mottram JC, North MJ, Barry JD, Coombs GH. A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension. FEBS Lett. 1989 Dec 4;258(2):211-5. doi: 10.1016/0014-5793(89)81655-2. PMID: 2599086.  A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C‐terminal extension (wiley.com) 

Nilvebrant J, Åstrand M, Georgieva-Kotseva M, Björnmalm M, Löfblom J, Hober, S.; (2014) Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin. PLoS ONE 9(8): e103094. doi:10.1371/journal.pone.0103094.

Panowksi S, Bhakta S, Raab H, Polakis P, Junutula JR.; Site-specific antibody drug conjugates for cancer therapy. MAbs. 2014 Jan-Feb;6(1):34-45. doi: 10.4161/mabs.27022.

Sassoon I, Blanc V.; Antibody-drug conjugate (ADC) clinical pipeline: a review. Methods Mol Biol. 2013;1045:1-27. doi: 10.1007/978-1-62703-541-5_1.

Turner S, Russell G, Williamson M, Guest J. Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli. Protein Eng. 1993;6:101–108. 

Whitley MJ, Arjunan P, Nemeria NS, Korotchkina LG, Park YH, Patel MS, Jordan F, Furey W. Pyruvate dehydrogenase complex deficiency is linked to regulatory loop disorder in the αV138M variant of human pyruvate dehydrogenase. J Biol Chem. 2018 Aug 24;293(34):13204-13213. doi: 10.1074/jbc.RA118.003996. Epub 2018 Jul 3. PMID: 29970614; PMCID: PMC6109939. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6109939/

Zhao HL, Xue C, Du JL, Ren M, Xia S, Liu ZM. Balancing the pharmacokinetics and pharmacodynamics of interferon-alpha2b and human serum albumin fusion protein by proteolytic or reductive cleavage increases its in vivo therapeutic efficacy. Mol Pharm. 2012;9:664–670.

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Bio-Synthesis provides a full spectrum of bio-conjugation services including high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
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The reaction of maleimides with thiol-containing biomolecules is a commonly used conjugation method. This conjugation reaction allows adding a label or cargo molecule to a protein, a peptide, or an oligonucleotide containing a linker with a thiol group.

Maleimide activated molecules modify thiol group-containing biomolecules at physiological pH conditions. An example is the conjugation of fluorescein-5-maleimide to a molecule with a thiol group.

Fluorescein-5-maleimide is a fluorophore with a sulfhydryl-reactive maleimide group on its lower-ring structure. This conjugation reaction results in a stable thioether bond. 

Figure  1:  Fluorescein-5-maleimide allows the modification of sulfhydryl groups to form thioester bonds.

Figure 2: The general reaction scheme of maleimides with thiol group used for bioconjugation and labeling of biomolecules.

Maleimides are electrophilic compounds with high selectivity towards thiols. Natural DNA does not contain thiols; therefore, linkers with thiol groups can be added to synthetic oligonucleotides. In proteins and peptides, thiol groups are very abundant as cysteine residues. Thiols can form disulfide bonds via oxidative dimerization. Cysteine residues in proteins form cystine bridges which stabilize tertiary structures. Disulfides do not react with maleimides. Therefore, it is necessary to reduce disulfides before conjugation. Conjugation protocols depend on the solubility of the starting components. For the conjugation of compounds with low solubility in water, organic solvents such as DMSO or DMF are essential.

Maleimide conjugation protocol

The following is a general protocol to conjugate fluorescent dye maleimides to proteins, peptides, and other thiolated biomolecules. 

1. Dissolve the biomolecule containing a thiol group to be labeled in a degassed buffer (PBS, Tris, HEPES are good, although others buffers containing no thiols can be used) at pH 7-7.5 in a reaction vial. The buffer can be degassed by applying a vacuum on it for several minutes, or by bubbling inert gas through it (nitrogen, argon, or helium may be used). For proteins, a good concentration range is between 1to10 mg/mL. 

2. Add an excess of TCEP (tris-carboxyethylphosphine) reagent to reduce disulfide bonds and flush with inert gas. Close the reaction vial. A 50 to 100x molar excess of TCEP will work well. Incubate the mixture for 20 minutes at room temperature. 

3. Dissolve the maleimide dye in DMSO or fresh DMF. Use 1 to10 mg in a volume of 100 uL. 

4. Add the dye solution to the thiol solution (If possible us a 10 to 20x fold excess of dye), flush vial with inert gas, and close the reaction vial tightly. 

5. Mix thoroughly, and keep overnight at room temperature, or 4 Celsius. 

6. Purify the conjugate using gel filteration, HPLC, FPLC, or electrophoresis. 

For maleimides with poor solubility in water, use a co-solvent such as DMF or DMSO. Maleimides with good aqueous solubility such as sulfo-Cyano maleimides can be dissolved in water. If a precipitation occurs, increase the content of organic solvent in the mixture to achieve better labeling. 

Please note: Dialysis is only useful for purification of maleimides with good water solubility.

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 Various gene therapies have been approved by FDA (U. S. Food & Drug Administration).  To treat cancer (ex. multiple myeloma, diffuse large B-cell lymphoma), several immunotherapies (ex. Novartis, Johnson & Johnson) have been approved since 2017, which involve administering T lymphocytes obtained from patients that have been genetically engineered to express 'chimeric antigen receptor' (CAR) recognizing tumor-specific proteins (https://www.fda.gov/vaccines-blood-biologics/cellular-gene-therapy-products/approved-cellular-and-gene-therapy-products).  In addition, virus therapy (Amgen), which involves injecting (into tumors) modified herpesvirus designed to replicate and rupture cancer cells selectively, has been approved to treat melanoma in 2015 (Fukuhara et al., 2016).  For treating 'Leber's congenital amaurosis' (inherited retinal condition progressively leading to blindness), recombinant AAV (adeno-associated virus), which has been modified to express the cDNA encoding RPE65 (retinal pigment that mediates phototransduction), has been approved in 2017 (Clarke et al., 2017).  To treat the neuromuscular disease 'spinal muscular atrophy', infusion (intravenous) of AAV9 virus (Novartis; 2.1M USD) that has been engineered to express SMN1 gene (functions in the biogenesis of splicing complex) was approved in 2019 (Mendell et al., 2021).

 However, the clinical use of viral vectors has faced several limitations.  Despite their ability to infect and transfer the genetic payload, the potential for insertional mutagenesis (ex. retrovirus, lentivirus) exists.  Earlier gene therapy trials of SCID (severe combined immunodeficiency caused by adenosine deaminase deficiency) patients documented the integration of the retroviral vector at an oncogenic locus in hematopoietic progenitor cell genome (Fischer et al., 2019).  Further, triggering immunological response may exclude the use of viruses (ex. adeno-associated virus type 2, adenovirus serotype 5) or limit repetitively administring viral vector (ex. adenovirus).  This has inspired pharmaceutical interest in using plasmids as a potential non-viral vector for gene therapy.

 The use of plasmids as gene delivery vector offers several advantages: easy to design, longer stability, inexpensive, less damaging to cells, minimal rate of genome integration, etc.  Nevertheless, the disadvantages include inefficient gene delivery, potential breakage of plamids, depletion of plasmids due to cell division, and immune recogntion of unmethylated bacterial CpG sequences.  It entailed making extensive modifications to plasmids to facilitate clinical application (Hardee et al., 2017).  For instance, to meet the FDA approval, the antibiotics-resistance gene used for selection had to be removed (to preempt dissemination into the environment).  As a substitute, plasmids harboring DNA sequences that titrate out repressors, which inhibit the replication of host bacterium by binding to the operator of ori (origin of replication), were developed (Marie et al., 2010).  Alternatively, plasmids expressing suppressor tRNA for the host bacteria (conditional mutant for ori) were designed. (Lilly et al., 2015).  Several plasmid-based gene therapies for infectious diseases (ex. HIV, HPV, hepatitis, malaria, influenza) or other disorder (ex. cardiac disease, cancer, diabetes) have progressed to phase I or II clinical trials (Hardee et al, 2017).

 Inefficient delivery could result in reduced drug activity as well as undesirable side effects.  Hence, equally significant for the development of plasmid-based gene delivery vectors is the ability to track them.  To trace, plasmids could be directly labeled with dyes to visually determine their location.  Ideally, labeling should occur without causing the degradation or altering the conformation of plasmids. 

For random direct labeling (not sequence directed), a previously developed method involves covalently labeling plasmid DNA via alkylating at the N⁷ position of (deoxy)guanine.  In Mirus 'Label IT' kit, a short linker was used to label DNA with a fluorophore.  Labeling of plasmids with the fluorscent dye cyanine using this approach did not affect the expression of the encoded genes (Watt et al. 2002) though it may affect transfection efficiency, endosomal escape, dissociation of DNA from a carrier, or nuclear retention of plasmid DNA.  In ULYSIS Nucleic Acid Labeling Kit (Molecular Probe), one of the coordination sites of the platinum-fluorophore complex was used for conjugating to the N⁷ position of (deoxy)guanine.   

An alternative method by Delvaux et al. (University of Iowa, USA) involves labeling plasmid DNA via forming a complex with 'cationic peptide-fluorphore' (containing aryl azide photolabel), followed by photoactivation (Devalux et al., 2022).  In one of their constructs, photolabel was added at the N-terminus while the sulfo-Cy5 fluorophore was conjugated to the C--teminus of NLS (nuclear localization signal) peptide from SV40 (simian virus 40)'s large T antigen for initial electrostatic interaction with DNA (double stranded).  In Photoprobe or FastTag Nucleic Acid Labeling System (Vector Laboratories), photoaffinity labeling was used to introduce fluorophore, biotin, primary amine, or a disulfide bond [upon reduction, it could react with thiol-reactive reagents, ex. maleimide-coupled fluorophore, hapten (detected using antibody)] (Watt et al. 2002). 

Other methods include incorporation of fluorophore or hapten-labeled nucleotide by DNA polymerase I (ex.  'nick translation'), TdT (terminal deoxynucleotidyl transferase), DNA ligase, DNA polymerase I's Knenow fragment (ex. fill-in reaction), etc.   For non-covalent labeling, DNA intercalating dyes such as DAPI or Hoechst 33258 could be used.   For efficiency, click-chemistry could be utilized by reacting alkyne [or dibenzocyclooctyne (DBCO)]-containing nucleotide (after incorporation) with azide-modified fluorophore, for instance [Rombouts et al., 2016).

For sequence-specific labeling, enzymes could be used to directly modify DNA.   DNA methyltransferases (MTases) could be used to catalyze the addition of fluorophore or functional groups (ex. alkenyl, alkynyl) (Rombouts et al., 2016; Deen et al., 2018).  Alternatively, fluorophore-conjugated oligonucleotide could hybridize to a specific complementary sequence [ex. triple-forming oligonucleotide (Molecular Beacons) utilizing FRET (fluorescence resonance energy transport)].  Or, proteins binding to specific DNA (or RNA) sequence (ex. transcription factors like LacI) fused to GFP (green fluorescence protein) could be expressed intracellularly to track administered polynucleotide. 

For vaccination, both plasmid DNA and mRNA have been developed.  For mRNA vaccine,  recognition by the innate system (TLR7, TLR8, TLR3) or cytoplasmic signaling [ex. RIG-1, Melanoma Differentiation-Associated protein 5 (MDA5), 2'-5'-oligoadenylate synthetase 1 (OAS), interferon-induced, double-stranded RNA-activated protein kinase (PKR)] entailed substituting with modified nucleosides.  Purification of other components associated with generating mRNA (ex. DNA template, unused nucleoside, double stranded RNA) is necessary to obviate immunogenicity, which could dampen antigen translation efficacy.  For repeated administration, potential issues associated with utilizing unnatural modified nucleosides or delivery vectors may need to be considered.  Compared to mRNA vaccine, plasmid DNA-based vaccine is lesser immunogenic and the concern over genomic integration or inciting autoimmune response against DNA has largely been subsided (Liu, 2019).  Other advantages of using plasmid DNA include greater stability, amplification via transcribing multiple mRNAs from each plasmid, simpler and speedier production, less costly, etc. albeit plasmid DNA must travel to the nucleus to generate mRNA.  A 'minimalistic, immunologically defined gene expression' (MIDGE) vector was used to express hTNF (human tumor necrosis factor) to sensitize melanoma cells to the anticancer drug vindesine (Kobelt et al., 2014).

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis.  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophoreseither terminally or internally as well as to conjugate to peptidesor antibodies. It provides custom conjugation of small molecules such as chemical drugs, metabolites and labeled compounds with synthetic or natural polymers (enzymes, peptide, protein, oligonucleotide, antibody, dendrimer, nanoparticle, etc).  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNA^NC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  It has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/covid-19.aspx

https://www.biosyn.com/mrna.aspx

https://www.biosyn.com/bioconjugation.aspx

https://www.biosyn.com/tew/Drug-Conjugation-Synthesis-Strategies.aspx

https://www.biosyn.com/tew/Basic-Bioconjugation-Chemistry-of-Reactive-Groups-in-Biomolecules.aspx

https://www.biosyn.com/tew/Maleimide-labeling-of-thiolated-biomolecules.aspx

References

Clarke T. Gene Therapy for Blindness Appears Initially Effective, Says U.S. FDA. Scientific American. October (2017).

Deen J, Neely RK. et al. A general strategy for direct, enzyme-catalyzed conjugation of functional compounds to DNA.  Nucleic Acids Res.  46 :e64 (2018).  PMID: 29546351

Delvaux NA, Mathew B, Rice KG. Fluorescent labeling of plasmid DNA for gene delivery: Implications of dye hydrophobicity on labeling efficiencies and nanoparticle size.  Anal Biochem. 2022 May 1;644:113895.  PMID: 32783899

Fischer A, Hacein-Bey-Abina S. Gene therapy for severe combined immunodeficiencies and beyond. J Exp Med.  217:e20190607 (2020).  PMID: 31826240

Rombouts K, Remaut K.  Fluorescent Labeling of Plasmid DNA and mRNA: Gains and Losses of Current Labeling Strategies.   Bioconjug Chem.  27:280-97 (2016).  PMID: 26670733

Fukuhara H, Ino Y, et al.  Oncolytic virus therapy: A new era of cancer treatment at dawn.  Cancer Sci. 107:1373-1379 (2016).  PMID: 27486853

Hardee CL, Arévalo-Soliz LM, et al. Advances in Non-Viral DNA Vectors for Gene Therapy.  Genes (Basel) 8 :65 (2017).  PMID: 28208635

Kobelt D, Walther W. et al. Preclinical study on combined chemo- and nonviral gene therapy for sensitization of melanoma using a human TNF-alpha expressing MIDGE DNA vector.  Mol Oncol. 8: 609-19 (2014).  PMID: 24503218

Lilly, J.; Camps, M. Mechanisms of theta plasmid replication. Microbiol. Spectr. 3, 1–18 (2015). PMID: 26104556

Liu MA.  Comparison of Plasmid DNA and mRNA as Vaccine Technologies.  Vaccines (Basel). 7:37 (2019).  PMID: 31022829

Marie, C.; Scherman, D. pFARs, plasmids free of antibiotic resistance markers, display high-level transgene expression in muscle, skin and tumour cells. J. Gene Med. 12, 323–332 (2010).  PMID: 20209487

Mendell JR, Al-Zaidy SA, et al. Current Clinical Applications of In Vivo Gene Therapy with AAVs.  Mol Ther.  29:464-488 (2021).  PMID: 33309881

Watt M, Hagstrom JE.  Intracellular Localization and Expression of Labeled Plasmid DNA using  Label IT Tracker™ Nucleic Acid Labeling Kits.  Technical Report(Mirus). (2002).