In Situ Forming, Enzyme-Responsive Peptoid-Peptide Hydrogels: An Advanced Long-Acting Injectable Drug Delivery System

Long-acting drug delivery systems are promising platforms to improve patient adherence to medication by delivering drugs over sustained periods and removing the need for patients to comply with oral regimens. This research paper provides a proof-of-concept for the development of a new optimized in situ forming injectable depot based on a tetrabenzylamine-tetraglycine-d-lysine-O-phospho-d-tyrosine peptoid-D-peptide formulation ((NPhe)4GGGGk(AZT)y(p)-OH). The chemical versatility of the peptoid-peptide motif allows low-molecular-weight drugs to be precisely and covalently conjugated. After subcutaneous injection, a hydrogel depot forms from the solubilized peptoid-peptide-drug formulation in response to phosphatase enzymes present within the skin space. This system is able to deliver clinically relevant concentrations of a model drug, the antiretroviral zidovudine (AZT), for 35 days in Sprague–Dawley rats. Oscillatory rheology demonstrated that hydrogel formation began within ∼30 s, an important characteristic of in situ systems for reducing initial drug bursts. Gel formation continued for up to ∼90 min. Small-angle neutron scattering data reveal narrow-radius fibers (∼0.78–1.8 nm) that closely fit formation via a flexible cylinder elliptical model. The inclusion of non-native peptoid monomers and D-variant amino acids confers protease resistance, enabling enhanced biostability to be demonstrated in vitro. Drug release proceeds via hydrolysis of an ester linkage under physiological conditions, releasing the drug in an unmodified form and further reducing the initial drug burst. Subcutaneous administration of (NPhe)4GGGGk(AZT)y(p)-OH to Sprague–Dawley rats resulted in zidovudine blood plasma concentrations within the 90% maximal inhibitory concentration (IC90) range (30–130 ng mL–1) for 35 days

Synthesis and Development of Long-Acting Abacavir Prodrug Nanoformulations

Over the past decade, work from our laboratory has demonstrated the potential of targeted nanoformulated antiretroviral therapy (nanoART) to produce sustained high plasma and tissue drug concentrations for weeks following a single intramuscular (IM) administration that can suppress ongoing viral replication and mitigate dose associated viral resistance. While progress has occurred towards developing long-acting nanoformulations for protease and nonnucleoside reverse transcriptase (RT) inhibitors, development of nanoformulations of hydrophilic nucleoside RT inhibitor drugs have remained elusive. Abacavir (ABC); a hydrophilic molecule exhibited limited utilities to develop into long-acting nanoformulation platform. Furthermore, inefficient conversion of ABC to its biological active metabolites; carbovir triphosphate jeopardizes its therapeutic index. Thus, improving bioavailability and the therapeutic index of the ABC is urgently needed. To this end, a phosphoramidate prodrug of ABC (PABC), and a myristoylated prdrug of ABC (MABC) was synthesized to improve the therapeutic index of its native hydrophilic counterpart. The notion of PABC synthesis was to increase intracellular nucleoside 5’-O- triphosphate levels by bypassing rate-limiting monophosphorylation of the parent drug. We reasoned that long-acting PABC nanoformulations could improve ABC’s Pharmacokinetic (PK) and pharmacodynamics (PD). Herein, PABC was successfully synthesized and characterized by 1H-NMR and FTIR spectroscopy. PABC was incorporated into a PLGA-lipid nanoformulation. In vitro and in vivo viral efficacy of PABC and PABC encased nanoformulation were evaluated in human monocytes derived macrophages (MDM) and Hu-PBL reconstituted NSG mice respectively. Concomitantly, a platform was constructed to convert the hydrophilic ABC into a hydrophobic derivative through esterification at 5’-OH position of ABC (MABC). MABC was loaded with high concentration into a polymer and decorated with appropriate targeting ligands for improvement in biodistribution, half-life and antiretroviral efficacy. Antiretroviral activity, uptake, retention and cellular trafficking of both the pro-drug and MABC encased in poloxamer nanoformulations were assessed in MDM. Drug PK was evaluated over 14 days for ABC and nanoformulated MABC after intramuscular injection in Balb/c mice

Activity Profiles & Mechanisms of Resistance of 3’-Azido-2’,3’-Dideoxynucleoside Analog Reverse Transcriptase Inhibitors of HIV-1

To investigate mechanisms of HIV-1 resistance to 3’-azidonucleoside analog reverse transcriptase inhibitors, in vitro selection experiments were conducted by serial passage of HIV-1LAI in MT-2 cells in increasing concentrations of 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG), 3′-azido-2′,3′-dideoxycytidine (3′-azido-ddC), or 3′-azido-2′,3′-dideoxyadenosine (3′-azido-ddA). 3′-Azido-ddG selected for virus 5.3-fold resistant to 3′-azido-ddG. Population sequencing of the reverse transcriptase (RT) gene identified L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain. Site-directed mutagenesis showed that these 5 mutations only conferred ~2.0-fold resistance. Single-genome sequencing analyses revealed a complex population of mutants that all contained L74V and L214F linked to other mutations, including ones not identified during population sequencing. Recombinant HIV-1 clones containing RT derived from single sequences exhibited 3.2- to 4.0-fold 3′-azido-ddG resistance. By contrast, 3′-azido-ddC selected for the V75I mutation in HIV-1 RT that conferred 5.9-fold resistance. We were unable to select HIV-1 resistant to 3′-azido-ddA, even at concentrations of 3′-azido-ddA that yielded high intracellular 3′-azido-ddA-5′-triphosphate levels. We have also defined the molecular mechanisms of 3’-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V/F77L/V106I/L214F/R277K/K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3’-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows HIV-1 RT to effectively discriminate between the natural nucleotide (dGTP) and 3’-azido-ddG-triphosphate (3’-azido-ddGTP). 3’-azido-ddGTP discrimination was primarily driven by a decrease in 3’-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3’-azido-ddG-monophosphate (3’-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3’-azido-ddGMP on an RNA/DNA, but not on DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Taken together, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3’-azido-2’,3’-dideoxynucleosides that can be exploited in the design of novel nucleoside RT inhibitors

Structure-Activity-Resistance Relationships of Novel Nucleoside and Nucleotide HIV-1 Reverse Transcriptase Inhibitors

Nucleos(t)ide reverse transcriptase inhibitors (N(t)RTI) are essential components of combination antiretroviral therapy for treatment of human immunodeficiency virus type-1 (HIV-1) infection. N(t)RTI are analogs of natural 2’-deoxyribonucleos(t)ides that lack a 3’-hydroxyl. Once metabolized by host kinases to the active form, their incorporation into viral DNA by HIV-1 reverse transcriptase (RT) results in chain termination of DNA synthesis. N(t)RTI efficacy is undermined primarily by rapid selection of resistant/cross-resistant HIV-1 variants. Consequently, the development of novel N(t)RTI with activity against a broad range of N(t)RTI-resistant HIV-1 is of critical importance. Rational design of novel N(t)RTI with knowledge of analog structure-activity-resistance relationships with the RT target enzyme is the most promising approach. We hypothesized that uncovering knowledge of how N(t)RTI base, sugar, and phosphate structures influence activity and resistance phenotypes would aid in the rational design of new N(t)RTI with improved activity and resistance profiles. Therefore, a combination of biochemical, antiviral, molecular modeling, and cellular pharmacology analyses provided a detailed characterization of structure-activity-resistance relationships for inhibition of wild-type and NRTI-resistant HIV-1 by novel N(t)RTI. First, we studied two novel nucleoside phosphonate NtRTI, (R)-6-[2-phosphonylmethoxy]propoxy]-2,4-diaminopyrimidine (PMEO-DAPym) and (5-(6-amino-purin-9-yl)-4-fluoro-2,5-dihydro-furan-2-yloxymethyl)phosphonate (GS-9148). We showed the diphosphate (-DP) form, PMEO-DAPym-DP acts as a purine mimetic that is recognized by RT as an adenosine analog and unambiguously incorporated across from thymine (DNA) or uracil (RNA). Studies indicated that PMEO-DAPym-DP and GS-9148-DP were superior to tenofovir-DP against both discrimination and excision RT resistance mechanisms. Next, we examined structure-activity-resistance relationships of 6-modified, 3’-azido-2’,3’-dideoxyguanosine (3’-azido-ddG) NRTI analogs. In RT-mediated DNA synthesis assays the triphosphate (-TP) form of each analog behaved as an adenosine mimetic for incorporation by HIV-1 RT. Importantly, the structure-activity relationships for incorporation and ATP-mediated excision were different, suggesting that new analogs can be designed that are efficiently incorporated but poorly excised by RT. RS-788, a 5’-monophosphate prodrug of 3’-azido-2’,3’-dideoxy-2,6-diaminopurine (3’-azido-2,6-DA-P), displayed potent activity against multi-NRTI-resistant HIV-1 and unique cellular metabolism. RS-788 was metabolized ~1:1 to both 3’-azido-2,6-DA-P and 3’-azido-ddG, thus delivering two distinct metabolites, each of which are potent RT chain-terminators that are incorporated opposite different bases, thymine and cytosine, respectively. Combinations of 3’-azido-2,6-DA-P+3’-azido-ddG synergistically inhibited multi-NRTI-resistant RT DNA synthesis

HIV genotyping

ThesisThe development of viral resistance to antiretroviral drugs used for treatment of human immunodeficiency virus 1 (HIV) infection is an important cause of antiretroviral treatment (ARV) failure and limits options for alternative antiretroviral regimens. Prevention, characterisation and clinical management of such resistance are receiving increasing attention. The primary objective of this project was to study the naturally occurring variants of HIV-1 present in a group of ARV-naiVe patients. The presence of pre-existing mutations may aid clinicians in designing optimal ARV combinations for the country. The secondary objective was to choose a suitable method for doing the analysis. Published primer sequences and inhouse methods for the different steps of the procedure were used, but this was changed to an established commercial system (viroseq) because of superior sensitivity and the fact that it is FDA approved. The study population consisted of 19 adult ARV-na·ive AIDS patients recruited from Tsepo House, and Medi Inn, Bloemfontein. The CD4 counts indicated that the immune systems of these patients were severely compromised, the highest count being 348 and the mean value 184 cells/mm3 whole blood. Therefore, according to the Department of Health's criteria, all of them qualified for ARV treatment. The viral loads were high, varying from 23 000 to >750 000 RNA copies/ml plasma. This demonstrates how people differ in their response to the viral infection. All patients were in the terminal stage of disease, yet displayed up to thirty-fold differences in viral load. After RT-PCR and sequencing, the sequences were trimmed to 99 codons of the full protease and the first 335 codons of the reverse transcriptase reading frames. These were translated to amino acids and used separately in phylogenetic analyses to study their relatedness to each other and to other isolates. A benefit of phylogenetic analysis is to indicate possible contamination of one sample by another as this will show tight clustering of some samples. The form and branch distances of the trees found clearly eliminated this possibility. The sequences were compared to 20 other South African isolates, randomly chosen from the Los Alamos (http://www.hiv.lanl.gov/content/index) HIV-1 sequence database. Both the protease and reverse transcriptase trees revealed that the Bloemfontein sequences do not differ appreciably from those found in the rest of the country, since they tend to diffuse through the tree rather than to cluster on their own. All the patients tested positive for subtype C, complying with the demographical data. Very few mutations were detected in the reverse transcriptase (RT) reading frame, although a mutation (K103N) which confers high level resistance against non-nucleoside RT inhibitors was found in one isolate. This may mean that a small percentage of patients may harbour a virus that is naturally resistant to these drugs. In the protease reading frame, mutations at nine amino acid positions have been designated primary or major resistance mutations. None of the primary mutations were found, although several secondary mutations (of lesser significance) contributing to reduced susceptibility (e.g., M361 and 193L) were found in 95% of our samples. This was not unexpected as these polymorphisms are extremely common in subtype C viruses. These genetic differences may be clinically relevant when considering long-term strategies for patients infected with nonS subtypes. As the public sector ARV rollouts gather momentum, the emergence of drugresistant isolates is sure to follow and the laboratory services must be geared to provide the backup needed by the clinicians to plan salvage therapy. This project has contributed to the baseline knowledge of the infected population enabling us to anticipate the emerging resistance mutations

Synthesis of modified D-allohexofuranosyl-uracil nucleoside analogs

Abstract The study of nucleosides, nucleotides, and their polymers is essential due to their critical roles in cellular processes such as DNA replication, RNA transcription, protein synthesis, signaling, and energy transfer. These molecules serve as the building blocks of life, making them fundamental to genetics, molecular biology, pharmacology, and other relevant scientific fields. Beyond their natural functions, chemically modified nucleosides, nucleotides, and their oligomers have emerged as powerful tools in medicine, biotechnology, and research. These advancements include the development of antiviral and anticancer therapies using modified nucleoside analogs, as well as therapies employing oligonucleotide-based treatments targeting pre-mRNA and mRNA. Furthermore, these modifications have enhanced diagnostic technologies and research tools. The ability to modify and efficiently synthesize these modified analogs and their oligomers opens new possibilities for therapeutic applications, offering improved stability, specificity, and efficacy. This work builds on an extensive body of literature exploring the roles of nucleotides, nucleosides, and their modified analogs. Initially, the review covers the physiological significance of natural nucleos(t)ides, emphasizing their central role in genetic information transfer and cellular metabolism. Then focus shifts toward chemically modified nucleos(t)ides, which have become increasingly important in antiviral, anticancer, gene therapies, and biotechnological tools. Various synthetic strategies for altering sugar, nucleobase, and phosphate moieties are critically reviewed, with a particular emphasis on methods that enable precise structural alterations. Special attention is also given to the utility of D-allofuranose, an atypical sugar that served as a scaffold for modified nucleoside analogs developed in the practical part of this work. These insights underscore the potential of developing novel therapeutic agents with enhanced properties and directly inform the synthetic approaches. This review guided the selection of synthetic routes, protecting group strategies, and targeted modifications that were further practically explored in this study. The experimental part of this research focused on synthesizing D-allofuranosyl-uracil analogs, with a special focus on modifying the 6’-hydroxyl group. The study explored the effectiveness of two different synthetic routes for the initial sugar configuration preparation, the separation of the α/β-anomer forms of the resulting uracil nucleosides, and the introduction of an azide group at the 6’OH-position. Despite encountering challenges, such as the unsuccessful addition of a triphosphate group, the research demonstrated the feasibility of synthesizing a modified nucleoside key intermediate. Further work is needed to optimize the phosphorylation process and fully evaluate the biological properties of consequently derived nucleotides’ antiviral properties

Antiviral Natural Products against Hepatitis-A Virus

The review on antiviral anti-hepatitis A virus agents is warranted given the importance of hepatitis A virus (HAV) as a human pathogen. Novel antiviral drugs have been sourced from natural agents and developed into products for management of viral infections. The role of purified natural products in treatment and as adjunctives in the management of HAV infections is clearly plausible. Treatments against Hepatitis A virus infection is currently limited. In this chapter, the antiviral natural products against hepatitis-A virus (HAV), their sources as well as their treatment approach and their application have been discussed. The antiviral natural products could be sourced generally from plants, herbs and animals. These natural agents have been shown to demonstrate substantial antiviral activity against HAV and could target various stages of the viral life cycle, replication, assemblage, release, as well as targeting virus-host specific interactions