Innovative anti-HIV strategies are urgently needed to address challenges in vaccine development and multidrug resistance. ProTides are a clinically validated prodrug strategy that improves nucleoside monophosphate delivery by bypassing the first phosphorylation step. Conventional ProTides employ phenol or 1-naphthol aryl groups, which release potentially toxic byproducts upon activation. We report the first use of coumarin-based fluorophores (4MU or 4TFMU) as aryl masking groups in stavudine (d4T) ProTides, creating hybrid profluorophores with dual antiviral and fluorescent tracking capabilities. Eight hybrid ProTides were synthesised and evaluated against HIV-1 (IIIB) and HIV-2 (ROD) in MT-4 cells. Five ProTides retained activity in thymidine kinase deficient C8166-TK- cells, confirming bypass of the first phosphorylation step. ProTide 21 showed potent activity (IC50: 80 nM for HIV-1, 140 nM for HIV-2) and high selectivity indices (1549 and 923), outperforming d4T. Enzymatic activation was verified by 31P NMR. Surprisingly, two phosphorodiamidate derivatives were isolated, revealing a new class of phosphorodiamidating reagents enabling efficient synthesis of diamidate prodrugs. This multifunctional ProTide platform combined enhanced potency, reduced toxicity, and built-in fluorescence, offering promising avenues for next generation nucleoside and non-nucleoside ProTide and diamidate based therapeutics.

Two aerobic, Gram-stain-negative, gliding motility and rod-shaped bacterial strains, designated as B11T and D4T, were isolated from rhizosphere soil of Litchi (Litchi chinensis Sonn.) in Guangzhou, Guangdong Province, P.R. China. Phylogenetic analysis based on the 16S rRNA gene sequences showed that strains B11T and D4T belonged to the genus Flavobacterium and shared the highest similarity to Flavobacterium ginsenosidimutans THG 01T (98.2%) and Flavobacterium daemonensis JCM 19455T (98.0%), respectively. The DNA G+C contents of strains B11T and D4T were 33.9 and 34.1 mol%, respectively. Both the genome-derived average nucleotide identity and digital DNA-DNA hybridization values between the two strains and their closely related type species Flavobacterium sharifuzzamanii KCTC 62405T and Flavobacterium denitrificans DSM 15936T were below 39.3 and 90.5%, respectively. They all took iso-C15:0 and summed feature 3 (C16:1 ω6c and/or C16:1  ω7c) as the major fatty acids. The polar lipids of strain B11T contained phosphatidylethanolamine (PE), two unidentified aminophospholipids (APL1-2) and four unidentified lipids (L1-4), while strain D4T took phosphatidylethanolamine (PE), three aminophospholipids (APL1-3) and four unidentified lipids (L1-4). Menaquinone-6 was their predominant quinone. Based on the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, strains B11T and D4T should be considered as two novel species of the genus Flavobacterium, for which the names Flavobacterium movens (type strain B11T=GDMCC 1.4103T=JCM 36423T) and Flavobacterium mesophilum (type strain D4T=GDMCC 1.4105T=JCM 36424T) are proposed.

In this work, we describe the design, synthesis, and biological evaluation of a series of novel dual-target prodrugs that simultaneously target HIV-1 viral infection factors (Vif) and reverse transcriptase (RT). Among them, the two most effective compounds, A1 and A7, were found to inhibit HIV-1IIIB at nanomolar concentrations (EC50 = 8.1 nM, EC50 = 9.4 nM) in C8166 cells, which were 95 and 81 times higher than the parent drug 6 m, respectively, and 2.7 and 2.3 times higher than that of stavudine (d4T). The stability of compound A1 in the medium suggests that it can effectively release the parent drugs 6 m and stavudine with a half-life of 6 h, suggesting that it is a potential dual-target prodrug targeting HIV Vif and reverse transcriptase.

I started my research career (in 1965) on interferon by identifying polyacrylic acid (PAA) as an interferon inducer. Poly(I).poly(C), discovered by Maurice Hilleman's group, proved to be more potent as an interferon inducer, and through its mRNA, we were able to clone and express human β-interferon. The discovery of the reverse transcriptase (RT) by Temin and Baltimore (in 1970) brought me to the detection of suramin as a powerful RT inhibitor and enabled Sam Broder and his colleagues to identify suramin as the first inhibitor of HIV replication. In this capacity, it was subsequently superseded by AZT and other 2',3'-dideoxynucleoside (ddN) analogs, including d4T. In collaboration with Antonín Holý, we discovered several acyclic nucleoside phosphonates as potent inhibitors of both HIV and HBV (hepatitis B virus) replication. In collaboration with Paul Janssen, we identified various non-nucleoside RT inhibitors (NNRTIs) of HIV-1 replication. Of the nucleotide RT inhibitors (NtRTTs), tenofovir emerged as the most promising congener. It was derivatized to its oral prodrugs TDF and TAF. To enhance their efficacy, they were combined with other anti-HIV drugs, and two of them were pursued (and found efficacious) in the Pre-Exposure Prophylaxis (PrEP) of HIV infections.

Published experience with stavudine during breastfeeding is limited. Stavudine is not a recommended agent during breastfeeding.[1,2]

We report on the synthesis and characterization of three types of nucleoside tetraphosphate derivatives 4-9 acting as potential prodrugs of d4T nucleotides: (i) the δ-phosph(on)ate is modified by two hydrolytically stable alkyl residues 4 and 5; (ii) the δ-phosph(on)ate is esterified covalently by one biodegradable acyloxybenzyl moiety and a nonbioreversible moiety 6 and 7; or (iii) the δ-phosphate of nucleoside tetraphosphate is masked by two biodegradable prodrug groups 8 and 9. We were able to prove the efficient release of d4T triphosphate (d4TTP, (i)), δ-monoalkylated d4T tetraphosphates (20 and 24, (ii)), and d4T tetraphosphate (d4T4P, (iii)), respectively, by chemical or enzymatic processes. Surprisingly, δ-dialkylated d4T tetraphosphates, δ-monoalkylated d4T tetraphosphates, and d4T4P were substrates for HIV-RT. Remarkably, the antiviral activity of TetraPPPPro-prodrug 7 was improved by 7700-fold (SI 5700) as compared to the parent d4T in CEM/TK[-] cells, denoting a successful cell membrane passage of these lipophilic prodrugs and an intracellular delivery of the nucleotide metabolites.

HIV-associated lipodystrophy has been reported in people taking anti-retroviral therapy (ART). Lipodystrophy can cause cardiovascular diseases, affecting the quality of life of HIV-infected individuals. In this study, we propose a pharmacological lipid index to estimate the risk of hyperlipidemia caused by anti-retroviral drugs. Lipid droplets were stained in cells treated with anti-retroviral drugs and cyclosporin A. Signal intensities of lipid droplets were plotted against the drug concentrations to obtain an isodose of 10 μM of cyclosporin A, which we call the Pharmacological Lipid Index (PLI). The PLI was then normalized by EC50. PLI/EC50 values were low in early proteinase inhibitors and the nucleoside reverse transcriptase inhibitor, d4T, indicating high risk of hyperlipidemia, which is consistent with previous findings of hyperlipidemia. In contrast, there are few reports of hyperlipidemia for drugs with high PLI/EC50 scores. Data suggests that PLI/EC50 is a useful index for estimating the risk of hyperlipidemia.

The behavior of four drugs from the family of nucleoside analog reverse-transcriptase inhibitors (zalcitabine, stavudine, didanosine, and apricitabine) in a membrane environment was traced using molecular dynamics simulations. The simulation models included bilayers and monolayers composed of POPC and POPG phospholipids. It was demonstrated that the drugs have a higher affinity towards POPG membranes than POPC membranes due to attractive long-range electrostatic interactions. The results obtained for monolayers were consistent with those obtained for bilayers. The drugs accumulated in the phospholipid polar headgroup region. Two adsorption modes were distinguished. They differed in the degree of penetration of the hydrophilic headgroup region. Hydrogen bonds between drug molecules and phospholipid heads were responsible for adsorption. It was shown that apricitabine penetrated the hydrophilic part of the POPC and POPG membranes more effectively than the other drugs. Van der Waals interactions between S atoms and lipids were responsible for this.

We report on the synthesis and evaluation of three different nucleotide prodrug systems: (i) nucleoside triphosphate analogues in which the γ-phosph(on)ate has two different lipophilic nonbioreversible alkyl residues with d4TDP as the released nucleotide analogue; (ii) nucleoside diphosphate analogues bearing a bioreversible and a stable β-alkyl group; or (iii) nucleoside diphosphate analogues bearing two nonhydrolysable lipophilic alkyl moieties. The delivery of d4TDP (for the triphosphate precursor) and d4TMP (for the diphosphate precursor) was demonstrated in CD4[+] T-lymphocyte CEM cell extracts as well as in phosphate buffer saline (PBS). In primer extension assay, we found that γ-dialkylated d4TTP derivatives and d4TDP were accepted as substrates by HIV-RT. Several of these compounds were observed to be extremely active against HIV-1/2 replication in HIV-infected cells. A more than 45,000-fold increase in the anti-HIV activity was detected for compound 18a as compared to the parent d4T which results in a selectivity index value of 37,000.

In this study, a total of 462 samples of whole blood were collected from 36 patients enrolled. During the entire course of antiretroviral therapy (ART) from 2003 to 2019, both the CD4 cell count and viral load (VL) of study patients were examined annually, and an HIV-1 genotypic drug resistance (DR) assay was carried out using an in-house method when the HIV-1 VL was >1,000 copies/mL. The results indicated that 13 (36.1%) experienced treatment failure and 23 (63.9%) experienced treatment success among 36 patients. The proportion of patients with effective treatment after the adjustment of ART regimens was significantly higher than before adjustment (χ[2] = 33.796, p < .001). Furthermore, the frequencies of HIV-1 DR mutations before adjustment were higher than those after adjustment (t = 3.345, p = .002). In particular, among 23 patients with effective treatment after adjustment, the means ± standard deviations of the VLs and CD4 cell counts before adjustment were 3.85 ± 0.65 log RNA copies/mL and 226.83 ± 106.06 cells/mm[3], respectively, compared with 2.19 ± 0.58 log RNA copies/mL and 367.68 ± 174.62 cells/mm[3], respectively, after adjustment. Obviously, there were statistically significant differences in the changes in VL (t = 8.728, p < .001) and CD4 cell count (t = -4.476, p < .001) before and after adjustment. Therefore, patients who received updated ART regimens containing LPV/r and TDF after adjustment achieved better therapeutic effects compared with patients who received initial ART regimens harboring D4T/AZT or NVP. This suggests that future research is needed to initiate the surveillance of DR, VLs, and CD4 cell counts immediately after HIV diagnosis, and dynamic changes in these indicators so as to optimize ART effects.