Discovery of Novel Aryl Triazolone Dihydropyridines (ATDPs) Targeting Highly Conserved Residue W229 as Promising HIV-1 NNRTIs.

NNRTI is an important component of the highly active antiretroviral therapy (HAART), but the rapid emergence of drug resistance and poor pharmacokinetics limited their clinical application. Herein, a series of novel aryl triazolone dihydropyridines (ATDPs) were designed by structure-guided design with the aim of improving drug resistance profiles and pharmacokinetic profiles. Compound 10n (EC50 = 0.009-17.7 µM) exhibited the most active potency, being superior to or comparable to that of doravirine (DOR) against the whole tested viral panel. Molecular docking was performed to clarify the reason for its higher resistance profiles. Moreover, 10n demonstrated excellent pharmacokinetic profile (T1/2 = 5.09 h, F = 108.96%) compared that of DOR (T1/2 = 4.4 h, F = 57%). Additionally, 10n was also verified to have no in vivo acute or subacute toxicity (LD50 > 2000 mg/kg), suggesting that 10n is worth further investigation as a novel oral NNRTIs for HIV-1 therapy.

Structure-guided design of novel biphenyl-quinazoline derivatives as potent non-nucleoside reverse transcriptase inhibitors featuring improved anti-resistance, selectivity, and solubility.

In pursuit of enhancing the anti-resistance efficacy and solubility of our previously identified NNRTI 1, a series of biphenyl-quinazoline derivatives were synthesized employing a structure-based drug design strategy. Noteworthy advancements in anti-resistance efficacy were discerned among some of these analogs, prominently exemplified by compound 7ag, which exhibited a remarkable 1.37 to 602.41-fold increase in potency against mutant strains (Y181C, L100I, Y188L, F227L + V106A, and K103N + Y181C) in comparison to compound 1. Compound 7ag also demonstrated comparable anti-HIV activity against both WT HIV and K103N, albeit with a marginal reduction in activity against E138K. Of significance, this analog showed augmented selectivity index (SI > 5368) relative to compound 1 (SI > 37764), Nevirapine (SI > 158), Efavirenz (SI > 269), and Etravirine (SI > 1519). Moreover, it displayed a significant enhancement in water solubility, surpassing that of compound 1, Etravirine, and Rilpivirine. To elucidate the underlying molecular mechanisms, molecular docking studies were undertaken to probe the critical interactions between 7ag and both WT and mutant strains of HIV-1 RT. These findings furnish invaluable insights driving further advancements in the development of DAPYs for HIV therapy.

Discovery of low-molecular-weight phenylalanine derivatives as novel HIV capsid modulators with improved antiretroviral activity and metabolic stability.

The HIV capsid (CA) protein is a promising target for anti-AIDS treatment due to its critical involvement in viral replication. Herein, we utilized the well-documented CA inhibitor PF74 as our lead compound and designed a series of low-molecular-weight phenylalanine derivatives. Among them, compound 7t exhibited remarkable antiviral activity with a high selection index (EC50 = 0.040 µM, SI = 2815), surpassing that of PF74 (EC50 = 0.50 µM, SI = 258). Furthermore, when evaluated against the HIV-2 strain, 7t (EC50 = 0.13 µM) demonstrated approximately 14-fold higher potency than that of PF74 (EC50 = 1.76 µM). Insights obtained from surface plasmon resonance (SPR) revealed that 7t exhibited stronger target affinity to the CA hexamer and monomer in comparison to PF74. The potential interactions between 7t and the HIV-1 CA were further elucidated using molecular docking and molecular dynamics simulations, providing a plausible explanation for the enhanced target affinity with 7t over PF74. Moreover, the metabolic stability assay demonstrated that 7t (T1/2 = 77.0 min) significantly outperforms PF74 (T1/2 = 0.7 min) in human liver microsome, exhibiting an improvement factor of 110-fold. In conclusion, 7t emerges as a promising drug candidate warranting further investigation.

Structure-based design and optimization lead to the identification of novel dihydrothiopyrano[3,2-d]pyrimidine derivatives as potent HIV-1 inhibitors against drug-resistant variants.

With our continuous endeavors in seeking potent anti-HIV-1 agents, we reported here the discovery, biological characterization, and druggability evaluation of a class of nonnucleoside reverse transcriptase inhibitors. To fully explore the chemical space of the NNRTI-binding pocket, novel series of dihydrothiopyrano [3,2-d]pyrimidines were developed by employing the structure-based design strategy. Most of the derivatives were endowed with prominent antiviral activities against HIV-1 wild-type and resistant strains at nanomolar levels. Among them, compound 23h featuring the aminopiperidine moiety was identified as the most potent inhibitor, with EC50 values ranging from 3.43 to 21.4 nmol/L. Especially, for the challenging double-mutants F227L + V106A and K103N + Y181C, 23h exhibited 2.3- to 14.5-fold more potent activity than the first-line drugs efavirenz and etravirine. Besides, the resistance profiles of 23h achieved remarkable improvement compared to efavirenz and etravirine. The binding target of 23h was further confirmed to be HIV-1 reverse transcriptase. Molecular modeling studies were also performed to elucidate the biological evaluation results and give guidance for the optimization campaign. Furthermore, no apparent inhibition of the major CYP450 enzymes and hERG channel was observed for 23h. Most importantly, 23h was characterized by good pharmacokinetic properties and excellent safety in vivo. Collectively, 23h holds great promise as a potential candidate for its effective antiviral efficacy and favorable drug-like profiles.

Design and synthesis of Fsp3-enriched spirocyclic-substituted diarylpyrimidine derivatives as novel HIV-1 NNRTIs.

In this study, a novel series of diarylpyrimidine derivatives with Fsp3-enriched spirocycles were designed and synthesized to further explore the chemical space of the hydrophobic channel of the NNRTI-binding pocket. The biological evaluation results showed that most of the compounds displayed effective inhibitory potency against the HIV-1 wild-type strain, with EC50 values ranging from micromolar to submicromolar levels. Among them, TT6 turned out to be the most effective inhibitor with an EC50 value of 0.17 µM, demonstrating up to 47 times more active than that of reference drug 3TC (EC50 = 8.01 µM). More encouragingly, TT6 was found to potently inhibit the HIV-1 mutant strain K103N with an EC50 value of 0.69 µM, being about 6-fold more potent than 3TC (EC50 = 3.68 µM) and NVP (EC50 = 4.62 µM). Furthermore, TT6 exhibited the most potent inhibitory activity toward HIV-1 reverse transcriptase with an IC50 value of 0.33 µM. Additionally, molecular simulation studies were conducted to investigate the binding modes between TT6 and NNRTI-binding pocket, which may provide valuable clues for the follow-up structural optimizations.

Structure-based design of diarylpyrimidines and triarylpyrimidines as potent HIV-1 NNRTIs with improved metabolic stability and drug resistance profiles.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) are an important component of anti-acquired immunodeficiency syndrome treatment regimen. In the present work, with the previously reported compound K-16c as lead, a series of novel 2,4,5-trisubstituted pyrimidine derivatives were designed based on the cocrystal structure of K-16c/RT, with the aim to improve the anti-human immunodeficiency virus type-1 (HIV-1) activities and metabolic stability properties. Compound 11b1 exhibited the most potent antiviral activity against wild-type (WT) and a panel of single mutant HIV-1 strains (EC50 = 2.4-12.4 nM), being superior to or comparable to those of the approved drug etravirine. Meanwhile, 11b1 exhibited moderate cytotoxicity (CC50 = 4.96 µM) and high selectivity index (SI = 1189) toward HIV-1 WT strain. As for HIV-1 RT inhibition test, 11b1 possessed excellent inhibitory potency (IC50 = 0.04 µM) and confirmed its target was RT. Moreover, the molecular dynamics simulation was performed to elucidate the improved drug resistance profiles. Moreover, 11b1 was demonstrated with favorable safety profiles and pharmacokinetic properties in vivo, indicating that 11b1 is a potential anti-HIV-1 drug candidate worthy of further development.

Selected Milestones in Antiviral Drug Development.

This review article will describe the (wide) variety of approaches that I envisaged to develop a specific therapy for viral infections: (i) interferon and its inducers, (ii) HSV, VZV and CMV inhibitors, (iii) NRTIs (nucleoside reverse transcriptase inhibitors), NtRTIs (nucleotide reverse transcriptase inhibitors) and NNRTIs (non-nucleoside reverse transcriptase inhibitors) as HIV inhibitors, (iv) NtRTIs as HBV inhibitors, and finally, (v) the transition of an HIV inhibitor to a stem cell mobilizer, as exemplified by AMD-3100 (Mozobil®).

Discovery, Crystallographic Studies, and Mechanistic Investigations of Novel Phenylalanine Derivatives Bearing a Quinazolin-4-one Scaffold as Potent HIV Capsid Modulators.

Optimization of compound 11L led to the identification of novel HIV capsid modulators, quinazolin-4-one-bearing phenylalanine derivatives, displaying potent antiviral activities against both HIV-1 and HIV-2. Notably, derivatives 12a2 and 21a2 showed significant improvements, with 2.5-fold over 11L and 7.3-fold over PF74 for HIV-1, and approximately 40-fold over PF74 for HIV-2. The X-ray co-crystal structures confirmed the multiple pocket occupation of 12a2 and 21a2 in the binding site. Mechanistic studies revealed a dual-stage inhibition profile, where the compounds disrupted capsid-host factor interactions at the early stage and promoted capsid misassembly at the late stage. Remarkably, 12a2 and 21a2 significantly promoted capsid misassembly, outperforming 11L, PF74, and LEN. The substitution of easily metabolized amide bond with quinolin-4-one marginally enhanced the stability of 12a2 in human liver microsomes compared to controls. Overall, 12a2 and 21a2 highlight their potential as potent HIV capsid modulators, paving the way for future advancements in anti-HIV drug design.

In situ click chemistry-based discovery of 1,2,3-triazole-derived diarylpyrimidines as novel HIV-1 NNRTIs by exploiting the tolerant region I in binding pocket.

HIV-1 reverse transcriptase (RT) is considered as one of the most significant targets for the anti-HIV-1 drug design due to their determined mechanism and well-decoded crystal structure. As a part of our continuous efforts towards the development of potent HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) by exploiting the tolerant region I of NNRTIs binding pocket (NNIBP), the miniaturized parallel synthesis via CuAAC click chemistry reaction followed by in situ biological screening have been performed in this work. The in situ enzyme inhibition screening results showed that 14 compounds exhibited higher or equivalent inhibitory activity compared to the lead K-5a2 and ETR. Anti-HIV-1 activity results indicated that C1N51 displayed the most potent activity (EC50 = 0.01-0.26 µM) against wild-type and a panel of NNRTIs-resistant strains. Moreover, the molecular simulation demonstrated that the newly introduced triazole ring could develop new hydrogen bonds with Lys103 and Pro236, which explained the feasibility of introducing triazole in the tolerant region I of the RT binding pocket.

Biktarvy for the treatment of HIV infection: Progress and prospects.

Bictegravir (BIC), a second-generation integrase strand-transfer inhibitor (INSTI) with high resilience to INSTI-resistance mutations, is integrated as a key component of Biktarvy® - a fixed-dose once-daily triple-drug regimen of bictegravir (BIC), emtricitabine (FTC) plus tenofovir alafenamide (TAF). Based on the accumulated evidence from HIV clinical trials and real-world studies, the clinical effectiveness of BIC + FTC + TAF has been proven non-inferior to other fixed-dose once-daily combinations such as dolutegravir + FTC + TAF and dolutegravir + abacavir + lamivudine. Biktarvy also shows limited drug-drug interactions and a high barrier to drug resistance. According to recent HIV guidelines, BIC + FTC + TAF is recommended as initial and long-term therapy for the treatment of HIV infection. For the pre-exposure prophylaxis, tenofovir disoproxil fumarate (TDF) or tenofovir alafenamide (TAF) remains advisable, but BIC may be possibly added to TDF or TAF. In the development of a long-acting once-monthly regimen, the novel nano-formulation of BIC + FTC + TAF could be possibly developed in the future.

Erratum: Author correction to 'Approved HIV reverse transcriptase inhibitors in the past decade' [Acta Pharmaceutica Sinica B 12 (2022) 1567-1590].

[This corrects the article DOI: 10.1016/j.apsb.2021.11.009.].

The magic of a methyl group: Biochemistry at the service of medicine.

A carbon-carbon linkage is created when a methyl group is implanted on dUMP, thus resulting in the formation of dTMP by thymidylate synthase. The methyl group is deleted by aromatase when androgens are converted to estrogens. The methyl group is rearranged with the help of vitamin B12 in the isomerization of methylmalonyl-CoA to succinyl-CoA. S-adenosylmethionine (SAM) serves as the universal methyl donor involved in the biosynthesis of adrenaline and creatine(phosphate). It also interferes with the 5'-mRNA capping and the degradation of catecholamines (i.e. adrenaline, noradrenaline). Cholesterol could be viewed as a conglomeration of methyl groups. Finally, as part of valine, two methyl functions participate in the origin of one of the most frequent hereditary diseases on earth, sickle cell anemia.

Improving the anti-HIV-1 activity and solubility of poorly water-soluble DAPYs by heteroaromatic replacement strategy: From naphthalene-DAPYs to quinoline-DAPYs.

To enhance the anti-HIV-1 efficacy and solubility of our previously documented NNRTI 1, a collection of innovative quinoline-substituted DAPY derivatives were devised using heteroaromatic replacement strategy. The results of biological evaluation revealed that the representative compound 5h possessed the highest inhibitory activity against wild-type HIV-1 and selectivity index (EC50 = 0.0018 µM, SI > 166667), which were obviously better than that of 1 (EC50 = 0.00978 µM, SI > 37764), NVP (EC50 = 0.059 µM, SI > 158), EFV (EC50 = 0.028 µM, SI > 269), and ETR (EC50 = 0.0029 µM, SI > 1519). The water solubility of compound 5h was remarkably improved, surpassing that of 1, ETR and RPV. Additionally, this compound exerted significantly enhanced anti-resistance potency, compared to 1, and displayed comparable activity to ETR against WT RT of HIV-1 (IC50 = 0.011 µM). To elucidate the underlying molecular mechanisms, molecular docking studies were conducted to investigate the crucial interactions between 5h and WT/mutant strains of HIV-1. These findings provide valuable insights and drive further advancements in the development of DAPYs for HIV therapy.

Development of novel HEPT analogs featuring significantly improved anti-resistance potency against HIV-1 through chemical space exploration of the tolerant region I.

Our recent great interest in developing 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine (HEPT) analogs for HIV therapy identified a potent non-nucleoside reverse transcriptase inhibitor (NNRTI) 3 (EC50 = 0.01681 µM), but its therapeutic efficacy was limited by its poor anti-resistance potency. This prompted us to search for potential HEPT analogs with broad-spectrum activities, leading to the generation of a series of novel HEPT analogs through exploring the chemical space of the solvent - protein interface. Encouraging improvements in anti-resistance efficacy were observed in some of these analogs, with the most promising compound 7 g being 3 to 26 - fold more potent than 3 against five mutant strains (E138K, Y181C, L100I, K103N, and Y188L). This analog surpassed the activity and selectivity of compound 3 by approximately 2-fold (EC50 = 0.007468 µM, SI = 4260). Furthermore, it was found to demonstrate feeble inhibition of CYP and hERG in vitro, and no in vivo acute toxicity. This study will further enrich the structure-activity relationships (SARs) of the HEPT scaffold, providing new guidance for the development of NNRTIs.

5-Cyano substituted diarylpyridines as potent HIV-1 NNRTIs: Rational design, synthesis, and activity evaluation.

To develop more potent HIV-1 inhibitors against a variety of NNRTIs-resistant strains, a series of 5-cyano substituted diarylpyridines was designed based on the cocrystal structural analysis. Among them, I-5b showed the greatest potency (EC50 = 5.62-171 nM) against the wild-type (WT) and mutant HIV-1 strains. Especially for K103 N, I-5b exhibited outstanding activity with EC50 values of 9.37 nM, being much superior to that of NVP (EC50 = 5128 nM) and EFV (EC50 = 114 nM) and comparable to that of ETR (EC50 = 3.45 nM). In addition, the target of all compounds was turned out to be HIV-1 RT with moderate RT enzyme inhibitory activity (IC50 = 0.094-12.0 µM). Moreover, the binding mode of representative compounds with RT was elaborated via molecular docking.

Discovery of novel diarypyrimidine derivatives bearing six-membered non-aromatic heterocycles as potent HIV-1 NNRTIs with improved anti-resistance and drug-like profiles.

Taking our previously reported HIV-1 NNRTIs BH-11c and XJ-10c as lead compounds, series of novel diarypyrimidine derivatives bearing six-membered non-aromatic heterocycles were designed to improve anti-resistance and drug-like profiles. According to the three rounds of in vitro antiviral activity screening, compound 12g was the most active inhibitor against wild-type and five prevalent NNRTI-resistant HIV-1 strains with EC50 values ranging from 0.024 to 0.0010 µM. This is obviously better than the lead compound BH-11c and the approved drug ETR. Detailed structure-activity relationship was investigated to provide valuable guidance for further optimization. The MD simulation study indicated that 12g could form additional interactions with residues around the binding site in HIV-1 RT, which provided reasonable explanations for its improved anti-resistance profile compared to ETR. Furthermore, 12g showed significant improvement in water solubility and other drug-like properties compared to ETR. The CYP enzymatic inhibitory assay indicated that 12g was unlikely to induce CYP-mediated drug-drug interactions. 12g pharmacokinetics parameters were investigated and it displayed a long half-life of 6.59 h in vivo. The properties of compound 12g make it a promising lead compound for the development of new generation of antiretroviral drugs.

Discovery of novel sulfonamide substituted indolylarylsulfones as potent HIV-1 inhibitors with better safety profiles.

Indolylarylsulfones (IASs) are classical HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a unique scaffold and possess potent antiviral activity. To address the high cytotoxicity and improve safety profiles of IASs, we introduced various sulfonamide groups linked by alkyl diamine chain to explore the entrance channel of non-nucleoside inhibitors binding pocket. 48 compounds were designed and synthesized to evaluate their anti-HIV-1 activities and reverse transcriptase inhibition activities. Especially, compound R10L4 was endowed with significant inhibitory activity towards wild-type HIV-1 (EC50(WT) = 0.007 µmol/L, SI = 30,930) as well as a panel of single-mutant strains exemplified by L100I (EC50 = 0.017 µmol/L, SI = 13,055), E138K (EC50 = 0.017 µmol/L, SI = 13,123) and Y181C (EC50 = 0.045 µmol/L, SI = 4753) which were superior to Nevirapine and Etravirine. Notably, R10L4 was characterized with significantly reduced cytotoxicity (CC50 = 216.51 µmol/L) and showed no remarkable in vivo toxic effects (acute and subacute toxicity). Moreover, the computer-based docking study was also employed to characterize the binding mode between R10L4 and HIV-1 RT. Additionally, R10L4 presented an acceptable pharmacokinetic profile. Collectively, these results deliver precious insights for next optimization and indicate that the sulfonamide IAS derivatives are promising NNRTIs for further development.

Picomolar inhibitor of reverse transcriptase featuring significantly improved metabolic stability.

Considering the undesirable metabolic stability of our recently identified NNRTI 5 (t1/2 = 96 min) in human liver microsomes, we directed our efforts to improve its metabolic stability by introducing a new favorable hydroxymethyl side chain to the C-5 position of pyrimidine. This strategy provided a series of novel methylol-biphenyl-diarylpyrimidines with excellent anti-HIV-1 activity. The best compound 9g was endowed with remarkably improved metabolic stability in human liver microsomes (t1/2 = 2754 min), which was about 29-fold longer than that of 5 (t1/2 = 96 min). This compound conferred picomolar inhibition of WT HIV-1 (EC50 = 0.9 nmol/L) and low nanomolar activity against five clinically drug-resistant mutant strains. It maintained particularly low cytotoxicity (CC50 = 264 µmol/L) and good selectivity (SI = 256,438). Molecular docking studies revealed that compound 9g exhibited a more stable conformation than 5 due to the newly constructed hydrogen bond of the hydroxymethyl group with E138. Also, compound 9g was characterized by good safety profiles. It displayed no apparent inhibition of CYP enzymes and hERG. The acute toxicity assay did not cause death and pathological damage in mice at a single dose of 2 g/kg. These findings paved the way for the discovery and development of new-generation anti-HIV-1 drugs.

Design, synthesis, and mechanistic study of 2-piperazineone-bearing peptidomimetics as novel HIV capsid modulators.

HIV-1 capsid (CA) is an attractive target for its indispensable roles in the viral life cycle. We report the design, synthesis, and mechanistic study of a novel series of 2-piperazineone peptidomimetics as HIV capsid modulators by mimicking the structure of host factors binding to CA. F-Id-3o was the most potent compound from the synthesized series, with an anti-HIV-1 EC50 value of 6.0 µM. However, this series of compounds showed a preference for HIV-2 inhibitory activity, in which Id-3o revealed an EC50 value of 2.5 µM (anti-HIV-2 potency), an improvement over PF74. Interestingly, F-Id-3o did bind HIV-1 CA monomers and hexamers with comparable affinity, unlike PF74, consequently showing antiviral activity in the early and late stages of the HIV-1 lifecycle. Molecular dynamics simulations shed light on F-Id-3o and Id-3o binding modes within the HIV-1/2 CA protein and provide a possible explanation for the increased anti-HIV-2 potency. Metabolic stability assays in human plasma and human liver microsomes indicated that although F-Id-3o has enhanced metabolic stability over PF74, further optimization is necessary. Moreover, we utilized computational prediction of drug-like properties and metabolic stability of F-Id-3o and PF74, which correlated well with experimentally derived metabolic stability, providing an efficient computational pipeline for future preselection based on metabolic stability prediction. Overall, the 2-piperazineone-bearing peptidomimetics are a promising new chemotype in the CA modulators class with considerable optimization potential.

Escaping from Flatland: Multiparameter Optimization Leads to the Discovery of Novel Tetrahydropyrido[4,3-d]pyrimidine Derivatives as Human Immunodeficiency Virus-1 Non-nucleoside Reverse Transcriptase Inhibitors with Superior Antiviral Activities against Non-nucleoside Reverse Transcriptase Inhibitor-Resistant Variants and Favorable Drug-like Profiles.

In the current landscape of antiretroviral options, there remains an urgent need for novel non-nucleoside reverse transcriptase inhibitors (NNRTIs) with improved resistance profiles and safety properties. Herein, a series of novel tetrahydropyrido[4,3-d]pyrimidine derivatives were discovered utilizing the "escape from flatland" strategy. The most potent inhibitor 10c was endowed with broad-spectrum antiviral activity and improved resistance profiles against NNRTI-resistant variants compared to efavirenz and etravirine. Molecular simulations were investigated to furnish insights into the biological results. Drug-likeness assessment showed that 10c exhibited desirable physicochemical properties and in vitro metabolic stability. Notably, lower cytochrome P450 inhibition and human ether-à-go-go-related gene blockade liability were observed for 10c than those for etravirine and rilpivirine. Besides, 10c was characterized by excellent in vivo safety properties without acute/subacute toxicity and organ pathological damage. Overall, our multiparameter optimization campaign led to the identification of 10c with excellent antiviral activities and favorable drug-like profiles that could serve as an ideal drug candidate for further development.