Synthesis, in vitro Anti-HIV-1RT evaluation, molecular modeling, DFT and acute oral toxicity studies of some benzotriazole derivatives.

This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound 3 h, followed closely by 6 h, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. In vivo oral toxicity studies were conducted for the most active compound 3 h, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of 3 h, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.

Structure-based design, synthesis, and biological characterization of indolylarylsulfone derivatives as novel human immunodeficiency virus type 1 inhibitors with potent antiviral activities and favorable drug-like profiles.

In the current antiretroviral landscape, continuous efforts are still needed to search for novel chemotypes of human immunodeficiency virus type 1 (HIV-1) inhibitors with improved drug resistance profiles and favorable drug-like properties. Herein, we report the design, synthesis, biological characterization, and druggability evaluation of a class of non-nucleoside reverse transcriptase inhibitors. Guided by the available crystallographic information, a series of novel indolylarylsulfone derivatives were rationally discovered via the substituent decorating strategy to fully explore the chemical space of the entrance channel. Among them, compound 11h bearing the cyano-substituted benzyl moiety proved to be the most effective inhibitor against HIV-1 wild-type and mutant strains (EC50 = 0.0039-0.338 µM), being far more potent than or comparable to etravirine and doravirine. Besides, 11h did not exhibit cytotoxicity at the maximum test concentration. Meanwhile, the binding target of 11h was further confirmed to be reverse transcriptase (IC50 = 0.055 µM). Preliminary structure-activity relationship were discussed to guide further optimization work. Molecular docking and dynamics simulation studies were investigated in detail to rationalize the biological evaluation results. Further drug-likeness assessment indicated that 11h possessed excellent physicochemical properties. Moreover, no apparent hERG blockade liability and cytochrome P450 inhibition were observed for 11h. Notably, 11h was characterized by favorable in vitro metabolic stability with moderate clearance rates and long half-lives in human plasma and liver microsomes. Overall, 11h holds great promise as an ideal Anti-HIV-1 lead compound due to its potent antiviral efficacy, low toxicity, and favorable drug-like profiles.

From Intercalation to External Binding: Ru(II) Complexes with a Spiro Ligand for TAR RNA Selective Binding and HIV-1 Reverse Transcriptase Inhibition.

As a typical RNA virus, the genetic information on HIV-1 is entirely stored in RNA. The reverse transcription activity of HIV-1 reverse transcriptase (RT) plays a crucial role in the replication and transmission of the virus. Non-nucleoside RT inhibitors (NNRTIs) block the function of RT by binding to the RNA binding site on RT, with very few targeting viral RNA. In this study, by transforming planar conjugated ligands into a spiro structure, we convert classical Ru(II) DNA intercalators into a nonintercalator. This enables selective binding to HIV-1 transactivation response (TAR) RNA on the outer side of nucleic acids through dual interactions involving hydrogen bonds and electrostatic attraction, effectively inhibiting HIV-1 RT and serving as a selective fluorescence probe for TAR RNA.

2'-ß-Methylselenyl nucleos(t)ide analogs as reverse transcriptase inhibitors against diverse HIV mutants.

Nucleoside reverse transcriptase inhibitors (NRTIs) have been extensively studied as drugs targeting HIV RT. However, the practice or use of approved NRTIs lacking the 3'-hydroxy group often promotes frequent HIV mutations and generates drug-resistance. Here, we describe a novel NRTI with 2'-ß-methylselenyl modification. We found that this modification inhibited the DNA elongation reaction by HIV-1 RT despite having a 3'-hydroxy group. Moreover, the conformation of this nucleoside analog is controlled at C3'-endo, a conformation that resists excision from the elongating DNA by HIV RT. Accordingly, the designed analogs exhibited activity against both wild-type HIV and multidrug-resistant HIV mutants.

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.

Tri-substituted 1,3,5-triazine-based analogs as effective HIV-1 non-nucleoside reverse transcriptase inhibitors (NNRTIs): A systematic review.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) have significantly impacted the HIV-1 wild-type due to their high specificity and superior potency. As well as different combinations of NNRTIs have been used on clinically approved combining highly active antiretroviral therapy (HAART) to resist the growth of HIV-1 and decrease the mortality rate of HIV/AIDS. Although the feeble strength against the drug-resistant mutant strains and the long-term damaging effects have been reducing the effectiveness of HAART, it could be a crucial challenge to develop novel Anti-HIV leads with a vital mode of action and the least side effects. The extensive chemical reactivity and the diverse chemotherapeutic applications of the 1,3,5-triazine have provided a wide scope of research in medicinal chemistry via a structural modification. In this review, we focused on the Anti-HIV profile of the tri-substituted s-triazine derivatives with structure-based features and also discussed the active mode of action to evaluate the significant findings. The tri-substituted 1,3,5-triazine derivatives have been found more promising to inhibit the growth of the drug-sensitive and drug-resistant variants of HIV-1, especially HIV-1 wild-type, HIV-1 K103N/Y181C, and HIV-1 Tyr181Cys. It has been observed that these derivatives have interacted with the enzyme protein residues via a significant π $\pi $ - π $\pi $ interaction and hydrogen bonding to resist the proliferation of the viral genomes. Further, the SAR and the active binding modes are critically described and highlight the role of structural variations with functional groups along with the binding affinity of targeted enzymes, which may be beneficial for rational drug discovery to develop highly dynamic Anti-HIV agents.

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.

Proposal of pharmacophore model for HIV reverse transcriptase inhibitors: Combined mutational effect analysis, molecular dynamics, molecular docking and pharmacophore modeling study.

OBJECTIVES: Antiretroviral therapy (ART) efficacy is jeopardized by the emergence of drug resistance mutations in HIV, compromising treatment effectiveness. This study aims to propose novel analogs of Effavirenz (EFV) as potential direct inhibitors of HIV reverse transcriptase, employing computer-aided drug design methodologies. METHODS: Three key approaches were applied: a mutational profile study, molecular dynamics simulations, and pharmacophore development. The impact of mutations on the stability, flexibility, function, and affinity of target proteins, especially those associated with NRTI, was assessed. Molecular dynamics analysis identified G190E as a mutation significantly altering protein properties, potentially leading to therapeutic failure. Comparative analysis revealed that among six first-line antiretroviral drugs, EFV exhibited notably low affinity with viral reverse transcriptase, further reduced by the G190E mutation. Subsequently, a search for EFV-similar inhibitors yielded 12 promising molecules based on their affinity, forming the basis for generating a pharmacophore model. RESULTS: Mutational analysis pinpointed G190E as a crucial mutation impacting protein properties, potentially undermining therapeutic efficacy. EFV demonstrated diminished affinity with viral reverse transcriptase, exacerbated by the G190E mutation. The search for EFV analogs identified 12 high-affinity molecules, culminating in a pharmacophore model elucidating key structural features crucial for potent inhibition. CONCLUSION: This study underscores the significance of EFV analogs as potential inhibitors of HIV reverse transcriptase. The findings highlight the impact of mutations on drug efficacy, particularly the detrimental effect of G190E. The generated pharmacophore model serves as a pivotal reference for future drug development efforts targeting HIV, providing essential structural insights for the design of potent inhibitors based on EFV analogs identified in vitro.

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.

Comparison of feline and human immunodeficiency virus reverse transcriptase enzymes through chemical screening and computational analysis.

Feline immunodeficiency virus (FIV) is a common infection found in domesticated and wild cats worldwide. Despite the wealth of therapeutic understanding of the disease in humans, considerably less information exists regarding the treatment of the disease in felines. Current treatment relies on drugs developed for the related human immunodeficiency virus (HIV) and includes compounds of the popular non-nucleotide reverse transcriptase (NNRTI) class. This is despite FIV-RT being only 67% similar to HIV-1 RT at the enzyme level, increasing to 88% for the allosteric pocket targeted by NNRTIs. The goal of this project was to try to quantify how well the more extensive pharmacological knowledge available for human disease translates to felines. To this end we screened known NNRTIs and 10 diverse pyrimidine analogs identified virtually. We use this chemo-centric probe approach to (a) assess the similarity between the two related RT targets based on the observed experimental inhibition values, (b) try to identify more potent inhibitors at FIV, and (c) gain a better appreciation of the structure-activity relationships (SAR). We found the correlation between IC50s at the two targets to be strong (r2 = 0.87) and identified compound 1 as the most potent inhibitor of FIV with IC50 of 0.030 µM ± 0.009. This compared to FIV IC50 values of 0.22 ± 0.17 µM, 0.040 ± 0.010 µM and >160 µM for known anti HIV-1 RT drugs Efavirenz, Rilpivirine, and Nevirapine, respectively. This knowledge, along with an understanding of the structural origin that give rise to any differences could improve the way HIV drugs are repurposed for FIV.

Profiling the Interaction between Human Serum Albumin and Clinically Relevant HIV Reverse Transcriptase Inhibitors.

Tenofovir (TFV) is the active form of the prodrugs tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF), both clinically prescribed as HIV reverse transcriptase inhibitors. The biophysical interactions between these compounds and human serum albumin (HSA), the primary carrier of exogenous compounds in the human bloodstream, have not yet been thoroughly characterized. Thus, the present study reports the interaction profile between HSA and TFV, TDF, and TAF via UV-Vis, steady-state, and time-resolved fluorescence techniques combined with isothermal titration calorimetry (ITC) and in silico calculations. A spontaneous interaction in the ground state, which does not perturb the microenvironment close to the Trp-214 residue, is classified as weak. In the case of HSA/TFV and HSA/TDF, the binding is both enthalpically and entropically driven, while for HSA/TAF, the binding is only entropically dominated. The binding constant (Ka) and thermodynamic parameters obtained via ITC assays agree with those obtained using steady-state fluorescence quenching measurements, reinforcing the reliability of the data. The small internal cavity known as site I is probably the main binding pocket for TFV due to the low steric volume of the drug. In contrast, most external sites (II and III) can better accommodate TAF due to the high steric volume of this prodrug. The cross-docking approach corroborated experimental drug-displacement assays, indicating that the binding affinity of TFV and TAF might be impacted by the presence of different compounds bound to albumin. Overall, the weak binding capacity of albumin to TFV, TDF, and TAF is one of the main factors for the low residence time of these antiretrovirals in the human bloodstream; however, positive cooperativity for TAF and TDF was detected in the presence of some drugs, which might improve their residence time (pharmacokinetic profile).

TAR RNA selective targeting ruthenium(II) complexes as HIV-1 reverse transcriptase inhibitors: On exploring structure-activity relationships of multiple positions.

HIV-1 reverse transcriptase (RT) inhibitors play a crucial role in the treatment of HIV by preventing the activity of the enzyme responsible for the replication of the virus. The HIV-1 Tat protein binds to transactivation response (TAR) RNA and recruits host factors to stimulate HIV-1 transcription. We have created a small library consisting of 4 × 6 polypyridyl Ru(II) complexes that selectively bind to TAR RNA, with targeting groups specific to HIV-1 TAR RNA. The molecule design was conducted by introducing hydroxyl or methoxy groups into an established potent TAR binder. The potential TAR binding ability was analysis from nature charge population and electrostatic potential by quantum chemistry calculations. Key modifications were found to be R1 and R3 groups. The most potent and selective TAR RNA binder was a3 with R1 = OH, R2 = H and R3 = Me. Through molecular recognition of hydrogen bonds and electrostatic attraction, they were able to firmly and selectively bind HIV-1 TAR RNA. Furthermore, they efficiently obstructed the contact between TAR RNA and Tat protein, and inhibited the reverse transcription activity of HIV-1 RT. The polypyridyl Ru(II) complexes were chemical and photo-stable, and sensitive and selective spectroscopic responses to TAR RNA. They exhibited little toxicity towards normal cells. Hence, this study might offer significant drug design approaches for researching AIDS and other illnesses associated with RT, including HCV, EBOV, and SARS-CoV-2. Moreover, it could contribute to fundamental research on the interactions of inorganic transition metal complexes with biomolecules.

Discovery of Benzisothiazolone Derivatives as Bifunctional Inhibitors of HIV-1 Reverse Transcriptase DNA Polymerase and Ribonuclease H Activities.

The ribonuclease H (RNase H) active site of HIV-1 reverse transcriptase (RT) is the only viral enzyme not targeted by approved antiretroviral drugs. Using a fluorescence-based in vitro assay, we screened 65,239 compounds at a final concentration of 10 µM to identify inhibitors of RT RNase H activity. We identified 41 compounds that exhibited 50% inhibitory concentration (i.e., IC50) values < 1.0 µM. Two of these compounds, 2-(4-methyl-3-(piperidin-1-ylsulfonyl)phenyl)benzo[d]isothiazol-3(2H)-one (1) and ethyl 2-(2-(3-oxobenzo[d]isothiazol-2(3H)-yl)thiazol-4-yl)acetate (2), which both share the same benzisothiazolone pharmacophore, demonstrate robust antiviral activity (50% effective concentrations of 1.68 ± 0.94 µM and 2.68 ± 0.54, respectively) in the absence of cellular toxicity. A limited structure-activity relationship analysis identified two additional benzisothiazolone analogs, 2-methylbenzo[d]isothiazol-3(2H)-one (3) and N,N-diethyl-3-(3-oxobenzo[d]isothiazol-2(3H)-yl)benzenesulfonamide (4), which also resulted in the inhibition of RT RNase H activity and virus replication. Compounds 1, 2 and 4, but not 3, inhibited the DNA polymerase activity of RT (IC50 values~1 to 6 µM). In conclusion, benzisothiazolone derivatives represent a new class of multifunctional RT inhibitors that warrants further assessment for the treatment of HIV-1 infection.

Deviated binding of anti-HBV nucleoside analog E-CFCP-TP to the reverse transcriptase active site attenuates the effect of drug-resistant mutations.

While certain human hepatitis B virus-targeting nucleoside analogs (NAs) serve as crucial anti-HBV drugs, HBV yet remains to be a major global health threat. E-CFCP is a 4'-modified and fluoromethylenated NA that exhibits potent antiviral activity against both wild-type and drug-resistant HBVs but less potent against human immunodeficiency virus type-1 (HIV-1). Here, we show that HIV-1 with HBV-associated amino acid substitutions introduced into the RT's dNTP-binding site (N-site) is highly susceptible to E-CFCP. We determined the X-ray structures of HBV-associated HIV-1 RT mutants complexed with DNA:E-CFCP-triphosphate (E-CFCP-TP). The structures revealed that exocyclic fluoromethylene pushes the Met184 sidechain backward, and the resultant enlarged hydrophobic pocket accommodates both the fluoromethylene and 4'-cyano moiety of E-CFCP. Structural comparison with the DNA:dGTP/entecavir-triphosphate complex also indicated that the cyclopentene moiety of the bound E-CFCP-TP is slightly skewed and deviated. This positioning partly corresponds to that of the bound dNTP observed in the HIV-1 RT mutant with drug-resistant mutations F160M/M184V, resulting in the attenuation of the structural effects of F160M/M184V substitutions. These results expand our knowledge of the interactions between NAs and the RT N-site and should help further design antiviral NAs against both HIV-1 and HBV.

Structure-based discovery of novel piperidine-biphenyl-DAPY derivatives as non-nucleoside reverse transcriptase inhibitors featuring improved potency, safety, and selectivity: From piperazine-biphenyl-DAPYs to piperidine-biphenyl-DAPYs.

Starting from our previously reported nonnucleoside reverse transcriptase inhibitor (NNRTI, 3), continuous efforts were made to enhance its potency and safety through a structure-based drug design strategy. This led to the discovery of a series of novel piperidine-biphenyl-diarylpyrimidines (DAPYs). Compound 10p, the most active compound in this series, exhibited an EC50 value of 6 nM against wide-type HIV-1 strain, which was approximately 560-fold more potent than the initial compound 3 (EC50 = 3.36 µM). Furthermore, significant improvements were observed in cytotoxicity and selectivity (CC50 > 202.17 µM, SI > 33144) compared to compound 3 (CC50 = 14.84 µM, SI = 4). Additionally, compound 10p demonstrated increased inhibitory activity against clinically mutant virus strains (EC50 = 7-63 nM). Further toxicity evaluation revealed that compound 10p exhibited minimal CYP enzyme and hERG inhibition. Importantly, single-dose acute toxicity testing did not result in any fatalities or noticeable pathological damage in mice. Therefore, compound 10p can be regarded as a lead candidate for guiding further development of biphenyl-diarylpyrimidine NNRTIs with favorable druggability for HIV therapy.

Computational drug design strategies applied to the modelling of human immunodeficiency virus-1 reverse transcriptase inhibitors

Reverse transcriptase (RT) is a multifunctional enzyme in the human immunodeficiency virus (HIV)-1 life cycle and represents a primary target for drug discovery efforts against HIV-1 infection. Two classes of RT inhibitors, the nucleoside RT inhibitors (NRTIs) and the nonnucleoside transcriptase inhibitors are prominently used in the highly active antiretroviral therapy in combination with other anti-HIV drugs. However, the rapid emergence of drug-resistant viral strains has limited the successful rate of the anti-HIV agents. Computational methods are a significant part of the drug design process and indispensable to study drug resistance. In this review, recent advances in computer-aided drug design for the rational design of new compounds against HIV-1 RT using methods such as molecular docking, molecular dynamics, free energy calculations, quantitative structure-activity relationships, pharmacophore modelling and absorption, distribution, metabolism, excretion and toxicity prediction are discussed. Successful applications of these methodologies are also highlighted.