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.

HIV-1 Resistance to Islatravir/Tenofovir Combination Therapy in Wild-Type or NRTI-Resistant Strains of Diverse HIV-1 Subtypes.

Tenofovir disoproxil fumarate (TDF) and islatravir (ISL, 4'-ethynyl-2-fluoro-2'-deoxyadensine, or MK-8591) are highly potent nucleoside reverse transcriptase inhibitors. Resistance to TDF and ISL is conferred by K65R and M184V, respectively. Furthermore, K65R and M184V increase sensitivity to ISL and TDF, respectively.

Development of Human Immunodeficiency Virus Type 1 Resistance to 4'-Ethynyl-2-Fluoro-2'-Deoxyadenosine Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor-Resistant Strains.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against wild-type (WT) and drug-resistant HIV-1 in phase III clinical trials. EFdA resistance is not well characterized. To study EFdA resistance patterns that may emerge in naive or tenofovir (TFV)-, emtricitabine/lamivudine (FTC/3TC)-, or zidovudine (AZT)-treated patients, we performed viral passaging experiments starting with WT, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1.

Conformational Changes in HIV-1 Reverse Transcriptase that Facilitate Its Maturation.

HIV-1 reverse transcriptase (RT) is translated as part of the Gag-Pol polyprotein that is proteolytically processed by HIV-1 protease (PR) to finally become a mature heterodimer, composed of a p66 and a p66-derived 51-kDa subunit, p51. Our previous work suggested that tRNA binding to p66/p66 introduces conformational changes in the ribonuclease (RNH) domain of RT that facilitate efficient cleavage of p66 to p51 by PR. In this study, we characterized the conformational changes in the RNH domain of p66/p66 imparted by tRNA using NMR.

Nanocrystal Formulation Improves Vaginal Delivery of CSIC for HIV Prevention.

5-Chloro-3-phenylsulfonylindole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) with potential for use in topical prophylaxis against HIV transmission. However, the hydrophobic nature of CSIC limits its administration through vaginal route. In this study, we developed nanocrystals of CSIC to potentially improve the aqueous solubility and intracellular uptake of CSIC in vitro and in vivo.

Pharmacophore-based design of novel 3-hydroxypyrimidine-2,4-dione subtypes as inhibitors of HIV reverse transcriptase-associated RNase H: Tolerance of a nonflexible linker.

The pharmacophore of active site inhibitors of human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated RNase H typically entails a flexible linker connecting the chelating core and the hydrophobic aromatics. We report herein that novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes with a nonflexible C-6 carbonyl linkage exhibited potent and selective biochemical inhibitory profiles with strong RNase H inhibition at low nM, weak to moderate integrase strand transfer (INST) inhibition at low μM, and no to marginal RT polymerase (pol) inhibition up to 10 μM. A few analogues also demonstrated significant antiviral activity without cytotoxicity.

6-Biphenylmethyl-3-hydroxypyrimidine-2,4-diones potently and selectively inhibited HIV reverse transcriptase-associated RNase H.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains an unvalidated drug target. Reported HIV RNase H inhibitors generally lack significant antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-biphenylmethyl subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype.

6-Arylthio-3-hydroxypyrimidine-2,4-diones potently inhibited HIV reverse transcriptase-associated RNase H with antiviral activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current drugs. Although a few chemotypes have been reported to inhibit HIV RNase H in biochemical assays, their general lack of significant antiviral activity in cell culture necessitates continued efforts in identifying highly potent RNase H inhibitors to confer antiviral activity. We report herein the design, synthesis, biochemical and antiviral evaluations of a new 6-arylthio subtype of the 3-hydroxypyrimidine-2,4-dione (HPD) chemotype.

Effect of tRNA on the Maturation of HIV-1 Reverse Transcriptase.

The mature HIV-1 reverse transcriptase is a heterodimer that comprises 66 kDa (p66) and 51 kDa (p51) subunits. The latter is formed by HIV-1 protease-catalyzed removal of a C-terminal ribonuclease H domain from a p66 subunit. This proteolytic processing is a critical step in virus maturation and essential for viral infectivity.

Preformulation and Vaginal Film Formulation Development of Microbicide Drug Candidate CSIC for HIV prevention.

Purpose: 5-chloro-3-[phenylsulfonyl] indole-2-carboxamide (CSIC) is a highly potent non-nucleoside reverse transcriptase inhibitor (NNRTI) of HIV-1 which has been shown to have a more desirable resistance profile than other NNRTIs in development as HIV prevention strategies. This work involves generation of preformulation data for CSIC and systematic development of a cosolvent system to effectively solubilize this hydrophobic drug candidate. This system was then applied to produce a polymeric thin film solid dosage form for vaginal administration of CSIC for use in prevention of sexual acquisition of HIV.

A 2-Hydroxyisoquinoline-1,3-Dione Active-Site RNase H Inhibitor Binds in Multiple Modes to HIV-1 Reverse Transcriptase.

The RNase H (RNH) function of HIV-1 reverse transcriptase (RT) plays an essential part in the viral life cycle. We report the characterization of YLC2-155, a 2-hydroxyisoquinoline-1,3-dione (HID)-based active-site RNH inhibitor. YLC2-155 inhibits both polymerase (50% inhibitory concentration [IC] = 2.

Double-Winged 3-Hydroxypyrimidine-2,4-diones: Potent and Selective Inhibition against HIV-1 RNase H with Significant Antiviral Activity.

Human immunodeficiency virus (HIV) reverse transcriptase (RT)-associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function yet to be exploited as an antiviral target. One of the possible challenges may be that targeting HIV RNase H is confronted with a steep substrate barrier. We have previously reported a 3-hydroxypyrimidine-2,4-dione (HPD) subtype that potently and selectively inhibited RNase H without inhibiting HIV in cell culture.

Synthesis, biological evaluation and molecular modeling of 2-Hydroxyisoquinoline-1,3-dione analogues as inhibitors of HIV reverse transcriptase associated ribonuclease H and polymerase.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not clinically validated as an antiviral target. 2-Hydroxyisoquinoline-1,3-dione (HID) is known to confer active site directed inhibition of divalent metal-dependent enzymatic functions, such as HIV RNase H, integrase (IN) and hepatitis C virus (HCV) NS5B polymerase. We report herein the synthesis and biochemical evaluation of a few C-5, C-6 or C-7 substituted HID subtypes as HIV RNase H inhibitors.

3-Hydroxypyrimidine-2,4-Diones as Novel Hepatitis B Virus Antivirals Targeting the Viral Ribonuclease H.

Hepatitis B virus (HBV) RNase H (RNH) is an appealing therapeutic target due to its essential role in viral replication. RNH inhibitors (RNHIs) could help to more effectively control HBV infections. Here, we report 3-hydroxypyrimidine-2,4-diones as novel HBV RNHIs with antiviral activity.

Structural basis of HIV inhibition by translocation-defective RT inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA).

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is the most potent nucleoside analog inhibitor of HIV reverse transcriptase (RT). It retains a 3'-OH yet acts as a chain-terminating agent by diminishing translocation from the pretranslocation nucleotide-binding site (N site) to the posttranslocation primer-binding site (P site). Also, facile misincorporation of EFdA-monophosphate (MP) results in difficult-to-extend mismatched primers.

Efficient Inhibition of HIV Replication in the Gastrointestinal and Female Reproductive Tracts of Humanized BLT Mice by EFdA.

Background: The nucleoside reverse transcriptase inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) in preclinical development exhibits improved safety and antiviral activity profiles with minimal drug resistance compared to approved NRTIs. However, the systemic antiviral efficacy of EFdA has not been fully evaluated. In this study, we utilized bone marrow/liver/thymus (BLT) humanized mice to investigate the systemic effect of EFdA treatment on HIV replication and CD4+ T cell depletion in the peripheral blood (PB) and tissues.

Design, Synthesis, and Biological Evaluations of Hydroxypyridonecarboxylic Acids as Inhibitors of HIV Reverse Transcriptase Associated RNase H.

Targeting the clinically unvalidated reverse transcriptase (RT) associated ribonuclease H (RNase H) for human immunodeficiency virus (HIV) drug discovery generally entails chemotypes capable of chelating two divalent metal ions in the RNase H active site. The hydroxypyridonecarboxylic acid scaffold has been implicated in inhibiting homologous HIV integrase (IN) and influenza endonuclease via metal chelation. We report herein the design, synthesis, and biological evaluations of a novel variant of the hydroxypyridonecarboxylic acid scaffold featuring a crucial N-1 benzyl or biarylmethyl moiety.

3-Hydroxypyrimidine-2,4-diones as Selective Active Site Inhibitors of HIV Reverse Transcriptase-Associated RNase H: Design, Synthesis, and Biochemical Evaluations.

Human immunodeficiency virus (HIV) reverse transcriptase (RT) associated ribonuclease H (RNase H) remains an unvalidated antiviral target. A major challenge of specifically targeting HIV RNase H arises from the general lack of selectivity over RT polymerase (pol) and integrase (IN) strand transfer (ST) inhibitions. We report herein the synthesis and biochemical evaluations of three novel 3-hydroxypyrimidine-2,4-dione (HPD) subtypes carefully designed to achieve selective RNase H inhibition.

Nucleotide Sugar Pucker Preference Mitigates Excision by HIV-1 RT.

A series of DNA primers containing nucleotides with various sugar pucker conformations at the 3'-terminus were chemically synthesized by solid-phase synthesis. The ability of wild-type (WT) HIV-1 reverse transcriptase (RT) and AZT-resistant (AZTr) RT to excise the 3'-terminal nucleotide was assessed. Nucleosides with a preference for the North conformation were more refractory to excision by both WT-RT and AZTr-RT.

Structural integrity of the ribonuclease H domain in HIV-1 reverse transcriptase.

The mature form of reverse transcriptase (RT) is a heterodimer comprising the intact 66-kDa subunit (p66) and a smaller 51-kDa subunit (p51) that is generated by removal of most of the RNase H (RNH) domain from a p66 subunit by proteolytic cleavage between residues 440 and 441. Viral infectivity is eliminated by mutations such as F440A and E438N in the proteolytic cleavage sequence, while normal processing and virus infectivity are restored by a compensatory mutation, T477A, that is located more than 10 Å away from the processing site. The molecular basis for this compensatory effect has remained unclear.

Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.

Vaginal Microbicide Film Combinations of Two Reverse Transcriptase Inhibitors, EFdA and CSIC, for the Prevention of HIV-1 Sexual Transmission.

Purpose: EFdA is a potent nucleoside reverse transcriptase inhibitor (NRTI) with activity against a wide spectrum of wild-type and drug resistant HIV-1 variants. CSIC is a tight-binding non-nucleoside reverse transcriptase inhibitor (NNRTI) with demonstrated anti-HIV properties important for use in topical prevention of HIV transmission. The objective of this study was to develop and characterize film-formulated EFdA and CSIC for use as a female-controlled vaginal microbicide to prevent sexual transmission of HIV.

Synthesis and properties of 2'-deoxy-2',4'-difluoroarabinose-modified nucleic acids.

We report the synthesis, thermal stability, and RNase H substrate activity of 2'-deoxy-2',4'-difluoroarabino-modified nucleic acids. 2'-Deoxy-2',4'-difluoroarabinouridine (2,'4'-diF-araU) was prepared in a stereoselective way in six steps from 2'-deoxy-2'-fluoroarabinouridine (2'-F-araU). NMR analysis and quantum mechanical calculations at the nucleoside level reveal that introduction of 4'-fluorine introduces a strong bias toward the North conformation, despite the presence of the 2'-βF, which generally steers the sugar pucker toward the South/East conformation.

Design, synthesis, biochemical, and antiviral evaluations of C6 benzyl and C6 biarylmethyl substituted 2-hydroxylisoquinoline-1,3-diones: dual inhibition against HIV reverse transcriptase-associated RNase H and polymerase with antiviral activities.

Reverse transcriptase (RT) associated ribonuclease H (RNase H) remains the only virally encoded enzymatic function not targeted by current chemotherapy against human immunodeficiency virus (HIV). Although numerous chemotypes have been reported to inhibit HIV RNase H biochemically, few show significant antiviral activity against HIV. We report herein the design, synthesis, and biological evaluations of a novel variant of 2-hydroxyisoquinoline-1,3-dione (HID) scaffold featuring a crucial C-6 benzyl or biarylmethyl moiety.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) inhibits HIV-1 reverse transcriptase with multiple mechanisms.

4'-Ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) is a nucleoside analog that, unlike approved anti-human immunodeficiency virus type 1 (HIV-1) nucleoside reverse transcriptase inhibitors, has a 3'-OH and exhibits remarkable potency against wild-type and drug-resistant HIVs. EFdA triphosphate (EFdA-TP) is unique among nucleoside reverse transcriptase inhibitors because it inhibits HIV-1 reverse transcriptase (RT) with multiple mechanisms. (a) EFdA-TP can block RT as a translocation-defective RT inhibitor that dramatically slows DNA synthesis, acting as a de facto immediate chain terminator.