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In the effort to identify and develop new HIV-1 inhibitors endowed with innovative mechanisms, we focused our attention on the possibility to target more than one viral encoded enzymatic function with a single molecule. In this respect, we have previously identified by virtual screening a new indolinone-based scaffold for dual allosteric inhibitors targeting both reverse transcriptase-associated functions: polymerase and RNase H. Pursuing with the structural optimization of these dual inhibitors, we synthesized a series of 35 new 3-[2-(4-aryl-1,3-thiazol-2-ylidene)hydrazin-1-ylidene]1-indol-2-one and 3-[3-methyl-4-arylthiazol-2-ylidene)hydrazine-1-ylidene)indolin-2-one derivatives, which maintain their dual inhibitory activity in the low micromolar range. Interestingly, compounds 1a, 3a, 10a, and 9b are able to block HIV-1 replication with EC 50 < 20 µM. Mechanism of action studies showed that such compounds could block HIV-1 integrase. In particular, compound 10a is the most promising for further multitarget compound development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Corona, Meleddu, Delelis, Subra, Cottiglia, Esposito, Distinto, Maccioni and Tramontano.)

Bioassay-guided fractionation of the ethyl acetate extract from Teucrium flavum subsp. glaucum , endowed with inhibitory activity towards the HIV-1 reverse transcriptase-associated RNase H function, led to the isolation of salvigenin ( 1 ), cirsimaritin ( 2 ) and cirsiliol ( 3 ) along with the neo -clerodanes teuflavin ( 4 ) and teuflavoside ( 5 ). Acid hydrolysis of the inactive teuflavoside provided three undescribed neo -clerodanes, flavuglaucins A-C ( 7-9 ) and one known neo -clerodane ( 10 ). Among all neo -clerodanes, flavuglaucin B showed the highest inhibitory activity towards RNase H function with a IC 50 value of 9.1 μM. Molecular modelling and site-directed mutagenesis analysis suggested that flavuglaucin B binds into an allosteric pocket close to RNase H catalytic site. This is the first report of clerodane diterpenoids endowed with anti-reverse transcriptase activity. Neo -clerodanes represent a valid scaffold for the development of a new class of HIV-1 RNase H inhibitors.

The paper focussed on a step-by-step structural modification of a cycloheptathiophene-3-carboxamide derivative recently identified by us as reverse transcriptase (RT)-associated ribonuclease H (RNase H) inhibitor. In particular, its conversion to a 2-aryl-cycloheptathienoozaxinone derivative and the successive thorough exploration of both 2-aromatic and cycloheptathieno moieties led to identify oxazinone-based compounds as new anti-RNase H chemotypes. The presence of the catechol moiety at the C-2 position of the scaffold emerged as critical to achieve potent anti-RNase H activity, which also encompassed anti-RNA dependent DNA polymerase (RDDP) activity for the tricyclic derivatives. Benzothienooxazinone derivative 22 resulted the most potent dual inhibitor exhibiting IC 50 s of 0.53 and 2.90 μM against the RNase H and RDDP functions. Mutagenesis and docking studies suggested that compound 22 binds two allosteric pockets within the RT, one located between the RNase H active site and the primer grip region and the other close to the DNA polymerase catalytic centre.

In this work we report a parallel application of both docking- and shape-based virtual screening (VS) methods, followed by Molecular Dynamics simulations (MDs), for discovering new compounds able to inhibit the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) RNA-dependent DNA polymerase activity. Specifically, we screened more than 143000 natural compounds commercially available in the ZINC database against the best five RT crystallographic models, taking into account the five approved NNRTIs as query compounds. As a result, 20 hit molecules were selected and tested on biochemical assays for the inhibition of the RNA dependent DNA polymerase RT function and, among them, an indoline pyrrolidine (hit1), an indonyl piperazine (hit2) and an indolyl indolinone (hit3) derivatives were identified as novel non-nucleoside RT inhibitors in the low micromolar range., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

A series of 3-3-{2-[2-3-methyl-4-phenyl-2,3-dihydro-1,3-thiazol-2-ylidene]hydrazin-1-ylidene-2,3-dihydro-1H-indol-2-one derivatives has been designed and synthesized to study their activity on both HIV-1 (Human Immunodeficiency Virus type 1) RT (Reverse Transcriptase) associated functions. These derivatives are analogs of previously reported series whose biological activity and mode of action have been investigated. In this work we investigated the influence of the introduction of a methyl group in the position 3 of the dihydrothiazole ring and of a chlorine atom in the position 5 of the isatin nucleus. The new synthesized compounds are active towards both DNA polymerase and ribonuclease H in the µM range. The nature of the aromatic group in the position 4 of the thiazole was relevant in determining the biological activity.

The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT.

The HIV-1 Reverse Transcriptase (RT) is a validated and deeply explored biological target for the treatment of AIDS. However, only drugs targeting the RT-associated DNA polymerase (DP) function have been approved for clinical use. We designed and synthesised a new generation of HIV-1 RT inhibitors, based on the (3Z)-3-(2-[4-(aryl)-1,3-thiazol-2-yl]hydrazin-1-ylidene)-2,3-dihydro-1H-indol-2-one scaffold. These compounds are active towards both RT-associated functions, DNA polymerase and ribonuclease H. The structure, biological activity and mode of action of the new derivatives have been investigated. In particular, the nature of the aromatic group in the position 4 of the thiazole ring plays a key role in the modulation of the activity towards the two RT-associated functions., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

A small library of 1,3-diarylpropenones was designed and synthesized as dual inhibitors of both HIV-1 reverse transcriptase (RT) DNA polymerase (DP) and ribonuclease H (RNase H) associated functions. Compounds were assayed on these enzyme activities, which highlighted dual inhibition properties in the low-micromolar range. Interestingly, mutations in the non-nucleoside RT inhibitor binding pocket strongly affected RNase H inhibition by the propenone derivatives without decreasing their capacity to inhibit DP activity, which suggests long-range RT structural effects. Biochemical and computational studies indicated that the propenone derivatives bind two different interdependent allosteric pockets., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The HIV-1 reverse transcriptase (RT) is one of the most attracting targets for the development of early phase infection inhibitors. Although many RT inhibitors have been approved for the treatment of HIV-1 infection, they all target the polymerase function of this enzyme. So far, no drugs are available for the inhibition of the RT associated ribonuclease H function (RNase H), which plays an essential role in the HIV replication cycle. Moreover it should be reported that many of the known RT inhibitors, targeting the polymerase function, enhance the RNase H activity, indicating that, although spatially distinct, a close relation occurs between the two functions. The aim of this review is to summarise the efforts in the design of new inhibitors either characterized by a novel mechanism of action or capable of blocking both RT associated functions, as well as pointing out the main binding features of the known RT inhibitors.

We report the first application of ligand-based virtual screening (VS) methods for discovering new compounds able to inhibit both human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT)-associated functions, DNA polymerase and ribonuclease H (RNase H) activities. The overall VS campaign consisted of two consecutive screening processes. In the first, the VS platform Rapid Overlay of Chemical Structures (ROCS) was used to perform in silico shape-based similarity screening on the NCI compounds database in which a hydrazone derivative, previously shown to inhibit the HIV-1 RT, was chosen. As a result, 34 hit molecules were selected and assayed on both RT-associated functions. In the second, the 4 most potent RT inhibitors identified were selected as queries for parallel VS performed by combining shape-based, 2D-fingerprint and 3D-pharmacophore VS methods. Overall, a set of molecules characterized by new different scaffolds were identified as novel inhibitors of both HIV-1 RT-associated activities in the low micromolar range., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

HIV-1 reverse transcriptase (RT) has two associated activities, DNA polymerase and RNase H, both essential for viral replication and validated drug targets. Although all RT inhibitors approved for therapy target DNA polymerase activity, the search for new RT inhibitors that target the RNase H function and are possibly active on RTs resistant to the known non-nucleoside inhibitors (NNRTI) is a viable approach for anti-HIV drug development. In this study, several alizarine derivatives were synthesized and tested for both HIV-1 RT-associated activities. Alizarine analogues K-49 and KNA-53 showed IC(50) values for both RT-associated functions of ∼ 10 μm. When tested on the K103N RT, both derivatives inhibited the RT-associated functions equally, whereas when tested on the Y181C RT, KNA-53 inhibited the RNase H function and was inactive on the polymerase function. Mechanism of action studies showed that these derivatives do not intercalate into DNA and do not chelate the divalent cofactor Mg(2+) . Kinetic studies demonstrated that they are noncompetitive inhibitors, they do not bind to the RNase H active site or to the classical NNRTI binding pocket, even though efavirenz binding negatively influenced K-49/KNA-53 binding and vice versa. This behavior suggested that the alizarine derivatives binding site might be close to the NNRTI binding pocket. Docking experiments and molecular dynamic simulation confirmed the experimental data and the ability of these compounds to occupy a binding pocket close to the NNRTI site., (© 2011 The Authors Journal compilation © 2011 FEBS.)