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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.

Current therapeutic protocols for the treatment of HIV infection consist of the combination of diverse anti-retroviral drugs in order to reduce the selection of resistant mutants and to allow for the use of lower doses of each single agent to reduce toxicity. However, avoiding drugs interactions and patient compliance are issues not fully accomplished so far. Pursuing on our investigation on potential anti HIV multi-target agents we have designed and synthesized a small library of biphenylhydrazo 4-arylthiazoles derivatives and evaluated to investigate the ability of the new derivatives to simultaneously inhibit both associated functions of HIV reverse transcriptase. All compounds were active towards the two functions, although at different concentrations. The substitution pattern on the biphenyl moiety appears relevant to determine the activity. In particular, compound 2-{3-[(2-{4-[4-(hydroxynitroso)phenyl]-1,3-thiazol-2-yl} hydrazin-1-ylidene) methyl]-4-methoxyphenyl} benzamide bromide ( EMAC2063 ) was the most potent towards RNaseH (IC 50 = 4.5 mM)- and RDDP (IC 50 = 8.0 mM) HIV RT-associated functions.

Ribonucleases H (RNases H) are endonucleolytic enzymes, evolutionarily related to retroviral integrases, DNA transposases, resolvases and numerous nucleases. RNases H cleave RNA in RNA/DNA hybrids and their activity plays an important role in the replication of prokaryotic and eukaryotic genomes, as well as in the replication of reverse-transcribing viruses. During reverse transcription, the RNase H activity of human immunodeficiency virus (HIV) and hepatitis B virus (HBV) degrades the viral genomic RNA to facilitate the synthesis of viral double-stranded DNA. HIV and HBV reverse transcriptases contain DNA polymerase and RNase H domains that act in a coordinated manner to produce double-stranded viral DNA. Although RNase H inhibitors have not been developed into licensed drugs, recent progress has led to the identification of a number of small molecules with inhibitory activity at low micromolar or even nanomolar concentrations. These compounds can be classified into metal-chelating active site inhibitors and allosteric inhibitors. Among them, α-hydroxytropolones, N-hydroxyisoquinolinediones and N-hydroxypyridinediones represent chemotypes active against both HIV and HBV RNases H. In this review we summarize recent developments in the field including the identification of novel RNase H inhibitors, compounds with dual inhibitory activity, broad specificity and efforts to decrease their toxicity., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Reverse transcriptase (RT)-associated DNA polymerase (RDDP) and ribonucleaser H (RNase H) functions are both essential for HIV-1 genome replication, and the identification of new inhibitors to block both of them is a goal actively pursued by the scientific community. In this field, natural extracts have shown a great potential as source of new antivirals. In the present work, we investigated the effect of Uvaria angolensis extracts on the HIV-1 reverse transcriptase-associated DNA polymerase and ribonuclease H activities. The U. angolensis stem bark methanol extract inhibit both HIV-1 RNase H function and RDDP activity with IC 50 values of 1.0 ± 0.2 and 0.62 ± 0.15 μg/mL, respectively and, after been fractionated with different solvents, its solid residue showed an IC 50 of 0.10 ± 0.03 and of 0.23 ± 0.04 μg/mL against RNase H and RDDP, respectively, hence laying the bases for further studies for identification of single active components.

In the continuous effort to identify new HIV-1 inhibitors endowed with innovative mechanisms, the dual inhibition of different viral functions would provide a significant advantage against drug-resistant variants. The HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) is the only viral-encoded enzymatic activity that still lacks an efficient inhibitor. We synthesized a library of 3,5-diamino-N-aryl-1H-pyrazole-4-carbothioamide and 4-amino-5-benzoyl-N-phenyl-2-(substituted-amino)-1H-pyrrole-3-carbothioamide derivatives and tested them against RNase H activity. We identified the pyrazolecarbothioamide derivative A15, able to inhibit viral replication and both RNase H and RNA-dependent DNA polymerase (RDDP) RT-associated activities in the low micromolar range. Docking simulations hypothesized its binding to two RT pockets. Site-directed mutagenesis experiments showed that, with respect to wt RT, V108A substitution strongly reduced A15 IC50 values (12.6-fold for RNase H inhibition and 4.7-fold for RDDP), while substitution A502F caused a 9.0-fold increase in its IC50 value for RNase H, not affecting the RDDP inhibition, reinforcing the hypothesis of a dual-site inhibition. Moreover, A15 retained good inhibition potency against three non-nucleoside RT inhibitor (NNRTI)-resistant enzymes, confirming a mode of action unrelated to NNRTIs and suggesting its potential as a lead compound for development of new HIV-1 RT dual inhibitors active against drug-resistant viruses., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Despite the availability of several anti-retrovirals, there is still an urgent need for developing novel therapeutic strategies and finding new drugs against underexplored HIV-1 targets. Among them, there are the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) function and the cellular α-glucosidase, involved in the control mechanisms of N-linked glycoproteins formation in the endoplasmic reticulum. It is known that many natural compounds, such as pentacyclic triterpenes, are a promising class of HIV-1 inhibitors. Hence, here we tested the pentacyclic triterpene Lupeol, showing that it inhibits the HIV-1 RT-associated RNase H function. We then performed combination studies of Lupeol and the active site RNase H inhibitor RDS1759, and blind docking calculations, demonstrating that Lupeol binds to an HIV-1 RT allosteric pocket. On the bases of these results and searching for potential multitarget active drug supplement, we also investigated the anti-HIV-1 activity of Hemidesmus indicus, an Ayurveda medicinal plant containing Lupeol. Results supported the potential of this plant as a valuable multitarget active drug source. In fact, by virtue of its numerous active metabolites, H. indicus was able to inhibit not only the RT-associated RNase H function, but also the HIV-1 RT-associated RNA-dependent DNA polymerase activity and the cellular α-glucosidase., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

HIV-1 integrase (IN) inhibitors are one of the most recent innovations in the treatment of HIV infection. The selection of drug resistance viral strains is however a still open issue requiring constant efforts to identify new anti-HIV-1 drugs. Pyrrolyl diketo acid (DKA) derivatives inhibit HIV-1 replication by interacting with the Mg 2+ cofactors within the HIV-1 IN active site or within the HIV-1 reverse-transcriptase associated ribonuclease H (RNase H) active site. While the interaction mode of pyrrolyl DKAs with the RNase H active site has been recently reported and substantiated by mutagenesis experiments, their interaction within the IN active site still lacks a detailed understanding. In this study, we investigated the binding mode of four pyrrolyl DKAs to the HIV-1 IN active site by molecular modeling coupled with site-directed mutagenesis studies showing that the DKA pyrrolyl scaffold primarily interacts with the IN amino residues P145, Q146 and Q148. Importantly, the tested DKAs demonstrated good effectiveness against HIV-1 Raltegravir resistant Y143A and N155H INs, thus showing an interaction pattern with relevant differences if compared with the first generation IN inhibitors. These data provide precious insights for the design of new HIV inhibitors active on clinically selected Raltegravir resistant variants. Furthermore, this study provides new structural information to modulate IN and RNase H inhibitory activities for development of dual-acting anti-HIV agents., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Despite the significant progress achieved with combination antiretroviral therapy in the fight against human immunodeficiency virus (HIV) infection, the difficulty to eradicate the virus together with the rapid emergence of multidrug-resistant strains clearly underline a pressing need for innovative agents, possibly endowed with novel mechanisms of action. In this context, owing to its essential role in HIV genome replication, the reverse transcriptase associated ribonuclease H (RNase H) has proven to be an appealing target. To identify new RNase H inhibitors, an in-house cycloheptathiophene-3-carboxamide library was screened; this led to compounds endowed with inhibitory activity, the structural optimization of which led to the catechol derivative 2-(3,4-dihydroxybenzamido)-N-(pyridin-2-yl)-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carboxamide (compound 33) with an IC50 value on the RNase H activity in the nanomolar range. Mechanistic studies suggested selective inhibition of the RNase H through binding to an innovative allosteric site, which could be further exploited to enrich this class of inhibitors., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The development of HIV-1 dual inhibitors is a highly innovative approach aimed at reducing drug toxic side effects as well as therapeutic costs. HIV-1 integrase (IN) and reverse transcriptase-associated ribonuclease H (RNase H) are both selective targets for HIV-1 chemotherapy, and the identification of dual IN/RNase H inhibitors is an attractive strategy for new drug development. We newly synthesized pyrrolyl derivatives that exhibited good potency against IN and a moderate inhibition of the RNase H function of RT, confirming the possibility of developing dual HIV-1 IN/RNase H inhibitors and obtaining new information for the further development of more effective dual HIV-1 inhibitors.

HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg(2+)-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

RNase H plays an essential role in the replication of human immunodeficiency virus type 1 (HIV-1). Therefore, it is a promising target for drug development. However, the identification of HIV-1 RNase H inhibitors (RHIs) has been hampered by the open morphology of its active site, the limited number of available RNase H crystal structures in complex with inhibitors, and the fact that, due to the high concentrations of Mg(2+) needed for protein stability, HIV-1 RNase H is not suitable for nuclear magnetic resonance (NMR) inhibitor studies. We recently showed that the RNase H domains of HIV-1 and prototype foamy virus (PFV) reverse transcriptases (RTs) exhibit a high degree of structural similarity. Thus, we examined whether PFV RNase H can serve as an HIV-1 RNase H model for inhibitor interaction studies. Five HIV-1 RHIs inhibited PFV RNase H activity at low-micromolar concentrations similar to those of HIV-1 RNase H, suggesting pocket similarity of the RNase H domains. NMR titration experiments with the PFV RNase H domain and the RHI RDS1643 (6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester) were performed to determine its binding site. Based on these results and previous data, in silico docking analysis showed a putative RDS1643 binding region that reaches into the PFV RNase H active site. Structural overlays were performed with HIV-1 and PFV RNase H to propose the RDS1643 binding site in HIV-1 RNase H. Our results suggest that this approach can be used to establish PFV RNase H as a model system for HIV-1 RNase H in order to identify putative inhibitor binding sites in HIV-1 RNase H., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A series of antiviral basic quinolinonyl diketo acid derivatives were developed as inhibitors of HIV-1 IN. Compounds 12d,f,i inhibited HIV-1 IN with IC50 values below 100 nM for strand transfer and showed a 2 order of magnitude selectivity over 3'-processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV-1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.

Despite the wealth of information available for the reverse transcriptase (RT)-associated ribonuclease H (RNaseH) domain of lentiviruses, gammaretroviruses and long terminal repeat containing retrotransposons, exploiting this information in the form of an RNaseH inhibitor with high specificity and low cellular toxicity has been disappointing. However, it is now becoming increasingly evident that the two-subunit HIV-1 RT is a highly versatile enzyme, undergoing major structural alterations in order to interact with, position and ultimately hydrolyze the RNA component of an RNA/DNA hybrid. Thus, in addition to targeting the RNaseH active site, identifying small molecules that bind elsewhere and disrupt catalysis allosterically by impairing conformational flexibility is gaining increased attention. This review summarizes current progress towards development of both active site and allosteric RNaseH inhibitors.

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.

Background: The degradative activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), termed ribonuclease H (RNase H), which hydrolyzes the RNA component of the heteroduplex RNA:DNA replication intermediate, is an excellent target for drug discovery. Anthraquinones (AQs) and their derivatives, which are common secondary metabolites occurring in bacteria, fungi, lichens and a large number of families in higher plants, have been reported to have several biological activities including that of inhibiting HIV-1 RT activities in biochemical assays., Methods: We have assayed new AQ derivatives on HIV-1 RNase H activities in biochemical assays., Results: Six series of new AQ derivatives with various substituents at positions 1, 2, 3 and 4 of the AQ ring were tested, and new analogs able to inhibit HIV-1 RT-associated RNase H activity in the low micromolar range were found., Conclusions: Our results demonstrate that AQ derivatives are promising anti-RNase H inhibitors., (Copyright © 2012 S. Karger AG, Basel.)

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.)

Ribonuclease H (RNase H) was identified as an important target for HIV therapy. Currently, no RNase H inhibitors have reached clinical status. Herein, a series of novel thiazolone[3,2-a]pyrimidine-containing RNase H inhibitors were developed, based on the hit compound 10i, identified from screening our in-house compound library. Some of these derivatives exhibited low micromolar inhibitory activity. Among them, compound 12b was identified as the most potent inhibitor of RNase H (IC50 = 2.98 μM). The experiment of magnesium ion coordination was performed to verify that this ligand could coordinate with magnesium ions, indicating its binding ability to the catalytic site of RNase H. Docking studies revealed the main interactions of this ligand with RNase H. A quantitative structure activity relationship (QSAR) was also conducted to disclose several predictive mathematic models. A molecular dynamics simulation was also conducted to determine the stability of the complex. Taken together, thiazolone[3,2-a]pyrimidine can be regarded as a potential scaffold for the further development of RNase H inhibitors. [ABSTRACT FROM AUTHOR]

In this work we investigated, for the first time, the effect of Plagius flosculosus (L.) Alavi & Heywood, a Sardinian–Corsican endemic plant, on HIV-1 integrase (IN) activity. The phytochemical analysis of the leaves chloroform extract led us to isolate and characterize three compounds (SPK1, SPK2, and SPK3) belonging to the spiroketals, a group of naturally occurring metabolites of phytochemical relevance with interesting biological properties. Due to their structural diversity, these cyclic ketals have attracted the interest of chemists and biologists. SPK1, SPK2, and SPK3 were evaluated here for their ability to inhibit HIV-1 integrase activity in biochemical assays. The results showed that all the compounds inhibited HIV-1 IN activity. In particular, the most active one was SPK3, which interfered in a low molecular range (IC50 of 1.46 ± 0.16 µM) with HIV-1 IN activity in the presence/absence of the LEDGF cellular cofactor. To investigate the mechanism of action, the three spiroketals were also tested on HIV-1 RT-associated Ribonuclease H (RNase H) activity, proving to be active in inhibiting this function. Although SPK3 was unable to inhibit viral replication in cell culture, it promoted the IN multimerization. We hypothesize that SPK3 inhibited HIV-1 IN through an allosteric mechanism of action. [ABSTRACT FROM AUTHOR]

Viral replication often depends on RNA maturation and degradation processes catalyzed by viral ribonucleases, which are therefore candidate targets for antiviral drugs. Here, we synthesized and studied the antiviral properties of a novel nitrocatechol compound (1c) and other analogs that are structurally related to the catechol derivative dynasore. Interestingly, compound 1c strongly inhibited two DEDD box viral ribonucleases, HIV-1 RNase H and SARS-CoV-2 nsp14 3′-to-5′ exoribonuclease (ExoN). While 1c inhibited SARS-CoV-2 ExoN activity, it did not interfere with the mRNA methyltransferase activity of nsp14. In silico molecular docking placed compound 1c in the catalytic pocket of the ExoN domain of nsp14. Finally, 1c inhibited SARS-CoV-2 replication but had no toxicity to human lung adenocarcinoma cells. Given its simple chemical synthesis from easily available starting materials, these results suggest that 1c might be a lead compound for the design of new antiviral compounds that target coronavirus nsp14 ExoN and other viral ribonucleases. [ABSTRACT FROM AUTHOR]

Bioisosteric replacement and scaffold hopping are powerful strategies in drug design useful for rationally modifying a hit compound towards novel lead therapeutic agents. Recently, we reported a series of thienopyrimidinones that compromise dynamics at the p66/p51 HIV-1 reverse transcriptase (RT)-associated Ribonuclease H (RNase H) dimer interface, thereby allosterically interrupting catalysis by altering the active site geometry. Although they exhibited good submicromolar activity, the isosteric replacement of the thiophene ring, a potential toxicophore, is warranted. Thus, in this article, the most active 2-(3,4-dihydroxyphenyl)-5,6-dimethylthieno[2,3-d]pyrimidin-4(3H)-one 1 was selected as the hit scaffold and several isosteric substitutions of the thiophene ring were performed. A novel series of highly active RNase H allosteric quinazolinone inhibitors was thus obtained. To determine their target selectivity, they were tested against RT-associated RNA-dependent DNA polymerase (RDDP) and integrase (IN). Interestingly, none of the compounds were particularly active on (RDDP) but many displayed micromolar to submicromolar activity against IN. [ABSTRACT FROM AUTHOR]

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 IC50s 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. [ABSTRACT FROM AUTHOR]

[Display omitted] • 1 H -Indeno[2′,1′:5,6]dihydropyrido[2,3- d ]pyrimidine and 1 H -indeno[2′,1′:5,6]pyrido[2,3- d ]pyrimidine derivatives were prepared. • High activity against Toxoplasma gondii parasites and Leishmania major amastigotes was observed. • A catechol derivative inhibits HIV-1 RT-associated RNase H. • Docking and ADME-T calculations confirmed drug-like properties of active compounds. A series of 1 H -indeno[2′,1′:5,6]dihydropyrido[2,3- d ]pyrimidine and 1 H -indeno[2′,1′:5,6]pyrido[2,3- d ]pyrimidine derivatives was prepared and screened for antiparasitic and viral RNase H inhibitory activity. Several compounds showed considerable activity against Toxoplasma gondii parasites and Leishmania major amastigotes, which warrants further investigation. Based on the structural similarities of certain derivatives with common viral RNase H inhibitors, a HIV-1 RNase H assay was used to study the RNase H inhibition by selected test compounds. Docking of active derivatives into the active site of the HIV-1 RNase H enzyme was carried out. The new compound 2a , inactive in the antiparasitic tests, showed distinct HIV-1 RNase H inhibition. Thus, ring substitution determines antiparasitic or HIV-1 RNase H inhibitory activity of this promising compound class. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus type 1 (HIV-1) infection, still represent a serious global health emergency. The chronic toxicity derived from the current anti-retroviral therapy limits the prolonged use of several antiretroviral agents, continuously requiring the discovery of new antiviral agents with innovative strategies of action. In particular, the development of single molecules targeting two proteins (dual inhibitors) is one of the current main goals in drug discovery. In this contest, metal-chelating molecules have been extensively explored as potential inhibitors of viral metal-dependent enzymes, resulting in some important classes of antiviral agents. Inhibition of HIV Integrase (IN) is, in this sense, paradigmatic. HIV-1 IN and Reverse Transcriptase-associated Ribonuclease H (RNase H) active sites show structural homologies, with the presence of two Mg(II) cofactors, hence it seems possible to inhibit both enzymes by means of chelating ligands with analogous structural features. Here we present a series of N'-acylhydrazone ligands with groups able to chelate the Mg(II) hard Lewis acid ions in the active sites of both the enzymes, resulting in dual inhibitors with micromolar and even nanomolar activities. The most interesting identified N'-acylhydrazone analog, compound 18, shows dual RNase H-IN inhibition and it is also able to inhibit viral replication in cell-based antiviral assays in the low micromolar range. Computational modeling studies were also conducted to explore the binding attitudes of some model ligands within the active site of both the enzymes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The HIV-1 ribonuclease H (RNase H) function of the reverse transcriptase (RT) enzyme catalyzes the selective hydrolysis of the RNA strand of the RNA:DNA heteroduplex replication intermediate, and represents a suitable target for drug development. A particularly attractive approach is constituted by the interference with the RNase H metal-dependent catalytic activity, which resides in the active site located at the C-terminus p66 subunit of RT. Herein, we report results of an in-house screening campaign that allowed us to identify 4-[4-(aryl)-1H-1,2,3-triazol-1-yl]benzenesulfonamides, prepared by the "click chemistry" approach, as novel potential HIV-1 RNase H inhibitors. Three compounds (9d, 10c, and 10d) demonstrated a selective inhibitory activity against the HIV-1 RNase H enzyme at micromolar concentrations. Drug-likeness, predicted by the calculation of a panel of physicochemical and ADME properties, putative binding modes for the active compounds, assessed by computational molecular docking, as well as a mechanistic hypothesis for this novel chemotype are reported. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

In a search for natural compounds with anti-HIV-1 activity, we studied the effect of the ethanolic extract obtained from leaves, bark, and peels of Punica granatum L. for the inhibition of the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) and integrase (IN) LEDGF-dependent activities. The chemical analyses led to the detection of compounds belonging mainly to the phenolic and flavonoid chemical classes. Ellagic acid, flavones, and triterpenoid molecules were identified in leaves. The bark and peels were characterized by the presence of hydrolyzable tannins, such as punicalins and punicalagins, together with ellagic acid. Among the isolated compounds, the hydrolyzable tannins and ellagic acid showed a very high inhibition (IC50 values ranging from 0.12 to 1.4 µM and 0.065 to 0.09 µM of the RNase H and IN activities, respectively). Of the flavonoids, luteolin and apigenin were found to be able to inhibit RNase H and IN functions (IC50 values in the 3.7–22 μM range), whereas luteolin 7-O-glucoside showed selective activity for HIV-1 IN. In contrast, betulinic acid, ursolic acid, and oleanolic acid were selective for the HIV-1 RNase H activity. Our results strongly support the potential of non-edible P. granatum organs as a valuable source of anti-HIV-1 compounds. [ABSTRACT FROM AUTHOR]

Plants are the everlasting source of a wide spectrum of specialized metabolites, characterized by wide variability in term of chemical structures and different biological properties such antiviral activity. In the search for novel antiviral agents against Human Immunodeficiency Virus type 1 (HIV-1) from plants, the phytochemical investigation of Scrophularia trifoliata L. led us to isolate and characterize four flavonols glycosides along with nine iridoid glycosides, two of them, 5 and 13, described for the first time. In the present study, we investigated, for the first time, the contents of a methanol extract of S. trifoliata leaves, in order to explore the potential antiviral activity against HIV-1. The antiviral activity was evaluated in biochemical assays for the inhibition of HIV-1Reverse Transcriptase (RT)-associated Ribonuclease H (RNase H) activity and HIV-1 Integrase (IN). Three isolated flavonoids, rutin, kaempferol-7-O-rhamnosyl-3-O-glucopyranoside, and kaempferol-3-O-glucopyranoside, 8–10, inhibited specifically the HIV-1 IN activity at submicromolar concentration, with the latter being the most potent, showing an IC50 value of 24 nM. [ABSTRACT FROM AUTHOR]

HIV-1 infection requires life-long treatment and with 2.1 million new infections/year, faces the challenge of an increased rate of transmitted drug-resistant mutations. Therefore, a constant and timely effort is needed to identify new HIV-1 inhibitors active against drug-resistant variants. The ribonuclease H (RNase H) activity of HIV-1 reverse transcriptase (RT) is a very promising target, but to date, still lacks an efficient inhibitor. Here, we characterize the mode of action of N'-(2-hydroxy-benzylidene)-3,4,5-trihydroxybenzoylhydrazone (compound 13), an N-acylhydrazone derivative that inhibited viral replication (EC50 = 10 µM), while retaining full potency against the NNRTI-resistant double mutant K103N-Y181C virus. Time-of-addition and biochemical assays showed that compound 13 targeted the reverse-transcription step in cell-based assays and inhibited the RT-associated RNase H function, being >20-fold less potent against the RT polymerase activity. Docking calculations revealed that compound 13 binds within the RNase H domain in a position different from other selective RNase H inhibitors; site-directed mutagenesis studies revealed interactions with conserved amino acid within the RNase H domain, suggesting that compound 13 can be taken as starting point to generate a new series of more potent RNase H selective inhibitors active against circulating drug-resistant variants. [ABSTRACT FROM AUTHOR]

The persistence of the AIDS epidemic, and the life-long treatment required, indicate the constant need of novel HIV-1 inhibitors. In this scenario the HIV-1 Reverse Transcriptase (RT)-associated ribonuclease H (RNase H) function is a promising drug target. Here we report a series of compounds, developed on the 2-amino-6-(trifluoromethyl)nicotinic acid scaffold, studied as promising RNase H dual inhibitors. Among the 44 tested compounds, 34 inhibited HIV-1 RT-associated RNase H function in the low micromolar range, and seven of them showed also to inhibit viral replication in cell-based assays with a selectivity index up to 10. The most promising compound, 21, inhibited RNase H function with an IC50 of 14 µM and HIV-1 replication in cell-based assays with a selectivity index greater than 10. Mode of action studies revealed that compound 21 is an allosteric dual-site compound inhibiting both HIV-1 RT functions, blocking the polymerase function also in presence of mutations carried by circulating variants resistant to non-nucleoside inhibitors, and the RNase H function interacting with conserved regions within the RNase H domain. Proving compound 21 as a promising lead for the design of new allosteric RNase H inhibitors active against viral replication with not significant cytotoxic effects. [ABSTRACT FROM AUTHOR]

Despite great efforts have been made in the prevention and therapy of human immunodeficiency virus (HIV-1) infection, however the difficulty to eradicate latent viral reservoirs together with the emergence of multi-drug-resistant strains require the search for innovative agents, possibly exploiting novel mechanisms of action. In this context, the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H), which is one of the few HIV-1 encoded enzymatic function still not targeted by any current drug, can be considered as an appealing target. In this work, we repurposed in-house anti-influenza derivatives based on the 1,2,4-triazolo[1,5-a]-pyrimidine (TZP) scaffold for their ability to inhibit HIV-1 RNase H function. Based on the results, a successive multi-step structural exploration around the TZP core was performed leading to identify catechol derivatives that inhibited RNase H in the low micromolar range without showing RT-associated polymerase inhibitory activity. The antiviral evaluation of the compounds in the MT4 cells showed any activity against HIV-1 (IIIB strain). Molecular modelling and mutagenesis analysis suggested key interactions with an unexplored allosteric site providing insights for the future optimization of this class of RNase H inhibitors. [ABSTRACT FROM AUTHOR]

[Display omitted] • Novel thiazolo[3, 2- a ]pyrimidine derivatives were firstly identified as potent inhibitors of RNase H. • Compound A28 exhibited around 5-fold more potent (IC 50 = 4.14 μM) than that of the hit compound A1 (IC 50 = 21.49 μM). • Compound A28 can be used as a lead compound for the further development of novel thiazolo[3, 2- a ]pyrimidine-based RNase H inhibitors. Targeting Ribonuclease H (RNase H) has been considered a viable strategy for HIV therapy. In this study, a series of novel thiazolo[3, 2- a ]pyrimidine derivatives were firstly designed and synthesized as potential inhibitors of HIV-1 RNase H. Among these compounds, A28 exhibited the most potent inhibition against HIV-1 RNase H with an IC 50 value of 4.14 μM, which was about 5-fold increase in potency than the hit compound A1 (IC 50 = 21.49 μM). To gain deeper insights into the structure–activity relationship (SAR), a CoMFA model was constructed to yield reasonable statistical results (q2 = 0.658 and R2 = 0.969). Results from magnesium ion chelation experiments and molecular docking studies revealed that these thiazolopyrimidine inhibitors may exert their inhibitory activity by binding to an allosteric site on RNase H at the interface between subunits p51 and p66. Furthermore, this analog demonstrated favorable physicochemical properties. Our findings provide valuable groundwork for further development of allosteric inhibitors targeting HIV-1 RNase H. [ABSTRACT FROM AUTHOR]

Abstract: We report herein the synthesis of a series of 3-hydroxyquinolin-2(1H)-one derivatives. Esters and amide groups were introduced at position 4 of the basis scaffold and some modulations of the benzenic moiety were performed. Most compounds presented selective inhibitory properties in the 10–20μM range against HIV-1 reverse transcriptase associated ribonuclease H activity, without affecting the integrase and reverse transcriptase DNA polymerase activities. Unfortunately all tested compounds exhibited high cellular cytotoxicity in cell culture which limited their applications as antiviral agents. [Copyright &y& Elsevier]

The management of Human Immunodeficiency Virus type 1 (HIV-1) infection requires life-long treatment that is associated with chronic toxicity and possible selection of drug-resistant strains. A new opportunity for drug intervention is offered by antivirals that act as allosteric inhibitors targeting two viral functions (dual inhibitors). In this work, we investigated the effects of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) derivatives on both HIV-1 Integrase (IN) and Reverse Transcriptase associated Ribonuclease H (RNase H) activities. Among the tested compounds, the dihydroxyindole-carboxamide 5 was able to inhibit in the low micromolar range (1–18 μM) multiple functions of IN, including functional IN-IN interactions, IN-LEDGF/p75 binding and IN catalytic activity. Docking and site-directed mutagenesis studies have suggested that compound 5 binds to a previously described HIV-1 IN allosteric pocket. These observations indicate that 5 is structurally and mechanistically distinct from the published allosteric HIV-1 IN inhibitors. Moreover, compound 5 also inhibited HIV-1 RNase H function, classifying this molecule as a dual HIV-1 IN and RNase H inhibitor able to impair the HIV-1 virus replication in cell culture. Overall, we identified a new scaffold as a suitable platform for the development of novel dual HIV-1 inhibitors. • 5,6-dihydroxyindole-2-carboxylic acid derivatives are a new scaffold for the development of novel dual HIV-1 inhibitors. • The dihydroxyindole-carboxamide compound 5 inhibits multiple functions of the HIV-1 IN in the low micromolar range. • The dihydroxyindole-carboxamide compound 5 inhibits the HIV-1 virus replication in cell culture. • Dihydroxyindole-carboxamide compound 5 has a mode of action different from the known allosteric HIV-1 IN inhibitors. [ABSTRACT FROM AUTHOR]

A number of compounds targeting different processes of the Human Immunodeficiency Virus type 1 (HIV-1) life cycle have been developed in the continuing fight against AIDS. Coumarin-based molecules already proved to act as HIV-1 Protease (PR) or Integrase (IN) inhibitors and also to target HIV-1 reverse transcriptase (RT), blocking the DNA-dependent DNA-polymerase activity or the RNA-dependent DNA-polymerase activity working as common NNRTIs. In the present study, with the aim to exploit a coumarin-based scaffold to achieve the inhibition of multiple viral coded enzymatic functions, novel 4-hydroxy-2 H , 5H-pyrano (3, 2-c) chromene–2, 5–dione derivatives were synthesized. The modeling studies calculated the theoretical binding affinity of the synthesized compounds on both HIV-1 IN and RT-associated Ribonuclease H (RNase H) active sites, which was confirmed by biological assays. Our results provide a basis for the identification of dual HIV-1 IN and RT RNase H inhibitors compounds. Image 1 • Dual inhibitors are a new approach in the anti-HIV therapy. • 16 4-hydroxypyranobenzopyran derivatives were synthesized as dual inhibitors. • They showed anti-HIV activity in the low micromolar range. • Modeling studies were applied on both HIV-1 IN and RT RNase H active site. • Compound 7 was the most promising HIV-1 IN and RT RNase H dual inhibitor. [ABSTRACT FROM AUTHOR]