Design, synthesis and biological evaluation of Thiazolo[3, 2-a]Pyrimidine derivatives as novel 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 IC50 value of 4.14 µM, which was about 5-fold increase in potency than the hit compound A1 (IC50 = 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.

Structure-Based Design of Novel Thiazolone[3,2-a]pyrimidine Derivatives as Potent RNase H Inhibitors for HIV Therapy.

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.

Mutations in the 3'-PPT Lead to HIV-1 Replication without Integration.

Integration of the reverse-transcribed genome is a critical step of the retroviral life cycle. Strand-transfer inhibitors (INSTIs) used for antiretroviral therapy inhibit integration but can lead to resistance mutations in the integrase gene, the enzyme involved in this reaction. A significant proportion of INSTI treatment failures, particularly those with second-generation INSTIs, show no mutation in the integrase gene. Here, we show that replication of a selected dolutegravir-resistant virus with mutations in the 3'-PPT (polypurine tract) was effective, although no integrated viral DNA was detected, due to the accumulation of unintegrated viral DNA present as 1-LTR circles. Our results show that mutation in the 3'-PPT leads to 1-LTR circles and not linear DNA as classically reported. In conclusion, our data provide a molecular basis to explain a new mechanism of resistance to INSTIs, without mutation of the integrase gene and highlights the importance of unintegrated viral DNA in HIV-1 replication. IMPORTANCE Our work highlights the role of HIV-1 unintegrated viral DNA in viral replication. A virus, resistant to strand-transfer inhibitors, has been selected in vitro. This virus highlights a mutation in the 3'PPT region and not in the integrase gene. This mutation modifies the reverse transcription step leading to the accumulation of 1-LTR circles and not the linear DNA. This accumulation of 1-LTR circles leads to viral replication without integration of the viral genome.

Targeting SARS-CoV-2 Main Protease: A Successful Story Guided by an In Silico Drug Repurposing Approach.

The SARS-CoV-2 main protease (Mpro) is a crucial enzyme for viral replication and has been considered an attractive drug target for the treatment of COVID-19. In this study, virtual screening techniques and in vitro assays were combined to identify novel Mpro inhibitors starting from around 8000 FDA-approved drugs. The docking analysis highlighted 17 promising best hits, biologically characterized in terms of their Mpro inhibitory activity. Among them, 7 cephalosporins and the oral anticoagulant betrixaban were able to block the enzyme activity in the micromolar range with no cytotoxic effect at the highest concentration tested. After the evaluation of the degree of conservation of Mpro residues involved in the binding with the studied ligands, the ligands' activity on SARS-CoV-2 replication was assessed. The ability of betrixaban to affect SARS-CoV-2 replication associated to its antithrombotic effect could pave the way for its possible use in the treatment of hospitalized COVID-19 patients.

Versatile anti-infective properties of pyrido- and dihydropyrido[2,3-d]pyrimidine-based compounds.

A series of 1H-indeno[2',1':5,6]dihydropyrido[2,3-d]pyrimidine and 1H-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.

Unlocking STING as a Therapeutic Antiviral Strategy.

Invading pathogens have developed weapons that subvert physiological conditions to weaken the host and permit the spread of infection. Cells, on their side, have thus developed countermeasures to maintain cellular physiology and counteract pathogenesis. The cyclic GMP-AMP (cGAMP) synthase (cGAS) is a pattern recognition receptor that recognizes viral DNA present in the cytosol, activating the stimulator of interferon genes (STING) protein and leading to the production of type I interferons (IFN-I). Given its role in innate immunity activation, STING is considered an interesting and innovative target for the development of broad-spectrum antivirals. In this review, we discuss the function of STING; its modulation by the cellular stimuli; the molecular mechanisms developed by viruses, through which they escape this defense system; and the therapeutical strategies that have been developed to date to inhibit viral replication restoring STING functionality.

Dihydroxyphenyl- and Heteroaromatic-Based Thienopyrimidinones to Tackle HIV-1 LEDGF/p75-Dependent IN Activity.

The spread of Human Immunodeficiency Virus (HIV) still represents a global public health issue of major concern, and would benefit from unveiling unique viral features as targets for drug design. In this respect, HIV-1 integrase (IN), due to the absence of homologs in human cells, is a popular target for the synthesis of novel selective compounds. Moreover, as drug-resistant viral strains are rapidly evolving, the development of novel allosteric inhibitors is acutely required. Recently, we have observed that Kuwanon-L, quinazolinones and thienopyrimidinones containing at least one polyphenol unit, effectively inhibited HIV-1 IN activity. Thus, in the present research, novel dihydroxyphenyl-based thienopyrimidinone derivatives were investigated for their LEDGF/p75-dependent IN inhibitory activity. Our findings indicated a close correlation between the position of the OH group on the phenyl moiety and IN inhibitory activity of these compounds. As catechol may be involved in cytotoxicity, its replacement by other aromatic scaffolds was also exploited. As a result, compounds 21-23, 25 and 26 with enhanced IN inhibitory activity provided good lead candidates, with 25 being the most selective for IN. Lastly, UV spectrometric experiments suggested a plausible allosteric mode of action, as none of the thienopirimidinones showed Mg2+ chelation properties otherwise typical of IN strand transfer inhibitors (INSTIs).

Targeting SARS-CoV-2 nsp13 Helicase and Assessment of Druggability Pockets: Identification of Two Potent Inhibitors by a Multi-Site In Silico Drug Repurposing Approach.

The SARS-CoV-2 non-structural protein 13 (nsp13) helicase is an essential enzyme for viral replication and has been identified as an attractive target for the development of new antiviral drugs. In detail, the helicase catalyzes the unwinding of double-stranded DNA or RNA in a 5' to 3' direction and acts in concert with the replication-transcription complex (nsp7/nsp8/nsp12). In this work, bioinformatics and computational tools allowed us to perform a detailed conservation analysis of the SARS-CoV-2 helicase genome and to further predict the druggable enzyme's binding pockets. Thus, a structure-based virtual screening was used to identify valuable compounds that are capable of recognizing multiple nsp13 pockets. Starting from a database of around 4000 drugs already approved by the Food and Drug Administration (FDA), we chose 14 shared compounds capable of recognizing three out of four sites. Finally, by means of visual inspection analysis and based on their commercial availability, five promising compounds were submitted to in vitro assays. Among them, PF-03715455 was able to block both the unwinding and NTPase activities of nsp13 in a micromolar range.

Integration of SARS-CoV-2 RNA in infected human cells by retrotransposons: an unlikely hypothesis and old viral relationships.

Zhang et al. (Proc Natl Acad Sci 118:e2105968118, 2021) recently reported that SARS-CoV-2 RNA can be retrotranscribed and integrated into the DNA of human cells by the L1 retrotransposon machinery. This phenomenon could cause persistence of viral sequences in patients and may explain the prolonged PCR-positivity of SARS-CoV-2 infected patients, even long after the phase of active virus replication has ended. This commentary does critically review the available data on this topic and discusses them in the context of findings made for other exogenous viruses and ancestral endogenous retroviral elements.

RNA-Seq Transcriptome Analysis Reveals Long Terminal Repeat Retrotransposon Modulation in Human Peripheral Blood Mononuclear Cells after In Vivo Lipopolysaccharide Injection.

Human endogenous retroviruses (HERVs) and mammalian apparent long terminal repeat (LTR) retrotransposons (MaLRs) are retroviral sequences that integrated into germ line cells millions of years ago. Transcripts of these LTR retrotransposons are present in several tissues, and their expression is modulated in pathological conditions, although their function remains often far from being understood. Here, we focused on the HERV/MaLR expression and modulation in a scenario of immune system activation. We used a public data set of human peripheral blood mononuclear cells (PBMCs) RNA-Seq from 15 healthy participants to a clinical trial before and after exposure to lipopolysaccharide (LPS), for which we established an RNA-Seq workflow for the identification of expressed and modulated cellular genes and LTR retrotransposon elements.IMPORTANCE We described the HERV and MaLR transcriptome in PBMCs, finding that about 8.4% of the LTR retrotransposon loci were expressed and identifying the betaretrovirus-like HERVs as those with the highest percentage of expressed loci. We found 4,607 HERV and MaLR loci that were modulated as a result of in vivo stimulation with LPS. The HERV-H group showed the highest number of differentially expressed most intact proviruses. We characterized the HERV and MaLR loci as differentially expressed, checking their genomic context of insertion and observing a general colocalization with genes that are involved and modulated in the immune response, as a consequence of LPS stimulation. The analyses of HERV and MaLR expression and modulation show that these LTR retrotransposons are expressed in PBMCs and regulated in inflammatory settings. The similar regulation of HERVs/MaLRs and genes after LPS stimulation suggests possible interactions of LTR retrotransposons and the immune host response.

Flavonoids and Acid-Hydrolysis derivatives of Neo-Clerodane diterpenes from Teucrium flavum subsp. glaucum as inhibitors of the HIV-1 reverse transcriptase-associated RNase H function.

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 IC50 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.

Garcinol from Garcinia indica inhibits HIV-1 reverse transcriptase-associated ribonuclease H.

The bioactive components of Garcinia indica, garcinol (camboginol), and isogarcinol (cambogin), are suitable drug candidates for the treatment of various human diseases. HIV-1-RNase H assay was used to study the RNase H inhibition by garcinol and isogarcinol. Docking of garcinol into the active site of the enzyme was carried out to rationalize the difference in activities between the two compounds. Garcinol showed higher HIV-1-RNase H inhibition than the known inhibitor RDS1759 and retained full potency against the RNase H of a drug-resistant HIV-1 reverse transcriptase form. Isogarcinol was distinctly less active than garcinol, indicating the importance of the enolizable ß-diketone moiety of garcinol for anti-RNase H activity. Docking calculations confirmed these findings and suggested this moiety to be involved in the chelation of metal ions of the active site. On the basis of its HIV-1 reverse transcriptase-associated RNase H inhibitory activity, garcinol is worth being further explored concerning its potential as a cost-effective treatment for HIV patients.

Structural and Biochemical Analysis of the Dual Inhibition of MG-132 against SARS-CoV-2 Main Protease (Mpro/3CLpro) and Human Cathepsin-L.

After almost two years from its first evidence, the COVID-19 pandemic continues to afflict people worldwide, highlighting the need for multiple antiviral strategies. SARS-CoV-2 main protease (Mpro/3CLpro) is a recognized promising target for the development of effective drugs. Because single target inhibition might not be sufficient to block SARS-CoV-2 infection and replication, multi enzymatic-based therapies may provide a better strategy. Here we present a structural and biochemical characterization of the binding mode of MG-132 to both the main protease of SARS-CoV-2, and to the human Cathepsin-L, suggesting thus an interesting scaffold for the development of double-inhibitors. X-ray diffraction data show that MG-132 well fits into the Mpro active site, forming a covalent bond with Cys145 independently from reducing agents and crystallization conditions. Docking of MG-132 into Cathepsin-L well-matches with a covalent binding to the catalytic cysteine. Accordingly, MG-132 inhibits Cathepsin-L with nanomolar potency and reversibly inhibits Mpro with micromolar potency, but with a prolonged residency time. We compared the apo and MG-132-inhibited structures of Mpro solved in different space groups and we identified a new apo structure that features several similarities with the inhibited ones, offering interesting perspectives for future drug design and in silico efforts.

Natural and Nature-Derived Products Targeting Human Coronaviruses.

The ongoing pandemic of severe acute respiratory syndrome (SARS), caused by the SARS-CoV-2 human coronavirus (HCoV), has brought the international scientific community before a state of emergency that needs to be addressed with intensive research for the discovery of pharmacological agents with antiviral activity. Potential antiviral natural products (NPs) have been discovered from plants of the global biodiversity, including extracts, compounds and categories of compounds with activity against several viruses of the respiratory tract such as HCoVs. However, the scarcity of natural products (NPs) and small-molecules (SMs) used as antiviral agents, especially for HCoVs, is notable. This is a review of 203 publications, which were selected using PubMed/MEDLINE, Web of Science, Scopus, and Google Scholar, evaluates the available literature since the discovery of the first human coronavirus in the 1960s; it summarizes important aspects of structure, function, and therapeutic targeting of HCoVs as well as NPs (19 total plant extracts and 204 isolated or semi-synthesized pure compounds) with anti-HCoV activity targeting viral and non-viral proteins, while focusing on the advances on the discovery of NPs with anti-SARS-CoV-2 activity, and providing a critical perspective.

Chemical Characterization and Anti-HIV-1 Activity Assessment of Iridoids and Flavonols from Scrophularia trifoliata.

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.

Exploring New Scaffolds for the Dual Inhibition of HIV-1 RT Polymerase and Ribonuclease Associated Functions.

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 (IC50 = 4.5 mM)- and RDDP (IC50 = 8.0 mM) HIV RT-associated functions.

Quercetin Blocks Ebola Virus Infection by Counteracting the VP24 Interferon-Inhibitory Function.

Ebola virus (EBOV) is among the most devastating pathogens causing fatal hemorrhagic fever in humans. The epidemics from 2013 to 2016 resulted in more than 11,000 deaths, and another outbreak is currently ongoing. Since there is no FDA-approved drug so far to fight EBOV infection, there is an urgent need to focus on drug discovery. Considering the tight correlation between the high EBOV virulence and its ability to suppress the type I interferon (IFN-I) system, identifying molecules targeting viral protein VP24, one of the main virulence determinants blocking the IFN response, is a promising novel anti-EBOV therapy approach. Hence, in the effort to find novel EBOV inhibitors, a screening of a small set of flavonoids was performed; it showed that quercetin and wogonin can suppress the VP24 effect on IFN-I signaling inhibition. The mechanism of action of the most active compound, quercetin, showing a half-maximal inhibitory concentration (IC50) of 7.4 µM, was characterized to significantly restore the IFN-I signaling cascade, blocked by VP24, by directly interfering with the VP24 binding to karyopherin-α and thus restoring P-STAT1 nuclear transport and IFN gene transcription. Quercetin significantly blocked viral infection, specifically targeting EBOV VP24 anti-IFN-I function. Overall, quercetin is the first identified inhibitor of the EBOV VP24 anti-IFN function, representing a molecule interacting with a viral binding site that is very promising for further drug development aiming to block EBOV infection at the early steps.

Comprehensive Characterization of the Human Endogenous Retrovirus HERV-K(HML-6) Group: Overview of Structure, Phylogeny, and Contribution to the Human Genome.

Eight percent of the human genome is composed of human endogenous retroviruses (HERVs), remnants of ancestral germ line infections by exogenous retroviruses, which have been vertically transmitted as Mendelian characters. The HML-6 group, a member of the class II betaretrovirus-like viruses, includes several proviral loci with an increased transcriptional activity in cancer and at least two elements that are known for retaining an intact open reading frame and for encoding small proteins such as ERVK3-1, which is expressed in various healthy tissues, and HERV-K-MEL, a small Env peptide expressed in samples of cutaneous and ocular melanoma but not in normal tissues.IMPORTANCE We reported the distribution and genetic composition of 66 HML-6 elements. We analyzed the phylogeny of the HML-6 sequences and identified two main clusters. We provided the first description of a Rec domain within the env sequence of 23 HML-6 elements. A Rec domain was also predicted within the ERVK3-1 transcript sequence, revealing its expression in various healthy tissues. Evidence about the context of insertion and colocalization of 19 HML-6 elements with functional human genes are also reported, including the sequence 16p11.2, whose 5' long terminal repeat overlapped the exon of one transcript variant of a cellular zinc finger upregulated and involved in hepatocellular carcinoma. The present work provides the first complete overview of the HML-6 elements in GRCh37(hg19), describing the structure, phylogeny, and genomic context of insertion of each locus. This information allows a better understanding of the genetics of one of the most expressed HERV groups in the human genome.

Scaffold hopping and optimisation of 3',4'-dihydroxyphenyl- containing thienopyrimidinones: synthesis of quinazolinone derivatives as novel allosteric inhibitors of HIV-1 reverse transcriptase-associated ribonuclease H.

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.

Probing the Importance of the G-Quadruplex Grooves for the Activity of the Anti-HIV-Integrase Aptamer T30923.

In this paper, we report studies concerning four variants of the G-quadruplex forming anti-HIV-integrase aptamer T30923, in which specific 2'-deoxyguanosines have been singly replaced by 8-methyl-2'-deoxyguanosine residues, with the aim to exploit the methyl group positioned in the G-quadruplex grooves as a steric probe to investigate the interaction aptamer/target. Although, the various modified aptamers differ in the localization of the methyl group, NMR, circular dichroism (CD), electrophoretic and molecular modeling data suggest that all of them preserve the ability to fold in a stable dimeric parallel G-quadruplex complex resembling that of their natural counterpart T30923. However, the biological data have shown that the T30923 variants are characterized by different efficiencies in inhibiting the HIV-integrase, thus suggesting the involvement of the G-quadruplex grooves in the aptamer/target interaction.