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Influenza (flu) virus is a serious threat to global health with the potential to generate devastating pandemics. The availability of broad spectrum antiviral drugs is an unequaled weapon during pandemic events, especially when a vaccine is still not available. One of the most promising targets for the development of new antiflu therapeutics is the viral RNA-dependent RNA polymerase (RdRP). The assembly of the flu RdRP heterotrimeric complex from the individual polymerase acidic protein (PA), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) subunits is a prerequisite for RdRP functions, such as mRNA synthesis and genome replication. In this Review, we report the known protein–protein interactions (PPIs) occurring by RdRP that could be disrupted by small molecules and analyze their benefits and drawbacks as drug targets. An overview of small molecules able to interfere with flu RdRP functions exploiting the PPI inhibition approach is described. In particular, an update on the most recent inhibitors targeting the well-consolidated RdRP PA–PB1 subunit heterodimerization is mainly reported, together with pioneer inhibitors targeting other virus–virus or virus–host interactions involving RdRP subunits. As demonstrated by the PA–PB1 interaction inhibitors discussed herein, the inhibition of flu RdRP functions by PPI disrupters clearly represents a valid means to identify compounds endowed with a broad spectrum of action and a reduced propensity to develop drug resistance, which are the main issues of antiviral drugs.

In the search for new anti-influenza virus (IV) compounds, we have identified the 1,2,4-triazolo[1,5-a]pyrimidine (TZP) as a very suitable scaffold to obtain compounds able to disrupt IV RNA-dependent RNA polymerase (RdRP) PA-PB1 subunits heterodimerization. In this work, in order to acquire further SAR insights for this class of compounds and identify more potent derivatives, we designed and synthesized additional series of analogues to investigate the role of the substituents around the TZP core. To this aim, we developed four facile and efficient one-step procedures for the synthesis of 5-phenyl-, 6-phenyl- and 7-phenyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines, and 2-amino-5-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7-ol. Two analogues having the ethyl carboxylate moiety at the C-2 position of the TZP were also prepared in good yields. Then, the scaffolds herein synthesized and two previous scaffolds were functionalized and evaluated for their anti-IAV activity, leading to the identification of compound 22 that showed both anti-PA-PB1 (IC50 = 19.5 μM) and anti-IAV activity (EC50 = 16 μM) at non-toxic concentrations, thus resulting among the most active TZP derivatives reported to date by us. A selection of the synthesized compounds, along with a set of in-house available analogues, was also tested against SARS-CoV-2. The most promising compound 49 from this series displayed an EC50 value of 34.47 μM, highlighting the potential of the TPZ scaffold in the search for anti-CoV agents. ispartof: EUROPEAN JOURNAL OF MEDICINAL CHEMISTRY vol:221 ispartof: location:France status: published

Influenza viruses (Flu) are responsible for seasonal epidemics causing high rates of morbidity, which can dramatically increase during severe pandemic outbreaks. Antiviral drugs are an indispensable weapon to treat infected people and reduce the impact on human health, nevertheless anti-Flu armamentarium still remains inadequate. In search for new anti-Flu drugs, our group has focused on viral RNA-dependent RNA polymerase (RdRP) developing disruptors of PA-PB1 subunits interface with the best compounds characterized by cycloheptathiophene-3-carboxamide and 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamide scaffolds. By merging these moieties, two very interesting hybrid compounds were recently identified, starting from which, in this paper, a series of analogues were designed and synthesized. In particular, a thorough exploration of the cycloheptathiophene-3-carboxamide moiety led to acquire important SAR insight and identify new active compounds showing both the ability to inhibit PA-PB1 interaction and viral replication in the micromolar range and at non-toxic concentrations. For few compounds, the ability to efficiently inhibit PA-PB1 subunits interaction did not translate into anti-Flu activity. Chemical/physical properties were investigated for a couple of compounds suggesting that the low solubility of compound 14, due to a strong crystal lattice, may have impaired its antiviral activity. Finally, computational studies performed on compound 23, in which the phenyl ring suitably replaced the cycloheptathiophene, suggested that, in addition to hydrophobic interactions, H-bonds enhanced its binding within the PAC cavity., Graphical abstract Image 1, Highlights • New hybrid 1,2,4-triazolo[1,5-a]pyrimidine-2-carboxamides showed anti-flu activity by disrupting RdRP PA-PB1 interaction. • A simple phenyl ring can replace the cycloheptathiophene, with advantages in the synthesis and solubility of the compound. • Molecular docking study has rationalized the binding mode of the compounds within the PAC.

The mosquito-borne viruses belonging to the genus Flavivirus such as Dengue virus (DENV) and Zika virus (ZIKV) cause human infections ranging from mild flu-like symptoms to hemorrhagic fevers, hepatitis, and neuropathies. To date, there are vaccines only for few flaviviruses while no effective treatments are available. Pyridobenzothiazole (PBTZ) derivatives are a class of compounds endowed with a promising broad-spectrum anti-flavivirus activity and most of them have been reported as potent inhibitors of the flaviviral NS5 polymerase. However, synthesis of PBTZ analogues entails a high number of purification steps, the use of hazardous reagents and environmentally unsustainable generation of waste. Considering the promising antiviral activity of PBTZ analogues which require further exploration, in this work, we report the development of a new and sustainable three-component reaction (3CR) that can be combined with a basic hydrolysis in a one-pot procedure to obtain the PBTZ scaffold, thus reducing the number of synthetic steps, improving yields and saving time. 3CR was significantly explored in order to demonstrate its wide scope by using different starting materials. In addition, taking advantage of these procedures, we next designed and synthesized a new set of PBTZ analogues that were tested as anti-DENV-2 and anti-ZIKV agents. Compound 22 inhibited DENV-2 NS5 polymerase with an IC50 of 10.4 μM and represented the best anti-flavivirus compound of the new series by inhibiting DENV-2- and ZIKV-infected cells with EC50 values of 1.2 and 5.0 μM, respectively, that translates into attractive selectivity indexes (SI - 83 and 20, respectively). These results strongly reaffirm PBTZ derivatives as promising anti-flavivirus agents that now can be synthesized through a convenient and sustainable 3CR in order to obtain more potent compounds for further pre-clinical development studies.

Abstract: Viruses are a continuing threat to global health. The lack or limited therapeutic armamentarium against some viral infections and increasing drug resistance issues make the search for new antiviral agents urgent. In recent years, a growing literature highlighted the use of triazolopyrimidine (TZP) heterocycles in the development of antiviral agents, with numerous compounds that showed potent antiviral activities against different RNA and DNA viruses. TZP core represents a privileged scaffold for achieving biologically active molecules, thanks to: i) the synthetic feasibility that allows to variously functionalize TZPs in the different positions of the nucleus, ii) the ability of TZP core to establish multiple interactions with the molecular target, and iii) its favorable pharmacokinetic properties. In the present review, after mentioning selected examples of TZP-based compounds with varied biological activities, we will focus on those antivirals that appeared in the literature in the last 10 years. Approaches used for their identification, the hit-to-lead studies, and the emerged structure-activity relationship will be described. A mention of the synthetic methodologies to prepare TZP nuclei will also be given. In addition, their mechanism of action, the binding mode within the biological target, and pharmacokinetic properties will be analyzed, highlighting the strengths and weaknesses of compounds based on the TZP scaffold, which is increasingly used in medicinal chemistry.

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. ispartof: MOLECULES vol:25 issue:5 ispartof: location:Switzerland status: published

With the aim to identify small molecules able to disrupt PA-PB1 subunits interaction of influenza virus (flu) RNA-dependent RNA polymerase, and based on previous structural and computational information, in this paper we have designed and synthesized a new series of cycloheptathiophene-3-carboxamide (cHTC) derivatives. Their biological evaluation led to highlight important structural insights along with new interesting compounds, such as the 2-hydroxybenzamido derivatives 29, 31, and 32, and the 4-aminophenyl derivative 54, which inhibited viral growth in the low micromolar range (EC50 = 0.18–1.2 μM) at no toxic concentrations (CC50 > 250 μM).\ud \ud This study permitted to obtain among the most potent anti-flu compounds within the PA-PB1 interaction inhibitors, confirming the cHTC scaffold as particularly suitable to achieve innovative anti-flu agents.

Bacterial resistance to antimicrobial agents has become an increasingly serious health problem in recent years. Among the strategies by which resistance can be achieved, overexpression of efflux pumps such as NorA of Staphylococcus aureus leads to a sub-lethal concentration of the antibacterial agent at the active site that in turn may predispose the organism to the development of high-level target-based resistance. With an aim to improve both the chemical stability and potency of our previously reported 3-phenyl-1,4-benzothiazine NorA inhibitors, we replaced the benzothiazine core with different nuclei. None of the new synthesized compounds showed any appreciable intrinsic antibacterial activity, and, in particular, 2-(3,4-dimethoxyphenyl)quinoline (6 c) was able to decrease, in a concentration-dependent manner, the ciprofloxacin MIC against the norA-overexpressing strains S. aureus SA-K2378 (norA++) and SA-1199B (norA+/A116E GrlA).

Targeting energy metabolism in Mycobacterium tuberculosis (Mtb) is a new paradigm in the search for innovative anti-TB drugs. NADH:menaquinone oxidoreductase is a non-proton translocating type II NADH dehydrogenase (NDH-2) that is an essential enzyme in the respiratory chain of Mtb and is not found in mammalian mitochondria. Phenothiazines (PTZs) represent one of the most known class of NDH-2 inhibitors, but their use as anti-TB drugs is currently limited by the wide range of potentially serious off-target effects. In this work, we designed and synthesized a series of new PTZs by decorating the scaffold in an unconventional way, introducing various halogen atoms. By replacing the sulfur atom with selenium, a dibromophenoselenazine 20 was also synthesized. Among the synthesized poly-halogenated PTZs (HPTZs), dibromo and tetrachloro derivatives 9 and 11, along with the phenoselenazine 20, emerged with a better anti-TB profile than the therapeutic thioridazine (TZ). They targeted non-replicating Mtb, were bactericidal, and synergized with rifampin and bedaquiline. Moreover, their anti-TB activity was found to be related to the NDH-2 inhibition. Most important, they showed a markedly reduced affinity to dopaminergic and serotonergic receptors respect to the TZ. From this work emerged, for the first time, as the poly-halogenation of the PTZ core, while permitting to maintain good anti-TB profile could conceivably lead to fewer CNS side-effects risk, making more tangible the use of PTZs for this alternative therapeutic application.

Indoleamine 2,3-dioxygenase 1 (IDO1) catalyzes the first step in the kynurenine pathway of tryptophan (Trp) degradation that produces several biologically active Trp metabolites. L-kynurenine (Kyn), the first byproduct by IDO1, promotes immunoregulatory effects via activation of the Aryl hydrocarbon Receptor (AhR) in dendritic cells (DCs) and T lymphocytes. We here identified the nuclear coactivator 7 (NCOA7) as a molecular target of 3-hydroxyanthranilic acid (3-HAA), a Trp metabolite produced downstream of Kyn along the kynurenine pathway. In cells overexpressing NCOA7 and AhR, the presence of 3-HAA increased the association of the two molecules and enhanced Kyn-driven, AhR-dependent gene transcription. Physiologically, conventional (cDCs) but not plasmacytoid DCs or other immune cells expressed high levels of NCOA7. In cocultures of CD4+ T cells with cDCs, the co-addition of Kyn and 3-HAA significantly increased the induction of Foxp3+ regulatory T cells and the production of immunosuppressive transforming growth factor β in an NCOA7-dependent fashion. Thus, the co-presence of NCOA7 and the Trp metabolite 3-HAA can selectively enhance the activation of ubiquitary AhR in cDCs and consequent immunoregulatory effects. Because NCOA7 is often overexpressed and/or mutated in tumor microenvironments, our current data may provide evidence for a new immune check-point mechanism based on Trp metabolism and AhR.

Nontuberculous mycobacteria (NTM) are ubiquitous microbes belonging to the Mycobacterium genus. Among all NTM pathogens, M. avium is one of the most frequent agents causing pulmonary disease, especially in immunocompromised individuals and cystic fibrosis patients. Recently, we reported the first ad hoc designed M. avium efflux pump inhibitor (EPI; 1b) able to strongly boost clarithromycin (CLA) MIC against different M. avium strains. Since the 3-phenylquinolone derivative 1b suffered from toxicity issues toward human macrophages, herein we report a two-pronged medicinal chemistry workflow for identifying new potent and safe NTM EPIs. Initially, we followed a computational approach exploiting our pharmacophore models to screen FDA approved drugs and in-house compounds to identify "ready-to-use" NTM EPIs and/or new scaffolds to be optimized in terms of EPI activity. Although nicardipine 2 was identified as a new NTM EPI, all identified molecules still suffered from toxicity issues. Therefore, based on the promising NTM EPI activity of 1b, we undertook the design, synthesis, and biological evaluation of new 3-phenylquinolones differently functionalized at the C6/C7 as well as N1 positions. Among the 27 synthesized 3-phenylquinolone analogues, compounds 11b, 12b, and 16a exerted excellent NTM EPI activity at concentrations below their CC50 on human cells, with derivative 16a being the most promising compound. Interestingly, 16a also showed good activity in M. avium-infected macrophages both alone as well as in combination with CLA. The antimycobacterial activity observed for 16a only when tested in the ex vivo model suggests a high therapeutic potential of EPIs against M. avium, which seems to need functional efflux pumps to establish intracellular infections.

The current anti-HIV combination therapy does not eradicate the virus that persists mainly in quiescent infected CD4+ T cells as a latent integrated provirus that resumes after therapy interruption. The Tat-mediated transactivation (TMT) is a critical step in the HIV replication cycle that could give the opportunity to reduce the size of latent reservoirs. More than two decades of research led to the identification of various TMT inhibitors. While none of them met the criteria to reach the market, the search for a suitable TMT inhibitor is still actively pursued. Really promising compounds, including one in a Phase III clinical trial, have been recently identified, thus warranting an update.

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., Graphical Abstract

We report the design, synthesis, and biological evaluation of a class of 1H-pyrido[2,1-b][1,3]benzothiazol-1-ones originated from compound 1, previously identified as anti-flavivirus agent. Some of the new compounds showed activity in low ?M range with reasonable selectivity against Dengue 2, Yellow fever (Bolivia strain), and West Nile viruses. One of the most interesting molecules, compound 16, showed broad antiviral activity against additional flaviviruses such as Dengue 1, 3 and 4, Zika, Japanese encephalitis, several strains of Yellow fever, and tick-borne encephalitis viruses. Compound 16 did not exert any effect on alphaviruses and phleboviruses and its activity was maintained in YFV infected cells from different species. The activity of 16 appears specific for flavivirus with respect to other virus families, suggesting, but not proving, that it might be targeting a viral factor. We demonstrated that the antiviral effect of 16 is not related to reduced viral RNA synthesis or virion release. On the contrary, viral particles grown in the presence of 16 showed reduced infectivity, being unable to perform a second round of infection. The chemical class herein presented thus emerges as suitable to provide pan-flavivirus inhibitors.

Influenza viruses are major respiratory pathogens responsible for both seasonal epidemics and occasional pandemics worldwide. The current available treatment options have limited efficacy and thus the development of new antivirals is highly needed. We previously reported the identification of a series of cycloheptathiophene-3-carboxamide compounds as influenza A virus inhibitors that act by targeting the protein-protein interactions between the PA-PB1 subunits of the viral polymerase. In this study, we characterized the antiviral properties of the most promising compounds as well as investigated their propensity to induce drug resistance. Our results show that some of the selected compounds possess potent, broad-spectrum anti-influenza activity as they efficiently inhibited the replication of several strains of influenza A and B viruses, including an oseltamivir-resistant clinical isolate, with nanomolar or low-micromolar potency. The most promising compounds specifically inhibited the PA-PB1 binding in vitro and interfered with the influenza A virus polymerase activity in a cellular context, without showing cytotoxicity. The most active PA-PB1 inhibitors showed to possess a drug resistance barrier higher than that of oseltamivir. Indeed, no viral variants with reduced susceptibility to the selected compounds emerged after serial passages of influenza A virus under drug selective pressure. Overall, our studies identified potent PA-PB1 inhibitors as promising candidates for the development of new anti-influenza drugs.

The identification of efflux inhibitors to be used as adjuvants alongside existing drug regimens could have a tremendous value in the treatment of any mycobacterial infection. Here, we investigated the ability of four 2-(4'-propoxyphenyl)quinoline Staphylococcus aureus NorA efflux inhibitors (1-4) to reduce the efflux activity in Mycobacterium smegmatis and Mycobacterium avium strains. All four compounds were able to inhibit efflux pumps in both mycobacterial species; in particular, O-ethylpiperazinyl derivative 2 showed an efflux inhibitory activity comparable to that of verapamil, the most potent mycobacterial efflux inhibitor reported to date, and was able to significantly reduce the MIC values of macrolides against different M. avium strains. The contribution of the M. avium efflux pumps MAV_1406 and MAV_1695 to clarithromycin resistance was proved because they were found to be overexpressed in two M. avium 104 isogenic strains showing high-level clarithromycin resistance. These results indicated a correlation between increased expression of efflux pumps, increased efflux, macrolide resistance, and reduction of resistance by efflux pump inhibitors such as compound 2. Additionally, compound 2 showed synergistic activity with clarithromycin, at a concentration below the cytotoxicity threshold, in an ex vivo experiment against M. avium 104-infected macrophages. In summary, the 2-(4'-propoxyphenyl)quinoline scaffold is suitable to obtain compounds endowed with good efflux pump inhibitory activity against both S. aureus and nontuberculous mycobacteria.

Human Diphtheria toxin-like ADP-ribosyltranferases (ARTD) 10 is an enzyme carrying out mono-ADP-ribosylation of a range of cellular proteins and affecting their activities. It shuttles between cytoplasm and nucleus and influences signaling events in both compartments, such as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and S phase DNA repair. Furthermore, overexpression of ARTD10 induces cell death. We recently reported on the discovery of a hit compound, OUL35 (compound 1), with 330 nM potency and remarkable selectivity towards ARTD10 over other enzymes in the human protein family. Here we aimed at establishing a structure-activity relationship of the OUL35 scaffold, by evaluating an array of 4-phenoxybenzamide derivatives. By exploring modifications on the linker between the aromatic rings, we identified also a 4-(benzyloxy)benzamide derivative, compound 32, which is potent (IC50 = 230 nM) and selective, and like OUL35 was able to rescue HeLa cells from ARTD10-induced cell death. Evaluation of an enlarged series of derivatives produced detailed knowledge on the structural requirements for ARTD10 inhibition and allowed the discovery of further tool compounds with submicromolar cellular potency that will help in understanding the roles of ARTD10 in biological systems.

The alarming and rapid spread of antimicrobial resistance among bacteria represents a high risk for global health. Targeting factors involved in resistance to restore the activity of failing antibiotics is a promising strategy to overcome this urgent medical need. Efflux pump inhibitors are able to increase antibiotic concentrations in bacteria, thus they can be considered true antimicrobial resistance breakers. In this work, continuing our studies on inhibitors of the Staphylococcus aureus NorA pump, we designed, synthesized and biologically evaluated novel 2-phenylquinoline derivatives starting from our hits 1 and 2. Two of the synthesized compounds (6 and 7) bearing a C-6 benzyloxy group showed the best NorA inhibition activity, thereby providing an excellent starting point to direct future chemical optimizations.

The major internal component of the HIV virion core is the nucleocapsid protein 7 (NCp7), a small, highly basic protein that is essential for multiple stages of the viral replicative cycle, and whose structure is preserved in all viral strains, including clinical isolates from therapy-experienced patients. This key protein is recognised as a potential target for an effective next-generation antiretroviral therapy, because it could offer the possibility to develop broad-spectrum agents that are less prone to select for resistant strains. Here, we provide a comprehensive overview of the covalent NCp7 inhibitors that have emerged over the past 25 years of drug discovery campaigns, emphasising, where possible, their structure-activity relationships (SARs) and pharmacophoric features.

Chronic activation of plasmacytoid dendritic cells (pDCs) is an important contributor to the immunopathogenesis of HIV infection. The quinolone derivative chloroquine (CQ) prevents endosomal acidification, required for toll-like receptor sensing of HIV by pDCs, and is currently under clinical trial as an immunotherapeutic approach. We tested three different 6-desfluoroquinolones (6-DFQs), structurally related to CQ and endowed with antiretroviral activity, for their ability to inhibit HIV-induced pDC activation and interferon (IFN)-α production in peripheral blood mononuclear cells (PBMCs) in vitro. PBMCs from six healthy donors were cultured overnight with aldrithiol-2 (AT-2)-inactivated HIV-1MN in the presence or absence of 6-DFQs or CQ. IFN-α production was measured by ELISA; pDC and monocyte activation was analyzed by flow cytometry. Incubation with HIV labeled with the fluorescent dye DyLight-488 (DL488) was used to test virus uptake by flow cytometry. We found that the 6-DFQs effectively inhibited HIV-induced IFN-α similar to CQ, but only 6-DFQs also inhibited the upregulation of the pDC activation marker CD83. Interestingly, HIV-induced expression of the costimulatory molecule CD80 and, to a lesser extent CD86, was further enhanced on pDCs by 6-DFQs, but not CQ. Conversely, 6-DFQs and CQ had similar inhibitory effects on HIV-induced monocyte activation, consistent with the primary mechanism being associated with IFN-α signaling. Finally, 6-DFQs interfered with HIV interaction with pDCs and monocytes, but not myeloid DCs. Our data indicate that 6-DFQs may interfere with pDC-mediated and IFN-α-dependent immunopathogenesis while supporting pDC differentiation into mature antigen-presenting cells by favoring expression of costimulatory molecules.

The limited number of drug classes licensed for treatment of influenza virus (Flu), together with the continuous emergence of viral variants and drug resistant mutants, highlights the urgent need to find antivirals with novel mechanisms of action. In this context, the viral RNA-dependent RNA polymerase (RdRP) subunits assembly has emerged as an attractive target. Starting from a cycloheptathiophene-3-carboxamide derivative recently identified by us for its ability to disrupt the interaction between the PA and PB1 subunits of RdRP, we have designed and synthesized a series of analogues. Their biological evaluation led to the identification of more potent protein-protein interaction inhibitors, endowed with antiviral activity that also encompassed a number of clinical isolates of FluA, including an oseltamivir-resistant strain, and FluB, without showing appreciable toxicity. From this study, the cycloheptathiophene-3-carboxamide scaffold emerged as being particularly suitable to impart anti-Flu activity.

Mycobacterium avium is a difficult-to-treat pathogen able to quickly develop drug resistance. Like for other microbial species, overexpression of efflux pumps is one of the main mechanisms in developing multidrug resistance. Although the use of efflux pumps inhibitors (EPIs) represents a promising strategy to reverse resistance, to date few M. avium EPIs are known. Recently, we showed that in-house 2-phenylquinoline S. aureus NorA EPIs exhibited also a good activity against M. avium efflux pumps. Herein, we report a series of 3-phenylquinolones designed by modifying the isoflavone biochanin A, a natural EPI of the related M. smegmatis, taking into account some important SAR information obtained around the 2-phenylquinoline NorA EPIs. The 3-phenylquinolones inhibited M. avium efflux pumps with derivatives 1e and 1g that displayed the highest synergistic activity against all the strains considered in the study, bringing down (from 4- to 128-fold reduction) the MIC values of macrolides and fluoroquinolones.

Two facile and efficient one-step procedures for the regioselective synthesis of 7-aryl-5-methyl- and 5-aryl-7-methyl-2-amino-[1,2,4]triazolo[1,5-a]pyrimidines have been developed, via reactions of 3,5-diamino-1,2,4-triazole with variously substituted 1-aryl-1,3-butanediones and 1-aryl-2-buten-1-ones, respectively. The excellent yield and/or regioselectivity shown by the reactions decreased when ethyl 5-amino-1,2,4-triazole-3-carboxylate was used. [1,2,4]Triazolo[1,5-a]pyrimidine being a privileged scaffold, the procedures herein reported may be useful for the preparation of biologically active compounds. In this study, the preparation of a set of compounds based on the [1,2,4]triazolo[1,5-a]pyrimidine scaffold led to the identification of compound 20 endowed with a very promising ability to inhibit influenza virus RNA polymerase PA-PB1 subunit heterodimerization.

The limited therapeutic options against the influenza virus (flu) and increasing challenges in drug resistance make the search for next-generation agents imperative. In this context, heterotrimeric viral PA/PB1/PB2 RNA-dependent RNA polymerase is an attractive target for a challenging but strategic protein-protein interaction (PPI) inhibition approach. Since 2012, the inhibition of the polymerase PA-PB1 subunit interface has become an active field of research following the publication of PA-PB1 crystal structures. In this Perspective, we briefly discuss the validity of flu polymerase as a drug target and its inhibition through a PPI inhibition strategy, including a comprehensive analysis of available PA-PB1 structures. An overview of all of the reported PA-PB1 complex formation inhibitors is provided, and approaches used for identification of the inhibitors, the hit-to-lead studies, and the emerged structure-activity relationship are described. In addition to highlighting the strengths and weaknesses of all of the PA-PB1 heterodimerization inhibitors, we analyze their hypothesized binding modes and alignment with a pharmacophore model that we have developed.

The screening of an in-house quinolones library against Mycobacterium tuberculosis (Mtb) H(37) Rv, followed by a first cycle of optimization, yielded 6-hydrogen-8-methyl derivatives endowed with good potency. The antitubercular activity also encompassed the bacteria in a non-replicating state (NRP-TB) with minimum inhibitory concentration values lower than those of the reference agent, moxifloxacin. Among the best compounds, 11w and 11ai, characterized by a properly substituted piperidine at the C-7 position, were active against single-drug-resistant (SDR-TB) Mtb strains, maintaining overall good potency also against ciprofloxacin-resistant Mtb. This study expands the body of SAR around antitubercular quinolones leading to reconsider the role played by the usual fluorine atom at the C-6 position. Further elaboration of the 6-hydrogen-8-methylquinolone scaffold, with a particular focus on the C-7 position, is expected to give even more potent congeners holding promise for shortening the current anti-TB regimen.

We identified a 6-aminoquinolone compound, WC5, that inhibits human cytomegalovirus (HCMV) replication with a selectivity index of ∼500. WC5 also showed activity against drug-resistant HCMV strains. In contrast, it did not significantly affect the replication of human herpesvirus 6 and 8 and was ∼10-fold less active against murine cytomegalovirus. Thus, WC5 may represent a lead for the development of new, potent, and selective anti-HCMV compounds.

We report here a comparative study of the anti-hepatitis C virus (HCV) activities of selected (i) nucleoside polymerase, (ii) nonnucleoside polymerase, (iii) α,γ-diketo acid polymerase, (iv) NS3 protease, and (v) helicase inhibitors, as well as (vi) cyclophilin binding molecules and (vii) alpha 2b interferon in four different HCV genotype 1b replicon systems.

New targets for antiviral strategies are needed against human cytomegalovirus (HCMV), a major human pathogen. A cell-based screen aimed at finding inhibitors of the viral transcription factor Immediate-Early 2 (IE2) was performed in HCMV-infected cells expressing EGFP under the control of an IE2-inducible viral promoter. Screening of a library of bioactive small molecules led to the identification of several compounds able to inhibit EGFP expression and also HCMV replication with potency in the low-micromolar range. Follow-up studies with four selected hits indicated that they all block viral DNA synthesis as well as viral Early and Late gene expression. Furthermore, mechanistic studies confirmed that the compounds specifically act via inhibition of IE2 transactivating activity, thus blocking viral Early gene expression and the progression of virus replication. These results provide proof of concept for identifying small molecules that modulate the activity of a microbial transcription factor to control pathogen replication.

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.

Huntington's disease is a fatal and devastating neurodegenerative genetic disorder for which there is currently no cure. It is characterized by Huntingtin protein's mRNA transcripts with 36 or more CAG repeats. Inhibiting the formation of pathological complexes between these expanded transcripts and target proteins may be a valuable strategy against the disease. Yet, the rational design of molecules specifically targeting the expanded CAG repeats is limited by the lack of structural information. Here, we use well-tempered metadynamics-based free energy calculations to investigate pose and affinity of two ligands targeting CAG repeats for which affinities have been previously measured. The first consists of two 4-guanidinophenyl rings linked by an ester group. It is the most potent ligand identified so far, with Kd = 60(30) nM. The second consists of a 4-phenyl dihydroimidazole and 4-1H-indole dihydroimidazole connected by a C-C bond (Kd = 700(80) nM). Our calculations reproduce the experimental affinities and uncover the recognition pattern between ligands' and their RNA target. They also provide a molecular basis for the markedly different affinity of the two ligands for CAG repeats as observed experimentally. These findings may pave the way for a structure-based hit-to-lead optimization to further improve ligand selectivity toward CAG repeat-containing mRNAs.

Objectives A number of previous studies has provided evidence that the well-known anti-bacterial quinolones may have potential as anti-cancer drugs. The aim of this study was to evaluate potential anti-tumour activity and selectivity of a set of 6-aminoquinolones showing some chemical similarity to naphthyridone derivative CX-5461, recently described as innovative anti-cancer agent. Materials and methods In-house quinolones 1-8 and ad hoc synthesized derivatives 9-13 were tested on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and mesenchymal progenitor (MePR2B) cell lines, analysing their effects on the cell cycle and cell death using FACS methodology. Activation of p53 was evaluated by western blotting. Results Benzyl esters 4, 5 and their amide counterparts 12, 13 drastically modulated MCF-7 cell cycles inducing DNA fragmentation and cell death, thus proving to be potential anti-tumour compounds. When assayed in non-tumour MePR2B cells, compounds 4 and 5 were cytotoxic while 12 and 13 had a certain degree of selectivity, with compound 12 emerging as the most promising. Western blot analysis revealed that severe p53-K382ac activation was promoted by benzylester 5. In contrast, amide 12 exerted only a moderate effect which was, however, comparable to that of suberoylanilide hydoxamic acid (SAHA). Conclusions Taken together, these results further reinforce evidence that quinolones have potential as anti-cancer agents. Future work will be focused on understanding compound 12 mechanisms of action, and to obtain more potent and selective compounds.

In continuing our efforts to identify small molecules able to disrupt the interaction of the polymerase acidic protein–basic protein 1 (PA–PB1) subunits of influenza virus (Flu) RNA-dependent RNA polymerase, this paper is devoted to the optimization of a dihydrotriazolopyrimidine derivative, previously identified through structure-based drug discovery. The structure modifications performed around the bicyclic core led to the identification of compounds endowed with both the ability to disrupt PA–PB1 subunits interaction and anti-Flu activity with no cytotoxicity. Very interesting results were obtained with the hybrid molecules 36 and 37, designed by merging some peculiar structural features known to impart PA–PB1 interaction inhibition, with compound 36 that emerged as the most potent PA–PB1 interaction inhibitor (IC50 = 1.1 μM) among all the small molecules reported so far. Calculations showed a very favored H-bonding between the 2-amidic carbonyl of 36 and Q408, which seems to justify its potent ability to interfere with the interaction of the polymerase subunits.

In this study we report the design, synthesis, and activity against bovine viral diarrhea virus (BVDV) of a novel series of acridone derivatives. BVDV is responsible for major losses in cattle. The virus is also considered to be a valuable surrogate for the hepatitis C virus (HCV) in antiviral drug studies. Some of the synthesized acridones elicited selective anti-BVDV activity with EC(50) values ranging from 0.4 to 4 microg/mL and were not cytotoxic at concentrations that were 25- to 200-fold higher (CC(50)100 microg/mL). It was proven that the most potent acridone derivative 10 was able to not only protect cells from virus-induced cytopathic effect but also reduce the production of infectious virus and extracellular viral RNA. Furthermore, compound 10, as well as a number of other analogues, inhibited HCV replication to some extent. However, there was no direct correlation between anti-BVDV and anti-HCV activity. Thus, the acridone scaffold, when appropriately functionalized, can yield compounds with selective activity against pestiviruses and related viruses such as the HCV.

The identification, synthesis, biological activity, and binding mode prediction of a series of pyrazolobenzothiazines as novel p38α MAPK inhibitors are reported. Some of these compounds showed interesting activity in both p38α MAPK and TNF-α release assays. Derivative 6 emerged as the most interesting compound with IC50 (p38α) = 0.457 μm, IC50 (TNF-α) = 0.5 μm and a promising kinase selectivity profile. The obtained results strongly indicate the pyrazolobenzothiazine core as a new p38α inhibitor chemotype worthy of future chemical optimization efforts directed toward identifying a new generation of anti-inflammatory agents.

The only therapeutic drugs for combating dementia disease are acetylcholine esterase inhibitors (AChEI). However, the use of tacrine, the first AChEI to be launched as an Alzheimer's disease (AD) drug, has been limited by serious side effects. Therefore, efforts to search for more potent and selective inhibitors of AChE still remain highly significant in the therapeutic treatment of AD. In this work we modified the cyclohexyl ring of velnacrine, a less toxic analogue of tacrine, by synthesizing a series of thiopyranoquinolines in which the C-3 methylene unit was replaced by a sulphur atom. The anti-AChE data show that the activity was maintained with the bioisosteric substitution carried out. The introduction of a chlorine atom at different positions of the aromatic ring resulted in an array of different activities. In an attempt to understand the different behaviours displayed by the chlorine-substituted derivatives, a molecular docking study was performed.

The synthesis of new tricyclic quinolones, resulting from peri-annelation of 1,2,4-oxadiazine moiety at the N-1/C-8 position of the pharmacophoric quinolone nucleus, are described. None of the synthesized compounds showed interesting antibacterial activity in vitro against the tested strains, with the exception of Klebsiella pneumoniae which was susceptible to all the compounds at MIC values of 8 μg/ml.

The discovery that proinflammatory prostaglandins are produced by cyclooxygenase-2 (COX-2), an inducible isoform of the constitutive cyclooxygenase-1 (COX-1), opened a new frontier in the treatment of inflammatory diseases, because the selective inhibition of COX-2 can lead to therapeutically effective compounds which do not have the common side effects of classical non-steroidal antiinflammatory drugs (NSAIDs). Different crystallographic structures of both free COX-1 and COX-2 as well as complexes with inhibitors have been solved. Because of the great similarity between the two enzymes, it is difficult to detect the most important structural and physicochemical features that would be useful for designing inhibitors with an improved selectivity. In this paper we describe the application of a chemometric procedure to the study of COX-2 selective inhibition. This method, developed to reveal the most suitable regions of isoenzymes for the design of selective ligands, also has a very practical utility. GRID multivariate characterization of the enzymes and subsequent Principal Component Analysis (PCA) of the descriptor variables allow the identification of chemical groups that could be added to a core template structure to increase ligand selectivity.

We have previously identified the pyrazolobenzothiazine scaffold as a promising chemotype against hepatitis C virus (HCV) NS5B polymerase, a validated and promising anti-HCV target. Herein we describe the design, synthesis, enzymatic and cellular characterization of new pyrazolobenzothiazines as anti-HCV inhibitors. The binding site for a representative derivative was mapped to NS5B palm site I employing a mutant counter screen assay, thus validating our previous in silico predictions. Derivative 2b proved to be the best selective anti-HCV derivative within the new series, exhibiting IC50 value of 7.9 µM against NS5B polymerase and antiviral effect (EC50 = 8.1 µM, EC90 = 23.3 µM) coupled with the absence of any antimetabolic effect (CC50 >224 µM, SI >28) in a cell based HCV replicon system assay. Significantly, microscopic analysis showed that, unlike the parent compounds, derivative 2b did not show any significant cell morphological alterations. Furthermore, since most of the pyrazolobenzothiazines tested altered cell morphology, this undesired aspect was further investigated by exploring possible perturbation of lipid metabolism during compound treatment.

The NS5B RNA-dependent RNA polymerase is an attractive target for the development of novel and selective inhibitors of hepatitis C virus replication. In order to identify novel structural hits as anti-HCV agents, we performed structure-based virtual screening of our in-house library followed by rational drug design, organic synthesis and biological testing. These studies led to the identification of pyrazolobenzothiazine scaffold as a suitable template for obtaining novel anti-HCV agents targeting the NS5B polymerase. The best compound of this series was the meta-fluoro-N-1-phenyl pyrazolobenzothiazine derivative 4a, which exhibited an EC50= 3.6 µM, EC90= 25.6 µM and CC50 > 180 µM in the Huh 9–13 replicon system, thus providing a good starting point for further hit evolution.

In a furtherance of our SAR study on the C-6 position of quinolone antibacterials, a series of 6-desfluoro-8-methylquinolones were synthesized and evaluated for their in vitro antimicrobial activity. As a result of this study, compounds with strong activity against Gram-positive bacteria, including ciprofloxacin-resistant and methicillin-resistant Staphylococcus aureus, were identified. The best Gram-positive antibacterial activity was exhibited by piperidinyl derivative 6c, which was 17 times more potent than ciprofloxacin and displayed extremely high activity against Streptococcus pneumoniae with an MIC value of0.016 microg/mL. Thus, we have shown that substituent combinations in the quinolone ring, excluding the C-6 fluorine atom, might produce powerful antibacterial agents.

The activity of the cyclin-dependent kinase 9 (CDK9) is critical for HIV-1 Tat-mediated transcription and represents a promising target for antiviral therapy. Here we present computational studies that, along with preliminary synthetic efforts, allowed us to identify and characterize a new class of nontoxic anti-CDK9 chemotypes based on the 2-phenylquinazolinone scaffold. Inhibition of CDK9 translated into the ability to interfere selectively with Tat-mediated transactivation of the viral promoter and in the inhibition of HIV-1 reactivation from latently infected cells, with the most potent derivative 37 (2-(4-aminophenyl)-7-chloroquinazolin-4(3H)-one) showing an IC50 value of 4.0 μM. Because the herein reported 2-phenylquinazolinones are merely fragments, they are largely optimizable, paving the way to derivatives with improved potency.

HIV-1 infection can be effectively controlled by HAART, which improves the quality of lives of infected individuals, but fails to completely eradicate the virus, even after decades of treatment. This issue, together with the emergence of multi-drug-resistant viruses, clearly underscores the continuing need to find novel agents able to target vulnerable steps in the viral replication cycle. HIV transcriptional regulation is a crucial step required to re-initiate viral replication from post-integration latency after interruption of therapy and to keep the virus in circulation. In this step, the viral protein Tat plays a central role by dramatically increasing the production of elongated transcripts through its unique interaction with the viral TAR RNA and the cellular cofactor P-TEFb, together with a myriad of other host factors which are recruited to the viral promoter to ensure efficient transcription. The transcriptional machinery, involving an intricate interplay of many viral and cellular components, offers a plethora of potential therapeutic targets that have not yet been exploited by any of the antiretroviral drugs used in therapy. In this review we explore the state-of-the-art of Tat-mediated transcription inhibitors which target the well-consolidated Tat/TAR/PTEFb axis, together with novel therapeutics that interfere with various host-cell factors, including some pioneer inhibitors designed on the basis of recent molecular and structural studies.

Although human cytomegalovirus (HCMV) infection is mostly asymptomatic for immunocompetent individuals, it remains a serious threat for those who are immunocompromised, in whom it is associated with various clinical manifestations. The therapeutic utility of the few available anti-HCMV drugs is limited by several drawbacks, including cross-resistance due to their common mechanism of action, i.e., inhibition of viral DNA polymerase. Therefore, compounds that target other essential viral events could overcome this problem. One example of this is the 6-aminoquinolone WC5, which acts by directly blocking the transactivation of essential viral Early genes by the Immediate-Early 2 (IE2) protein. In this study, the quinolone scaffold of the lead compound WC5 was investigated in depth, defining more suitable substituents for each of the scaffold positions explored and identifying novel, potent and nontoxic compounds. Some compounds showed potent anti-HCMV activity by interfering with IE2-dependent viral E gene expression. Among them, naphthyridone 1 was also endowed with potent anti-HIV activity in latently infected cells. Their antiviral profile along with their innovative mechanism of action make these anti-HCMV quinolones a very promising class of compounds to be exploited for more effective antiviral therapeutic treatment.

The current anti-HIV treatments fail to completely eradicate the virus in HIV-infected individuals, mainly as a result of a small pool of latently infected cells. This issue, together with the emergence of multidrug-resistant viruses, clearly highlights the need to find additional strategies. An overview of the Tat-mediated transcription inhibitors 6-desfluoroquinolones (6-DFQs), identified by our group, is given in this review along with a critical appraisal of their advantages and drawbacks. Attempts are also made to place them within the context of new potential anti-HIV therapeutics. Due to their innovative mechanism of action, the 6-DFQs could be interesting candidates for use in association with the currently used cocktail of drugs. Their potential as antivirals deserves further investigation.

In a furtherance of our SAR study on the C-6 position of quinolone antibacterials, a series of 6-desfluoro-8-methylquinolones were synthesized and evaluated for their in vitro antimicrobial activity. As a result of this study, compounds with strong activity against Gram-positive bacteria, including ciprofloxacin-resistant and methicillin-resistant Staphylococcus aureus, were identified. The best Gram-positive antibacterial activity was exhibited by piperidinyl derivative 6c, which was 17 times more potent than ciprofloxacin and displayed extremely high activity against Streptococcus pneumoniae with an MIC value of ≤0.016 μg/mL. Thus, we have shown that substituent combinations in the quinolone ring, excluding the C-6 fluorine atom, might produce powerful antibacterial agents.

A new 6-desfluoroquinolone derivative, characterized by the presence of a 6-hydroxyl group instead of the usual fluorine atom at the C-6 position, was synthesized with the aim to better understand the mechanistic role of the C-6 substituent in the quinolone/DNA/DNA-gyrase interaction. The antibacterial activity unambiguously shows that the hydroxyl group is a good substitute for the C-6 fluorine atom, especially against Gram-positive bacteria. On the contrary, it is a very weak inhibitor of the target DNA gyrase, displaying the highest IC50 value observed for all the C-6 substituted analogues. This behaviour could be explained on the basis of its DNA binding properties.

WC5 is a 6-aminoquinolone that potently inhibits the replication of human cytomegalovirus (HCMV) but has no activity, or significantly less activity, against other herpesviruses. Here we investigated the nature of its specific anti-HCMV activity. Structure-activity relationship studies on a small series of analogues showed that WC5 possesses the most suitable pattern of substitutions around the quinolone scaffold to give potent and selective anti-HCMV activity. Studies performed to identify the possible target of WC5 indicated that it prevents viral DNA synthesis but does not significantly affect DNA polymerase activity. In yield reduction experiments with different multiplicities of infection, the anti-HCMV activity of WC5 appeared to be highly dependent on the viral inoculum, suggesting that WC5 may act at an initial stage of virus replication. Consistently, time-of-addition and time-of-removal studies demonstrated that WC5 affects a phase of the HCMV replicative cycle that precedes viral DNA synthesis. Experiments to monitor the effects of the compound on virus attachment and entry showed that it does not inhibit either process. Evaluation of viral mRNA and protein expression revealed that WC5 targets an event of the HCMV replicative cycle that follows the transcription and translation of immediate-early genes and precedes those of early and late genes. In cell-based assays to test the effects of WC5 on the transactivating activity of the HCMV immediate-early 2 (IE2) protein, WC5 markedly interfered with IE2-mediated transactivation of viral early promoters. Finally, WC5 combined with ganciclovir in checkerboard experiments exhibited highly synergistic activity. These findings suggest that WC5 deserves further investigation as a candidate anti-HCMV drug with a novel mechanism of action.