Dimerization inhibitors of HIV-1 reverse transcriptase, protease and integrase: A single mode of inhibition for the three HIV enzymes?

The genome of human immunodeficiency virus type 1 (HIV-1) encodes 15 distinct proteins, three of which provide essential enzymatic functions: a reverse transcriptase (RT), an integrase (IN), and a protease (PR). Since these enzymes are all homodimers, pseudohomodimers or multimers, disruption of protein-protein interactions in these retroviral enzymes may constitute an alternative way to achieve HIV-1 inhibition. A growing number. of dimerization inhibitors for these enzymes is being reported. This mini review summarizes some approaches that have been followed for the development of compounds that inhibit those three enzymes by interfering with the dimerization interfaces between the enzyme subunits. (c) 2006 Elsevier B.V. All rights reserved

TSAO compounds: The comprehensive story of a unique family of HIV-1 specific inhibitors of reverse transcriptase

Emergence of drug-resistant viral strains is one of the major milestones and the main cause for the failure of antiretroviral therapy. Combination of different anti-HIV agents has E become the standard clinical practice to keep the viral load at low or even undetectable levels and to prevent emergence of virus-drug resistance. Among the human immunodeficiency virus (HIV) reverse transcriptase (RT) inhibitors, the so called nonnucleoside RT inhibitors (NNRTIs) have gained a definitive place in the treatment of HIV infections in combination with nucleoside analogue RT inhibitors (NRTIs) and HIV protease inhibitors (PIs). The virus can be markedly suppressed for a relatively long period of time when exposed to multiple drug combination therapy (highly active antiretroviral therapy, HAART). TSAO derivatives are a peculiar group of highly functionalized nucleosides that belong to the so-called nonnucleoside RT inhibitors (NNRTIs). They exert their unique selectivity for HIV-1 through a specific interaction with the p51 subunit of HIV-1 RT. They are the first small molecules that seem to interfere with the dimerization process of the enzyme. This review covers the work carried out with this unique class of specific inhibitors of HIV-1 reverse transcriptase, including structure activity relationship studies (SAR), its mechanism of action, resistance studies, model of interaction with the enzyme, etc

N-benzyl 4,4-disubstituted piperidines as a potent class of influenza H1N1 virus inhibitors showing a novel mechanism of hemagglutinin fusion peptide interaction

The influenza virus hemagglutinin (HA) is an attractive target for antiviral therapy due to its essential role in mediating virus entry into the host cell. We here report the identification of a class of N-benzyl- 4,4,-disubstituted piperidines as influenza A virus fusion inhibitors with specific activity against the H1N1 subtype. Using the highly efficient one-step Ugi four-component reaction, diverse library of piperidine-based analogues was synthesized and evaluated to explore the structure-activity relation- ships (SAR). Mechanistic studies, including resistance selection with the most active compound (2) demonstrated that it acts as an inhibitor of the low pH-induced HA-mediated membrane fusion process. Computational studies identified an as yet unrecognized fusion inhibitor binding site, which is located at the bottom of the HA2 stem in close proximity to the fusion peptide. A direct p-stacking interaction between the N-benzylpiperidine moiety of 2 and F9HA2 of the fusion peptide, reinforced with an addi- tional p-stacking interaction with Y119HA2, and a salt bridge of the protonated piperidine nitrogen with E120HA2, were identified as important interactions to mediate ligand binding. This site rationalized the observed SAR and provided a structural explanation for the H1N1-specific activity of our inhibitors. Furthermore, the HA1-S326V mutation resulting in resistance to 2 is close to the proposed new binding pocket. Our findings point to the N-benzyl-4,4,-disubstituted piperidines as an interesting class of influenza virus inhibitors, representing the first example of fusion peptide binders with great potential for anti-influenza drug development

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.

Pyrrolopyrimidine vs imidazole-phenyl-thiazole scaffolds in nonpeptidic dimerization inhibitors of leishmania infantum trypanothione reductase

Disruption of protein-protein interactions of essential oligomeric enzymes by small molecules represents a significant challenge. We recently reported some linear and cyclic peptides derived from an α-helical region present in the homodimeric interface of Leishmania infantum trypanothione reductase (Li-TryR) that showed potent effects on both dimerization and redox activity of this essential enzyme. Here, we describe our first steps toward the design of nonpeptidic small-molecule Li-TryR dimerization disruptors using a proteomimetic approach. The pyrrolopyrimidine and the 5-6-5 imidazole-phenyl-thiazole α-helix-mimetic scaffolds were suitably decorated with substituents that could mimic three key residues (K, Q, and I) of the linear peptide prototype (PKIIQSVGIS-Nle-K-Nle). Extensive optimization of previously described synthetic methodologies was required. A library of 15 compounds bearing different hydrophobic alkyl and aromatic substituents was synthesized. The imidazole-phenyl-thiazole-based analogues outperformed the pyrrolopyrimidine-based derivatives in both inhibiting the enzyme and killing extracellular and intracellular parasites in cell culture. The most active imidazole-phenyl-thiazole compounds 3e and 3f inhibit Li-TryR and prevent growth of the parasites at low micromolar concentrations similar to those required by the peptide prototype. The intrinsic fluorescence of these compounds inside the parasites visually demonstrates their good permeability in comparison with previous peptide-based Li-TryR dimerization disruptors.We thank the Spanish Government (MINECO/FEDER Projects SAF2015-64629-C2, BFU2017-90030-P), the Comunidad de Madrid (BIPEDD-2-CM ref S-2010/BMD-2457), and the Consejo Superior de Investigaciones Cientıfí cas (CSIC Project 201980E028) for financial support. We thank staff from ALBA Synchrotron (Barcelona, Spain) for support during data collection.Peer Reviewe

A Novel and Versatile Class of Coronavirus non-covalent Mpro Inhibitors based on 1,4,4-Trisubstituted Piperidines

This pilot study shows that a significant proportion of Long COVID-19 cases are positive for HERV-W ENV expression along with a subgroup of ME/CFS samples from a pre-COVID pandemia collection, raising the question of whether the presence of HERV-W ENV protein, known to induce TLR4-driven immuno- and neuro-pathogenicity, could be a common factor to their overlapping symptoms. Being this the case, HERV-W ENV could constitute a future therapeutic target, following the steps of other neurologic or autoimmune diseases such as multiple sclerosis or diabetes type I. Particularly since ongoing clinical trials assaying HERV-directional therapies based on antiretroviral agents or monoclonal antibodies are showing promising results.The COVID19 pandemia has greatly encouraged the development of vaccines and novel antivirals to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of anti-influenza H1N1 1,4,4-trisubstituted piperidines, developed in our goup, we performed a SAR analysis of these unique inhibitors that allowed to define the structural elements essential for antiHcoV-229E activity. Four of the best molecules were confirmed to be equally active against SARS-CoV-2. A TOA experiment indicated that these new CoV inhibitors interact at a post virus entry point lying at the stage of viral poly protein processing and the start of viral RNA synthesis. Enzymatic assays were performed with different CoV proteins involved in these processes. The compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was modest, the ability to bind to the catalytic site of Mpro was assessed by in silico studies. The combination of results from TOA, enzymatic assays, resistance selection and in silico molecular modeling allowed us to conclude that the 1,4,4-trisubstituted piperidines represent a structurally novel and unique class of compounds that inhibit CoV Mpro inhibitors via a non-covalent mechanism, making these inhibitors fundamentally different from other Mpro inhibitors represented by the approved drug nirmatrelvir. The five points of diversity make these N-substituted piperidinebased compounds highly versatile and amenable to further rational optimization to maximize their activity and selectivity and gain full insight their antiviral mechanism.6th Innovative Approaches for Identification of Antiviral Agents Summer School September 26th-30th 2022, Santa Margherita di Pula, Sardinia, Ital

Antivascular and antitumor properties of the tubulin-binding chalcone TUB091

We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3’’-amino-4’’-methoxyphenyl)-1-(5’-methoxy-3’,4’-methylendioxyphenyl)- 2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-tostraight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.M-DC thanks the Fondo Social Europeo (FSE) and the JAE Predoc Programme for a predoctoral fellowship. This work has received the Ramón Madroñero award for young researchers (to M-DC and OB) in the XVII call www.impactjournals.com/oncotarget 17 Oncotarget sponsored by the Spanish Society of Medicinal Chemistry (SEQT). This project has been supported by the Spanish Ministerio de Economia y Competitividad (SAF2012- 39760-C02-01 to M-JC, M-JP-P, SV and E-MP; and BIO2013-42984-R to JFD), Comunidad de Madrid (BIPEDD2; ref. P2010/BMD-2457 to M-JC and J-FD), the Swiss National Science Foundation (310030B_138659 and 31003A_166608; to MOS). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature - from natural products chemistry to drug discovery” and COST action CM1470.Peer reviewe

Peptidoglycan editing in non-proliferating intracellular Salmonella as source of interference with immune signaling

Salmonella enterica causes intracellular infections that can be limited to the intestine or spread to deeper tissues. In most cases, intracellular bacteria show moderate growth. How these bacteria face host defenses that recognize peptidoglycan, is poorly understood. Here, we report a high-resolution structural analysis of the minute amounts of peptidoglycan puri- fied from S. enterica serovar Typhimurium (S. Typhimurium) infecting fibroblasts, a cell type in which this pathogen undergoes moderate growth and persists for days intracellularly. The peptidoglycan of these non-proliferating bacteria contains atypical crosslinked muropep- tides with stem peptides trimmed at the L-alanine-D-glutamic acid-(γ) or D-glutamic acid-(γ)- meso-diaminopimelic acid motifs, both sensed by intracellular immune receptors. This pepti- doglycan has a reduced glycan chain average length and ~30% increase in the L,D-cross- link, a type of bridge shared by all the atypical crosslinked muropeptides identified. The L,D- transpeptidases LdtD (YcbB) and LdtE (YnhG) are responsible for the formation of these L, D-bridges in the peptidoglycan of intracellular bacteria. We also identified in a fraction of muropeptides an unprecedented modification in the peptidoglycan of intracellular S. Typhi- murium consisting of the amino alcohol alaninol replacing the terminal (fourth) D-alanine. Alaninol was still detectable in the peptidoglycan of a double mutant lacking LdtD and LdtE, thereby ruling out the contribution of these enzymes to this chemical modification. Remark- ably, all multiple mutants tested lacking candidate enzymes that either trim stem peptides or form the L,D-bridges retain the capacity to modify the terminal D-alanine to alaninol and all attenuate NF-κB nuclear translocation. These data inferred a potential role of alaninol-con- taining muropeptides in attenuating pro-inflammatory signaling, which was confirmed with a synthetic tetrapeptide bearing such amino alcohol. We suggest that the modification of D- alanine to alaninol in the peptidoglycan of non-proliferating intracellular S. Typhimurium is an editing process exploited by this pathogen to evade immune recognition inside host cells.This work was funded by grants PID2020-112971GB-I00/10.13039/501100011033 (F.G-dP.) and PID2019-104070RB-C21 (S.V.) of the Spanish Ministry of Science and Innovation, VR2018-02823 of the Swedish Research Council (F.C.), KAW2012.0184 of the Knut and Alice Wallenberg Foundation (F.C.), and SMK2062 of the Kempe Foundation (F.C.). S.C. was recipient of an EMBO Short-Term Fellowship number 6426 for a stay in the lab of F.C. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscriptPeer reviewe

A Versatile Class of 1,4,4-Trisubstituted Piperidines Block Coronavirus Replication In Vitro

There is a clear need for novel antiviral concepts to control SARS-CoV-2 infection. Based on the promising anti-coronavirus activity observed for a class of 1,4,4-trisubstituted piperidines, we here conducted a detailed analysis of the structure–activity relationship of these structurally unique inhibitors. Despite the presence of five points of diversity, the synthesis of an extensive series of analogues was readily achieved by Ugi four-component reaction from commercially available reagents. After evaluating 63 analogues against human coronavirus 229E, four of the best molecules were selected and shown to have micromolar activity against SARS-CoV-2. Since the action point was situated post virus entry and lying at the stage of viral polyprotein processing and the start of RNA synthesis, enzymatic assays were performed with CoV proteins involved in these processes. While no inhibition was observed for SARS-CoV-2 nsp12-nsp7-nsp8 polymerase, nsp14 N7-methyltransferase and nsp16/nsp10 2’-O-methyltransferase, nor the nsp3 papain-like protease, the compounds clearly inhibited the nsp5 main protease (Mpro). Although the inhibitory activity was quite modest, the plausibility of binding to the catalytic site of Mpro was established by in silico studies. Therefore, the 1,4,4-trisubstituted piperidines appear to represent a novel class of non-covalent CoV Mpro inhibitors that warrants further optimization and development.This research was funded by grants from the Spanish MICINN (Projects PID2019-104070RB- C21 and PID2019-104070RB-C22); the Spanish Agencia Estatal Consejo Superior de Investigaciones Científicas (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028); the European Union’s Innovative Medicines Initiative (IMI) under Grant Agreement 101005077 [Corona Accelerated R&D in Europe (CARE) project]; and Fundació La Marató de TV3, Spain (Projects No. 201832-30 and No. 202135-30). B.V.L. holds an SB-PhD fellowship from the FWO Research Foundation Flanders (project: 1S66321N)Peer reviewe

Identification of L. infantum trypanothione synthetase inhibitors with leishmanicidal activity from a (non-biased) in-house chemical library

Redox homeostasis in trypanosomatids is based on the low-molecular-weight trypanothione, an essential dithiol molecule that is synthetized by trypanothione synthetase (TryS) and maintained in its reduced state by trypa- nothione disulfide reductase (TryR). The fact that both enzymes are indispensable for parasite survival and absent in the mammalian hosts makes them ideal drug targets against leishmaniasis. Although many efforts have been directed to developing TryR inhibitors, much less attention has been focused on TryS. The screening of an in-house library of 144 diverse molecules using two parallel biochemical assays allowed us to detect 13 inhibitors of L. infantum TryS. Compounds 1 and 3 were characterized as competitive inhibitors with Ki values in the low micromolar range and plausible binding modes for them were identified by automated ligand docking against refined protein structures obtained through computational simulation of an entire catalytic cycle. The proposed binding site for both inhibitors overlaps the polyamine site in the enzyme and, additionally, 1 also occupies part of the ATP site. Compound 4 behaves as a mixed hyperbolic inhibitor with a Ki of 0.8 μM. The activity of 5 is clearly dependent on the concentration of the polyamine substrate, but its kinetic behavior is clearly not compatible with a competitive mode of inhibition. Analysis of the activity of the six best inhibitors against intracellular amastigotes identified 5 as the most potent leishmanicidal candidate, with an EC50 value of 0.6 μM and a selectivity index of 35.This work has been supported by the Spanish MICINN (Projects PID2019-104070RB-C21 and PID2019-104070RBC22), the Spanish National Research Council (CSIC, Projects CSIC-PIE-201980E100 and CSIC-PIE-201980E028), and the Comunidad de Madrid (PLATESA2-CM ref S-2018/BAA-4370). The MCIN is also acknowledged for the pre- doctoral fellowship to M.A.C.Peer reviewe