Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials

IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7 Å resolution, respectively. The structure of the E. coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.     

A Covalent Inhibitor for Glutathione S-Transferase Pi (GSTP1-1) in Human Cells

Glutathione S-transferase π (GSTP_1-1) is overexpressed in many types of cancer and is involved in drug resistance. Therefore, GSTP_1-1 is an important target in cancer therapy, and many GST inhibitors have been reported. We had previously developed an irreversible inhibitor, GS-ESF, as an effective GST inhibitor; however, its cellular permeability was too low for it to be used in inhibiting intracellular GST. We have now developed new irreversible inhibitors by introducing sulfonyl fluoride (SF) into chloronitrobenzene (CNB). The mechanism of action was revealed to be that CNBSF first reacts with glutathione (GSH) through an aromatic substitution in the cell, then the sulfonyl group on the GSH conjugate with CNBSF reacts with Tyr108 of GST to form a sulfonyl ester bond. Our new inhibitor irreversible inhibited GSTP_1-1 both in vitro and in cellulo with a long duration of action.     

A Structure‐Based Virtual Screening Approach toward the Discovery of Histone Deacetylase Inhibitors: Identification of Promising Zinc‐Chelating Groups

The inhibitors of histone deacetylases (HDACs) have drawn a great deal of attention due to their promising potential as small‐molecule therapeutics for the treatment of cancer. By means of virtual screening with docking simulations under consideration of the effects of ligand solvation, we were able to identify six novel HDAC inhibitors with IC₅₀ values ranging from 1 to 100 μM . These newly identified inhibitors are structurally diverse and have various chelating groups for the active site zinc ion, including N‐[1,3,4]thiadiazol‐2‐yl sulfonamide, N‐thiazol‐2‐yl sulfonamide, and hydroxamic acid moieties. The former two groups are included in many drugs in current clinical use and have not yet been reported as HDAC inhibitors. Therefore, they can be considered as new inhibitor scaffolds for the development of anticancer drugs by structure–activity relationship studies to improve the inhibitory activities against HDACs. Interactions with the HDAC1 active site residues responsible for stabilizing these new inhibitors are addressed in detail.     

A Potent N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide Inhibitor of Adenylyl Cyclase of G. lamblia: Biological Evaluation and Molecular Modelling Studies

In this work, we report a derivative of N-(piperidin-4-yl)-1H-pyrrole-2-carboxamide as a new inhibitor for adenylyl cyclase of Giardia lamblia which was obtained from a study using structural data of the nucleotidyl cyclase 1 (gNC1) of this parasite. For such a study, we developed a model for this specific enzyme by using homology techniques, which is the first model reported for gNC1 of G. lamblia. Our studies show that the new inhibitor has a competitive mechanism of action against this enzyme. 2-Hydroxyestradiol was used as the reference compound for comparative studies. Results in this work are important from two points of view. on the one hand, an experimentally corroborated model for gNC1 of G. lamblia obtained by molecular modelling is presented; on the other hand, the new inhibitor obtained is an undoubtedly excellent starting structure for the development of new metabolic inhibitors for G. lamblia.     

Direct Staudinger–Phosphonite Reaction Provides Methylphosphonamidates as Inhibitors of CE4 De‐N‐acetylases

De‐N‐acetylases of β‐(1→6)‐D ‐N‐acetylglucosamine polymers (PNAG) and β‐(1→4)‐D ‐N‐acetylglucosamine residues in peptidoglycan are attractive targets for antimicrobial agents. PNAG de‐N‐acetylases are necessary for biofilm formation in numerous pathogenic bacteria. Peptidoglycan de‐N‐acetylation facilitates bacterial evasion of innate immune defenses. To target these enzymes, transition‐state analogue inhibitors containing a methylphosphonamidate have been synthesized through a direct Staudinger–phosphonite reaction. The inhibitors were tested on purified PgaB, a PNAG de‐N‐acetylase from Escherichia coli, and PgdA, a peptidoglycan de‐N‐acetylase from Streptococcus pneumonia. Herein, we describe the most potent inhibitor of peptidoglycan de‐N‐acetylases reported to date (K_i=80 μM ). The minimal inhibition of PgaB observed provides insight into key structural and functional differences in these enzymes that will need to be considered during the development of future inhibitors.     

Design,Synthesis and Biological Evaluation of Rose Bengal Analogues as SecA Inhibitors

SecA, a key component of bacterial Sec‐dependent secretion pathway, is an attractive target for exploring novel antimicrobials. Rose bengal (RB), a polyhalogenated fluorescein derivative, was found from our previous study as a potent SecA inhibitor. Here we describe the synthesis and structure–activity relationships (SAR) of 23 RB analogues that were designed by systematical dissection of RB. Evaluation of these analogues allowed us to establish an initial SAR in SecA inhibition. The antimicrobial effects of these SecA inhibitors are confirmed in experiments using E. coli and B. subtilis.     

Design,Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Substituted Tricyclic Hexahydro-Furofuran Derivatives as P2 Ligands

The design, synthesis, X-ray structural, and biological evaluation of a series of highly potent HIV-1 protease inhibitors are reported herein. These inhibitors incorporate novel cyclohexane-fused tricyclic bis-tetrahydrofuran as P2 ligands in combination with a variety of P1 and P2′ ligands. The inhibitor with a difluoromethylphenyl P1 ligand and a cyclopropylaminobenzothiazole P2′ ligand exhibited the most potent antiviral activity. Also, it maintained potent antiviral activity against a panel of highly multidrug-resistant HIV-1 variants. The corresponding inhibitor with an enantiomeric ligand was significantly less potent in these antiviral assays. The new P2 ligands were synthesized in optically active form using enzymatic desymmetrization of meso-diols as the key step. To obtain molecular insight, two high-resolution X-ray structures of inhibitor-bound HIV-1 protease were determined and structural analyses have been highlighted.     

Benzodioxane-Benzamides as Antibacterial Agents: Computational and SAR Studies to Evaluate the Influence of the 7-Substitution in FtsZ Interaction

FtsZ is a crucial prokaryotic protein involved in bacterial cell replication. It recently arose as a promising target in the search for antimicrobial agents able to fight antimicrobial resistance. In this work, going on with our structure-activity relationship (SAR) study, we developed variously 7-substituted 1,4-benzodioxane compounds, linked to the 2,6-difluorobenzamide by a methylenoxy bridge. Compounds exhibit promising antibacterial activities not only against multidrug-resistant Staphylococcus aureus, but also on mutated Escherichia coli strains, thus enlarging their spectrum of action toward Gram-negative bacteria as well. Computational studies elucidated, through a validated FtsZ binding protocol, the structural features of new promising derivatives as FtsZ inhibitors.     

Glucocerebrosidase Enhancers for Selected Gaucher Disease Genotypes by Modification of α‐1‐C‐Substituted Imino‐D‐xylitols (DIXs) by Click Chemistry

A series of hybrid analogues was designed by combination of the iminoxylitol scaffold of parent 1C9‐DIX with triazolylalkyl side chains. The resulting compounds were considered potential pharmacological chaperones in Gaucher disease. The DIX analogues reported here were synthesized by CuAAC click chemistry from scaffold 1 (α‐1‐C‐propargyl‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol) and screened as imiglucerase inhibitors. A set of selected compounds were tested as β‐glucocerebrosidase (GBA1) enhancers in fibroblasts from Gaucher patients bearing different genotypes. A number of these DIX compounds were revealed as potent GBA1 enhancers in genotypes containing the G202R mutation, particularly compound DIX‐28 (α‐1‐C‐[(1‐(3‐trimethylsilyl)propyl)‐1H‐1,2,3‐triazol‐4‐yl)methyl]‐1,5‐dideoxy‐1,5‐imino‐D ‐xylitol), bearing the 3‐trimethylsilylpropyl group as a new surrogate of a long alkyl chain, with approximately threefold activity enhancement at 10 nM . Despite their structural similarities with isofagomine and with our previously reported aminocyclitols, the present DIX compounds behaved as non‐competitive inhibitors, with the exception of the mixed‐type inhibitor DIX‐28.     

Targeted Synthesis of Complex Spiro[3H-indole-3,2′-pyrrolidin]-2(1H)-ones by Intramolecular Cyclization of Azomethine Ylides: Highly Potent MDM2–p53 Inhibitors

Mouse double minute 2 (MDM2) is a main and direct inhibitor of the crucial tumor suppressor p53. Reports from initial clinical trials showed that blocking this interaction with a small-molecule inhibitor can have great value in the treatment of cancer for patients with p53 wild-type tumors; however, it also revealed dose-limiting hematological toxicities and drug-induced resistance as main issues. To overcome the former, an inhibitor with superior potency and pharmacokinetic properties to ultimately achieve full efficacy with less-frequent dosing schedules is required. Toward this aim, we optimized our recently reported spiro-oxindole inhibitors by focusing on the crucial interaction with the amino acid side chain of His96_MDM2. The designed molecules required the targeted synthesis of structurally complex spiro[indole-3,2′-pyrrolo[2,3-c]pyrrole]-2,4′-diones for which we developed an unprecedented intramolecular azomethine ylide cycloaddition and investigated the results by computational methods. One of the new compounds showed superior cellular potency over previously reported BI-0252. This finding is a significant step toward an inhibitor suitable to potentially mitigate hematological on-target adverse effects.     

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PL^pro) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide ( 2 b ), which is known to bind into the S3 and S4 pockets of the SARS-CoV PL^pro. Moreover, we report the discovery of isoindolines as a new class of potent PL^pro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PL^pro are valuable starting points for the development of new pan-coronaviral inhibitors.     

Discovery of a Histidine-Based Scaffold as an Inhibitor of Gut Microbial Choline Trimethylamine-Lyase

Anaerobic choline metabolism by human gut microbiota to produce trimethylamine (TMA) has recently evolved as a potential therapeutic target because of its association with chronic kidney disease and increased cardiovascular risks. Limited examples of choline analogues have been reported as inhibitors of bacterial enzyme choline TMA-lyase (CutC), a key enzyme regulating choline anaerobic metabolism. We used a new workflow to discover CutC inhibitors based on focused screening of a diversified library of small molecules for intestinal metabolic stability followed by in vitro CutC inhibitory assay. This workflow identified a histidine-based scaffold as a CutC inhibitor with an IC₅₀ value of 1.9±0.2 μM. Remarkably, the identified CutC inhibitor was able to reduce the production of TMA in whole-cell assays using various bacterial strains as well as in complex gut microbiota environment. The improved efficiency of the new scaffold identified in this study in comparison to previously reported CutC inhibitors would enable optimization of potential leads for in vivo screening and clinical translation. Finally, docking studies and molecular-dynamic simulations were used to predict putative interactions created between inhibitor and CutC.     

A Fluorescence-Based Assay for N5-Carboxyaminoimidazole Ribonucleotide Mutase

The enzyme N⁵-carboxylaminoinidazole ribonucleotide (N⁵-CAIR) mutase is found in microbial de novo purine biosynthesis but is absent in humans making it an attractive antimicrobial target. N⁵-CAIR mutase catalyzes the synthesis of carboxyaminoimidazole ribonucleotide (CAIR) from N⁵-CAIR which is itself prepared from aminoimidazole ribonucleotide (AIR) by the enzyme N⁵-CAIR synthetase. During our research on identifying inhibitors of N⁵-CAIR mutase, we developed an innovative, fluorescence-based assay to measure the activity of this enzyme. This assay relies upon our recent serendipitous observation that AIR reversibly reacts with the compound isatin. Reaction of a fluorescently-tagged isatin with AIR resulted in a large increase in fluorescence intensity allowing a measurement of the concentration of AIR in solution. From this observation, we developed a reproducible, non-continuous assay that can replicate the known kinetic parameters of the enzyme and can readily detect a recognized inhibitor of the enzyme. This assay should find utility in screening for inhibitors targeting N⁵-CAIR mutase.     

Novel Quinic Acid Glycerates from Tussilago farfara Inhibit Polypeptide GalNAc-Transferase

The discovery of a bioactive inhibitor tool for human polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts), the initiating enzyme for mucin-type O-glycosylation, remains challenging. In the present study, we identified an array of quinic acid derivatives, including four new glycerates ( 1 – 4 ) from Tussilago farfara, a traditional Chinese medicinal plant, as active inhibitors of GalNAc-T2 using a combined screening approach with a cell-based T2-specific sensor and purified enzyme assay. These inhibitors dose-dependently inhibited human GalNAc-T2 but did not affect O-linked N-acetylglucosamine transferase (OGT), the other type of glycosyltransferase. Importantly, they are not cytotoxic and retain inhibitory activity in cells lacking elongated O-glycans, which are eliminated by the CRISPR/Cas9 gene editing tool. A structure-activity relationship study unveiled a novel quinic acid-caffeic acid conjugate pharmacophore that directs inhibition. Overall, these new natural product inhibitors could serve as a basis for developing an inhibitor tool for GalNAc-T2.     

Inhibition of Human α‐Methylacyl CoA Racemase (AMACR): a Target for Prostate Cancer

The enzyme α‐methylacyl CoA racemase (AMACR) is involved in the metabolism of branched‐chain fatty acids and has been identified as a promising therapeutic target for prostate cancer. By using the recently available human AMACR from HEK293 kidney cell cultures, we tested a series of new rationally designed inhibitors to determine the structural requirements in the acyl component. An N‐methylthiocarbamate (K_i=98 nM ), designed to mimic the proposed enzyme‐bound enolate, was found to be the most potent AMACR inhibitor reported to date.     

Evaluation of 4-(4-Fluorobenzyl)piperazin-1-yl]-Based Compounds as Competitive Tyrosinase Inhibitors Endowed with Antimelanogenic Effects

There is a considerable attention for the development of inhibitors of tyrosinase (TYR) as therapeutic strategy for the treatment of hyperpigmentation disorders in humans. Continuing in our efforts to identify TYR inhibitors, we describe the design, synthesis and pharmacophore exploration of new small molecules structurally characterized by the presence of the 4-fluorobenzylpiperazine moiety as key pharmacophoric feature for the inhibition of TYR from Agaricus bisporus (AbTYR). Our investigations resulted in the discovery of the competitive inhibitor [4-(4-fluorobenzyl)piperazin-1-yl]-(3-chloro-2-nitro-phenyl)methanone 26 (IC₅₀=0.18 μM) that proved to be ∼100-fold more active than reference compound kojic acid (IC₅₀=17.76 μM). Notably, compound 26 exerted antimelanogenic effect on B16F10 cells in absence of cytotoxicity. Docking analysis suggested its binding mode into AbTYR and into modelled human TYR.     

Induced‐Fit Docking Approach Provides Insight into the Binding Mode and Mechanism of Action of HIV‐1 Integrase Inhibitors

A three‐dimensional model of a complex between HIV‐1 integrase (IN), viral DNA, and metal ions that we recently built was used as a target for a docking method (induced‐fit docking, IFD) that accurately predicts ligand binding modes and concomitant structural changes in the receptor. Six different well‐known integrase strand transfer inhibitors (INSTIs): L‐708,906, L‐731,988, S‐1360, L‐870,810, raltegravir, and elvitegravir were thus used as ligands for our docking simulations. The obtained IFD results are consistent with the mechanism of action proposed for this class of IN inhibitors, that is, metal chelating/binding agents. This study affords new insight into the possible mechanism of inhibition and binding conformations for INSTIs. The impact on our hypothesis of specific mutations associated with IN inhibitor resistance was also evaluated. All these findings might have implications for integrase‐directed HIV‐1 drug discovery efforts.     

MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)‐Indatraline as Native Marker

We herein present label‐free, mass‐spectrometry‐based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis‐configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)‐indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)‐indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)‐indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT.     

Search for Shorter Portions of the Proline-Rich Antimicrobial Peptide Fragment Bac5(1–25) That Retain Antimicrobial Activity by Blocking Protein Synthesis

The spread of antibiotic-resistant pathogens has boosted the search for new antimicrobial drugs. Proline-rich antimicrobial peptides are promising lead compounds for the development of next-generation antibiotics, given their very low cytotoxicity and their good antimicrobial activity targeting the bacterial ribosome. Bac5(1–25) is an N-terminal fragment of the bovine proline-rich antimicrobial peptide Bac5, whose mode of action has been recently described. In this work we tested a number of Bac5(1–25) fragments, and we characterized their antimicrobial activity against Escherichia coli, Acinetobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Salmonella enterica, and Pseudomonas aeruginosa. We evaluated their cytotoxicity toward human cells and their efficacy in inhibiting bacterial protein synthesis. This allowed us to identify some shorter fragments of Bac5(1–25) with a good balance between antibacterial efficacy, protein synthesis inhibition, and ease/cost-effectiveness of synthesis, suitable as lead compounds to develop new antibacterials.