Analogue Inhibitors Research Articles

Comparison of three zinc binding groups for HDAC inhibitors – A potency, selectivity and enzymatic kinetics study

Hydroxamic acid and benzamide are the most commonly used zinc binding group (ZBG) for HDAC inhibitors both in clinic and pre-clinic. Recently, we discovered several analogs of new type HDAC inhibitors with hydrazide as ZBG. Representative compounds displayed high potency, class I HDAC selectivity and excellent pharmacokinetics profile. In this research, we synthesize tool compounds 4 and 6 by modifying the hydroxamic acid of SAHA with benzamide and hydrazide, respectively, and compare the potency, isoform selectivity, binding profile and enzymatic kinetics for the hydroxamate, benzamide and hydrazide-based inhibitors. It is well known that SAHA with hydroxamic acid is a pan-HDAC inhibitor with competitive binding and fast-on/fast-off profile. Compound 6 is a slow-binding class I selective inhibitor with mixed (competitive and non-competitive) binding mode, which is the same as the hydrazide inhibitors in our previous study. Compound 4 is a class I selective, fast-on/fast-off inhibitor with competitive binding mode to HDAC1/2/3, which is different with published benzamide MS275 and 106. Therefore, the kinetics profile of benzamide is not only due to the ZBG, but also rely on the cap and linker groups. To the best of our knowledge, this is the first report to compare the enzymatic profile of three promising ZBGs of HDAC inhibitors.

Transmembrane Protease Serine 2 Proteolytic Cleavage of the SARS-CoV-2 Spike Protein: A Mechanistic Quantum Mechanics/Molecular Mechanics Study to Inspire the Design of New Drugs To Fight the COVID-19 Pandemic.

Despite the development of vaccines against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus, there is an urgent need for efficient drugs to treat infected patients. An attractive drug target is the human transmembrane protease serine 2 (TMPRSS2) because of its vital role in the viral infection mechanism of SARS-CoV-2 by activation of the virus spike protein (S protein). Having in mind that the information derived from quantum mechanics/molecular mechanics (QM/MM) studies could be an important tool in the design of transition-state (TS) analogue inhibitors, we resorted to adiabatic QM/MM calculations to determine the mechanism of the first step (acylation) of proteolytic cleavage of the S protein with atomistic details. Acylation occurred in two stages: (i) proton transfer from Ser441 to His296 concerted with the nucleophilic attack of Ser441 to the substrate’s P1-Arg and (ii) proton transfer from His296 to the P1′-Ser residue concerted with the cleavage of the ArgP1-SerP1′ peptide bond, with a Gibbs activation energy of 17.1 and 15.8 kcal mol–1, relative to the reactant. An oxyanion hole composed of two hydrogen bonds stabilized the rate-limiting TS by 8 kcal mol–1. An analysis of the TMPRSS2 interactions with the high-energy, short-lived tetrahedral intermediate highlighted the limitations of current clinical inhibitors and pointed out specific ways to develop higher-affinity TS analogue inhibitors. The results support the development of more efficient drugs against SARS-CoV-2 using a human target, free from resistance development.

The structure and molecular dynamics of prolyl oligopeptidase from Microbulbifer arenaceous provide insights into catalytic and regulatory mechanisms.

Prolyl oligopeptidases (POPs) are atypical serine proteases that are unique in their involvement in the maturation and degradation of prolyl-containing peptide hormones and neuropeptides. They are potential pharmaceutical targets for the treatment of several neurodegenerative disorders, such as Alzheimer's disease. In this study, the catalytic and substrate-regulatory mechanisms of a novel bacterial POP from Microbulbifer arenaceous (MaPOP) were investigated. The crystal structure revealed that the catalytic triad of MaPOP was covered by the central tunnel of an unusual β-propeller domain. The tunnel not only provided the sole access to the active site for oligopeptides, but also protected large structured peptides or proteins from accidental proteolysis. The enzyme was able to cleave angiotensin I specifically at the carboxyl side of the internal proline residue, but could not hydrolyze long-chain bovine insulin B invitro. Like the ligand-free structure, MaPOP bound to the transition-state analog inhibitor ZPR was also in a closed state, which was not modulated by the common `latching loop' found in other POPs. The substrate-assisted catalytic mechanism of MaPOP reported here may represent a common mechanism for all POPs. These results may facilitate a better understanding of the catalytic behavior of POPs under physiological conditions.

Treatment of chronic hepatitis B: virological and pharmacological aspects

Tenofovir is a nucleotidic analog inhibitor used in monotherapy as first line treatment of chronic Hepatitis B virus (HBV) infection. This drug requires a two-step phosphorylation by cellular kinases. The active triphosphate form inhibits viral DNA polymerase. Tenofovir has a very low oral bioavailability following oral administration. Hence, its oral administration requires the use of prodrugs: tenofovir disoproxil fumararate (TDF) or tenofovir alafénamide (TAF). TAF demonstrated a lower kidney and bone toxicity than TDF. TAF and TDF treatments allow prevention of chronic hepatitis B complications, which are cirrhosis and hepatocellular carcinoma. Prevention of these complications requires a virological response to the treatment, defined as undetectable HBV DNA. TDF and TAF are associated with virological response rates from 64 to 94 % after one year of treatment. This rate depends on HBV viral load at diagnostic, HBe antigen status, mutations in the HBV polymerase gene (Pol/RT) and patient compliance to the treatment. Tenofovir has a high genetic barrier and resistance mutations to this drug have not yet been described. However, mutations rt181T/V and/or rtN236T have been associated with reduced susceptibility to TDF/TAF in vitro and delayed response in vivo. Recently described mutations CYEI and rtA194T have been associated with reduced susceptibility to TDF/TAF in vitro without any change in viral response in vivo. Patient compliance can be improved using cognitive behavioral therapy, supporter interventions and use of short message service. Finally, some genetic polymorphisms in MRP(multidrug resistance-associated protein)-2 and MRP4 efflux transporters could be associated with TDF toxicity and virological response to TDF or TAF. In the perspective of functional HBV cure, TDF and TAF are likely to be still used, in association with new class of antivirals. For this reason, it is important to further characterize the pharmacological and virological factors associated with partial virological response to tenofovir.

Investigation of the Potential of Bile Acid Methyl Esters as Inhibitors of Aldo-keto Reductase 1C2: Insight from Molecular Docking, Virtual Screening, Experimental Assays and Molecular Dynamics.

Human aldo-keto reductase 1C isoforms (AKR1C1-C4) catalyze reduction of endogenous and exogenous compounds, including therapeutic drugs, and are associated with chemotherapy resistance. AKR1C2 is involved in metastatic processes and is a target for the treatment of various cancers. Here we used molecular docking to explore the potential of a series of eleven bile acid methyl esters as AKR1C2 inhibitors. Autodock 4.2 ranked 10 of the 11 test compounds above a decoy set generated based on ursodeoxycholic acid, a known AKR1C2 inhibitor, while 5 of these 10 ranked above 94 % of decoys in Autodock Vina. Seven inactives reported in the literature not to inhibit AKR1C2 ranked below the decoy threshold: 5 of these are specific inhibitors of AKR1C3, a related isoform. Using the same parameters, Autodock Vina identified steroidal analogs of AKR1C substrates, bile acids, and AKR1C inhibitors in the top 5 % of a virtual screen of a natural product library. In experimental assays, 6 out of 11 of the tested bile acid methyl esters inhibited >50 % of AKR1C2 activity, while 2 compounds were strong AKR1C3 inhibitors. Potential off-target interactions with the glucocorticoid receptor were measured using a yeast-based fluorescence assay, where results suggest that the methyl ester could interfere with binding. The top ranking compound based on docking and experimental results showed dose-dependent inhibition of AKR1C2 with an IC50 of ∼3.6 μM. Molecular dynamics simulations (20 ns) were used to explore potential interactions between a bile acid methyl ester and residues in the AKR1C2 active site. Our molecular docking results identify AKR1C2 as a target for bile acid methyl esters, which combined with virtual screening results could provide new directions for researchers interested in synthesis of AKR1C inhibitors.

Targeting protein–protein interaction for immunomodulation: A sunflower trypsin inhibitor analog peptidomimetic suppresses RA progression in CIA model

Protein–protein interactions (PPI) of co-stimulatory molecules CD2-CD58 are important in the early stage of an immune response, and increased expression of these co-stimulatory molecules is observed in the synovial region of joints in rheumatoid arthritis (RA) patients. A CD2 epitope region that binds to CD58 was grafted on to sunflower trypsin inhibitor (SFTI) template structure to inhibit CD2-CD58 PPI. The peptide was incorporated with an organic moiety dibenzofuran (DBF) in its structure. The designed peptidomimetic was studied for its ability to inhibit CD2-CD58 interactions in vitro, and its thermal and enzymatic stability was evaluated. Stability studies indicated that the grafted peptidomimetic was stable against trypsin cleavage. In vivo studies using the collagen-induced arthritis (CIA) model in mice indicated that the peptidomimetic was able to slow down the progress of arthritis, an autoimmune disease in the mice model. These studies suggest that with the grafting of organic functional groups in the stable peptide template SFTI stabilizes the peptide structure, and these peptides can be used as a template to design stable peptides for therapeutic purposes.

In Silico Screening for Novel Tyrosine Kinase Inhibitors with Oxindole Scaffold as Anti-Cancer Agents: Design, QSAR Analysis, Molecular Docking and ADMET Studies

Recently, anti-cancer targeting drugs are directed against specific molecules and signaling pathways. These targeting agents have reasonable specificity, efficacy and less side effects. Tyrosine kinases, which play an essential role in growth factor signaling regulation, are significant targets in this type of therapy. Synthesized numerous tyrosine-kinase inhibitors (TKIs), such as substituted indolin-2-ones, are effective as anti-tumor and anti-leukemia agents. In this study, a series of novel substituted indolin-2-ones were studied as kinase inhibitor analogs through quantitative structure–activity relationship (QSAR) analysis. Two chemometrics methods, such as multiple linear regression (MLR) and partial least squares combined with genetic algorithm for variable selection (GA-PLS), were employed to establish relationships between structural characteristics and kinase inhibitory activity of used oxindole analogs. The GA-PLS was developed as the best predictor and validated QSAR model. The data set compounds were also studied by molecular docking to investigate their binding mechanism in the active site of tyrosine kinase enzyme. According to the information obtained from QSAR models and molecular docking analysis, 40 new potent lead compounds with novel structural features were introduced. Molecular docking, drug-likeness rules, ADMET analysis, bioavailability, toxicity prediction and target identification were carried out on the newly designed oxindoles to elucidate fundamental structural properties that affect their inhibitory activity.

Focused dilemmas in the diagnosis and treatment of digestive system neuroendocrine neoplasms

Neuroendocrine neoplasms (NENs) originate from neuroendocrine cells diffusely distributed throughout the body. NENs are liable to be misdiagnosed and missed clinically, which brings great difficulties to clinical diagnosis and treatment, and seriously affects their prognosis. This article will introduce the difficulties and hot spots in the diagnosis and treatment of NENs from the aspects of laboratory and pathological examinations, anatomical and functional imaging examinations, including endoscopy, peptide receptor radionuclide therapy, somatostatin analogue and immune checkpoint inhibitor therapy. It aims to provide more ideas for the early diagnosis and early treatment of NENs, standardized diagnosis and treatment of high-grade NENs in the middle and late stages, and to provide more strategies for clinical multidisciplinary experts towards the management of focused dilemmas in NENs.

Retrospective evaluation of microalbuminuria and GFR levels of diabetic patients taking DPP-4 Inhibitor, GLP-1 Analog, or SGLT-2 Inhibitor

Background: In our study, we determined the changes in microalbuminuria and gfr (glomerular filtration rate) values, which are important for diabetic nephropathy, within 1 year after starting treatment in our patients taking DPP-4 inhibitor (linagliptin), GLP-1 analog (exenatide) and SGLT-2 inhibitor (empagliflozin).
 
 Material and Methods: We evaluated the urea, creatinine, gfr and microalbuminuria levels of our patients who were treated with linagliptin, exenatide and empagliflozin on their 0th, 6th and 12th month visits. We included patients who were followed up for nephropathy for at least 1 year after starting treatment in each drug group.
 
 Results: When the 0th and 12th month GFR values of our 98 patients who were prescribed linagliptin were compared, an increase of 4.57% was detected (p0.05). While no statistically significant difference was found in the 0th and 12th month GFR follow-ups of our 97 patients using exenatide (p>0.05); in this group, it was determined that the microalbuminuria decreased significantly in 12 months in 33 of our patients who could be followed up in terms of microalbuminuria (p0.05); however, it was determined that microalbuminuria decreased significantly at the end of 1 year in our 79 patients who could be followed up for microalbuminuria in this group (p

405 Role of Glycans in Cancer Associated Fibroblast-Derived Exosome Immunoregulation

OBJECTIVES/GOALS: To identify the role of sialoglycans in the mechanisms underlying cancer-associated fibroblast-derived exosomes (CAFEX) immuno-regulatory properties. The central hypothesis is that CAFEX manipulates the immune response to allow immunoevasion and glycomic approaches can identify the signaling factors involved. METHODS/STUDY POPULATION: Cancer associated fibroblasts (CAFs) were isolated from primary head and neck tumors, expanded, characterized and cryopreserved prior to experimentation and isolation of CAFEX. Sialoglycan expression was determined using lectin-specific staining of cells and bead-captured CAFEX in combination with flow cytometry analysis. Siglec expression and expression of M2-macrophage markers were also determined by flow cytometry analysis and cytokine bead arrays. Inhibition studies involved either the enzymatic removal of cell-surface sialoglycans or alternatively, a specific small molecule analog inhibitor of sialoglycan transferases. RESULTS/ANTICIPATED RESULTS: Both CAFs and CAFEX expressed abundant levels of cell-surface sialoglycans. CAFEX induced an M2-macrophage phenotype in primary monocytes, based on surface marker expression and cytokine secretion profiles. The induction of the M2 phenotype in macrophages was attenuated upon the removal of sialoglycans from the surface of CAFEX either by enzymatic removal or via a small molecule inhibitor. CAFEX were also able to directly bind members of the Siglec family, which are sialoglycan specific lectin receptors expressed on immune cells, including macrophages. DISCUSSION/SIGNIFICANCE: Collectively, these studies suggest that surface presentation of sialoglycans by CAFEX may induce an immunosuppressive phenotype in monocytes/macrophages. Consequently, this may be a novel mechanism by which cells within the tumor bed facilitate immunoevasion during tumor progression.

Sofosbuvir adsorption onto activated carbon derived from argan shell residue: Optimization, kinetic, thermodynamic and theoretical approaches

Activated carbon (AC) obtained from argan shell residue (ASR) was studied for the removal of the drug sofosbuvir from the liquid phase. The operating parameters including pH, adsorbent dosage, and sofosbuvir concentration were optimized with a Box Behnken design (B.B.D) using the response surface methodology (R.S.M.) approach. The results show a maximum removal at room temperaure of 54.5 mg.g−1 (99.63%) of sofosbuvir at pH 7, AC mass 0.5 g.L−1, and sofosbuvir concentration 0.1 mM. The adsorption investigation indicated that the process is well described by Freundlish isotherm model with R2 = 98.2%. Thermodynamic parameters, including ΔG°, ΔH°, and ΔS°, were also studied. The adsorption kinetics follows pseudo 2nd order. The produced AC was characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), and differential thermal analysis (DTA). To better understand the adsorption mechanism and dynamic behavior of sofosbuvir, an oral nucleoside analog and potent inhibitor of the hepatitis C virus, we applied the density functional theory (DFT) approach, Monte Carlo simulations, and molecular dynamics (MD) simulations to examine the impact of different adsorption sites, adsorption energy, and mechanism. The moisture content H(%), volatile matter Vm, and iodine value in AC were also evaluated. The results indicate that argan shell-based AC is a promising candidate for the adsorption procedure due to its low cost and higher adsorption capacity for sofosbuvir removal from the liquid phase.

The possible ameliorative role of Moringa oleifera seed oil on sofosbuvir-induced nephrotoxicity in albino rats; histopathological, immunohistochemical and biochemical studies

BackgroundHepatitis C is a liver infection caused by the hepatitis C virus (HCV). It can cause both acute and chronic hepatitis. Sofosbuvir (sofo) is a nucleotide analog inhibitor of HCV NS5B polymerase used to treat chronic hepatitis C infection as a component of a combination of antiviral treatment regimen. Many side effects of sofo were reported in different mammalian organs including kidney. Moringa oleifera (MO) is one of the medicinal plants which have many pharmacological activities and nutritional applications due to its rich phytonutrients content. This study aimed to investigate the possible ameliorative effect of MO seed oil against nephrotoxicity induced by sofo in adult male albino rats. The experimental animals were divided equally into four groups. Group I: animals were served as control. Group II: animals were orally given MO oil (2 ml/kg/day). Group III: animals were orally administered with sofo (36 mg/kg/day). Group IV: animals were orally given sofo then after 2 h they were given MO oil (with the same previous doses). All doses were daily given to the animals for eight weeks. At the end of the experiments, animals were sacrificed and sera were collected to determine urea, creatinine and malondialdehyde levels and catalase activity. Kidneys were removed out and prepared for both the histological and immunohistochemical studies.ResultsSofo-treated animals showed many pathological changes; damaged glomeruli and degenerated renal tubules with vacuolated lining epithelial cells contain pyknotic nuclei. In addition, leukocytic infiltration, congested blood vessels and hemorrhage were seen. Caspase-3 and PCNA were expressed in a large number of cells in the same group. Moreover, a significant increase in urea, creatinine and malondialdehyde levels was recorded as well as a significant decrease in catalase activity. Co-treatment of MO oil with sofo effectively counteracted the observed adverse effects. It attenuated the histological picture of the kidney, significantly ameliorated urea, creatinine and malondialdehyde levels and catalase activity and restored the normal expressions of caspase-3 and PCNA.ConclusionsMoringa oleifera oil can ameliorate nephrotoxicity induced by sofo via its antioxidant, anti-inflammatory and anti-apoptotic properties.

Synthesis and Characterization of Transition-State Analogue Inhibitors against Human DNA Methyltransferase 1.

Hypermethylation of CpG regions by human DNA methyltransferase 1 (DNMT1) silences tumor-suppression genes, and inhibition of DNMT1 can reactivate silenced genes. The 5-azacytidines are approved inhibitors of DNMT1, but their mutagenic mechanism limits their utility. A synthon approach from the analogues of S-adenosylhomocysteine, methionine, and deoxycytidine recapitulated the chemical features of the DNMT1 transition state in the synthesis of 16 chemically stable transition-state mimics. Inhibitors causing both full and partial inhibition of purified DNMT1 were characterized. The inhibitors show modest selectivity for DNMT1 versus DNMT3b. Active-site docking predicts inhibitor interactions with S-adenosyl-l-methionine and deoxycytidine regions of the catalytic site, validated by direct binding analysis. Inhibitor action with purified DNMT1 is not reflected in cultured cells. A partial inhibitor activated cellular DNA methylation, and a full inhibitor had no effect on cellular DNA methylation. These compounds provide chemical access to a new family of noncovalent DNMT chemical scaffolds for use in DNA methyltransferases.

Backbone-Anchoring, Solid-Phase Synthesis Strategy To Access a Library of Peptidouridine-Containing Small Molecules.

Nucleoside diphosphate sugar (NDP-sugar) substrates provide the inspiration for nucleoside analogue inhibitor scaffolds. By employing solid-phase synthesis, we provide a method to access a library of peptidouridine inhibitors with both minimal compound handling and purification steps. Specifically, this strategy is exemplified by generating uridine diphosphate sugar (UDP-sugar) mimics, which allow for compound elaboration by altering the dipeptide composition, the N-terminal linkage, and a pendant aryl group. To exemplify the versatility, 41 unique nucleoside analogues are presented.

Modulation of the monomer-dimer equilibrium and catalytic activity of SARS-CoV-2 main protease by a transition-state analog inhibitor

The role of dimer formation for the onset of catalytic activity of SARS-CoV-2 main protease (MProWT) was assessed using a predominantly monomeric mutant (MProM). Rates of MProWT and MProM catalyzed hydrolyses display substrate saturation kinetics and second-order dependency on the protein concentration. The addition of the prodrug GC376, an inhibitor of MProWT, to MProM leads to an increase in the dimer population and catalytic activity with increasing inhibitor concentration. The activity reaches a maximum corresponding to a dimer population in which one active site is occupied by the inhibitor and the other is available for catalytic activity. This phase is followed by a decrease in catalytic activity due to the inhibitor competing with the substrate. Detailed kinetics and equilibrium analyses are presented and a modified Michaelis-Menten equation accounts for the results. These observations provide conclusive evidence that dimer formation is coupled to catalytic activity represented by two equivalent active sites.

Hybrid QM/MM Free-Energy Evaluation of Drug-Resistant Mutational Effect on the Binding of an Inhibitor Indinavir to HIV-1 Protease.

A human immunodeficiency virus-1 (HIV-1) protease is a homodimeric aspartic protease essential for the replication of HIV. The HIV-1 protease is a target protein in drug discovery for antiretroviral therapy, and various inhibitor molecules of transition state analogues have been developed. However, serious drug-resistant mutants have emerged. For understanding the molecular mechanism of the drug resistance, an accurate examination of the impacts of the mutations on ligand binding and enzymatic activity is necessary. Here, we present a molecular simulation study on the ligand binding of indinavir, a potent transition state analogue inhibitor, to the wild-type protein and a V82T/I84V drug-resistant mutant of the HIV-1 protease. We employed a hybrid ab initio quantum mechanical/molecular mechanical (QM/MM) free-energy optimization technique which combines a highly accurate QM description of the ligand molecule and its interaction with statistically ample conformational sampling of the MM protein environment by long-time molecular dynamics simulations. Through the free-energy calculations of protonation states of catalytic groups at the binding pocket and of the ligand-binding affinity changes upon the mutations, we successfully reproduced the experimentally observed significant reduction of the binding affinity upon the drug-resistant mutations and elucidated the underlying molecular mechanism. The present study opens the way for understanding the molecular mechanism of drug resistance through the direct quantitative comparison of ligand binding and enzymatic reaction with the same accuracy.

Positional scanning of natural product hispidol’s ring-B: discovery of highly selective human monoamine oxidase-B inhibitor analogues downregulating neuroinflammation for management of neurodegenerative diseases

Multifunctional molecules might offer better treatment of complex multifactorial neurological diseases. Monoaminergic pathways dysregulation and neuroinflammation are common convergence points in diverse neurodegenerative and neuropsychiatric disorders. Aiming to target these diseases, polypharmacological agents modulating both monoaminergic pathways and neuroinflammatory were addressed. A library of analogues of the natural product hispidol was prepared and evaluated for inhibition of monoamine oxidases (MAOs) isoforms. Several molecules emerged as selective potential MAO B inhibitors. The most promising compounds were further evaluated in vitro for their impact on microglia viability, induced production of proinflammatory mediators and MAO-B inhibition mechanism. Amongst tested compounds, 1p was a safe potent competitive reversible MAO-B inhibitor and inhibitor of microglial production of neuroinflammatory mediators; NO and PGE2. In-silico study provided insights into molecular basis of the observed selective MAO B inhibition. This study presents compound 1p as a promising lead compound for management of neurodegenerative disease.

Discovery of Dual TAF1-ATR Inhibitors and Ligand-Induced Structural Changes of the TAF1 Tandem Bromodomain.

Bromodomains regulate chromatin remodeling and gene transcription through recognition of acetylated lysines on histones and other proteins. Bromodomain-containing protein TAF1, a subunit of general transcription factor TFIID, initiates preinitiation complex formation and cellular transcription. TAF1 serves as a cofactor for certain oncogenic transcription factors and is implicated in regulating the p53 tumor suppressor. Therefore, TAF1 is a potential target to develop small molecule therapeutics for diseases arising from dysregulated transcription, such as cancer. Here, we report the ATR kinase inhibitor AZD6738 (Ceralasertib) and analogues thereof as bona fide inhibitors of TAF1. Crystallographic and small-angle X-ray scattering studies established that newly identified and previously reported inhibitors stabilize distinct structural states of the TAF1 tandem bromodomain through "open-closed" transitions and dimerization. Combined with functional studies on p53 signaling in cancer cell lines, the data provide new insights into the feasibility and challenges of TAF1 inhibitors as chemical probes and therapeutics.

Termination of Poly-N-acetylglucosamine (PNAG) Polymerization with N-Acetylglucosamine Analogues.

Bacteria require polysaccharides for structure, survival, and virulence. Despite their central role in microbiology, few tools are available to manipulate their production. In E. coli, the glycosyltransferase complex PgaCD produces poly-N-acetylglucosamine (PNAG), an extracellular matrix polysaccharide required for biofilm formation. We report that C6-substituted (H, F, N3, SH, NH2) UDP-GlcNAc substrate analogues are inhibitors of PgaCD. In vitro, the inhibitors cause PNAG chain termination, consistent with the mechanism of PNAG polymerization from the nonreducing terminus. In vivo, expression of the GlcNAc-1-kinase NahK in E. coli provided a non-native GlcNAc salvage pathway that produced the UDP-GlcNAc analogue inhibitors in situ. The 6-fluoro and 6-deoxy derivatives were potent inhibitors of biofilm formation in the transformed strain, providing a tool to manipulate this key exopolysaccharide. Characterization of the UDP-GlcNAc pool and quantification of PNAG generation support PNAG termination as the primary in vivo mechanism of biofilm inhibition by 6-fluoro UDP-GlcNAc.

Pyrazole and Triazole Derivatives as Mycobacterium tuberculosis UDP-Galactopyranose Inhibitors.

UDP-galactopyranose mutase (UGM) is an essential enzyme involved in the bacterial cell wall synthesis, and is not present in mammalian cells. Thus, UGM from Mycobacterium tuberculosis (Mtb) represents a novel and attractive drug target for developing antituberculosis agents. A pyrazole-based compound, MS208, was previously identified as a mixed inhibitor of MtbUGM which targets an allosteric site. To understand more about the structure activity relationship around the MS208 scaffold as a MtbUGM inhibitor, thirteen pyrazoles and triazole analogues were synthesized and tested against both MtbUGM and Mycobacterium tuberculosis in vitro. While the introduced structural modifications to MS208 did not improve the antituberculosis activity, most of the compounds showed MtbUGM inhibitory activity. Interestingly, the pyrazole derivative DA10 showed a competitive model for MtbUGM inhibition with improved Ki value of 51 ± 4 µM. However, the same compound did not inhibit the growth of Mycobacterium tuberculosis.