Mechanism-based Inhibitors Research Articles

Sulfoxides of sulfur-containing amino acids are suicide substrates of Citrobacter freundii methionine γ-lyase. Structural bases of the enzyme inactivation

Interactions of Citrobacter freundii methionine γ-lyase (MGL) with sulfoxides of typical substrates were investigated. It was found that sulfoxides are suicide substrates of the enzyme. The products of the β- and γ-elimination reactions of sulfoxides, thiosulfinates, oxidize three cysteine residues of the enzyme. Three-dimensional structures of MGL inactivated by dimethyl thiosulfinate and diethyl thiosulfinate were determined at 1.46 Å and 1.59 Å resolution. Analysis of the structures identified SH groups oxidized by thiosulfinates and revealed the structural bases of MGL inactivation. The extent of inactivation of MGL in the catalysis of the β-elimination reaction depends on the length of the «tail» at oxidized Cys115. Oxidation of Cys115 results in MGL incapable to catalyze the stage of methyl mercaptan elimination of the physiological reaction.

Two-Step Bioorthogonal Activity-Based Protein Profiling of Individual Human Proteasome Catalytic Sites.

Bioorthogonal chemistry allows the selective modification of biomolecules in complex biological samples. One application of this methodology is in two-step activity-based protein profiling (ABPP), a methodology that is particularly attractive where direct ABPP using fluorescent or biotinylated probes is ineffective. Herein we describe a set of norbornene-modified, mechanism-based proteasome inhibitors aimed to be selective for each of the six catalytic sites of human constitutive proteasomes and immunoproteasomes. The probes developed for β1i, β2i, β5c, and β5i proved to be useful two-step ABPs that effectively label their developed proteasome subunits in both Raji cell extracts and living Raji cells through inverse-electron-demand Diels-Alder (iEDDA) ligation. The compound developed for β1c proved incapable of penetrating the cell membrane, but effectively labels β1c in vitro. The compound developed for β2c proved not selective, but its azide-containing analogue LU-002c proved effective in labeling of β2c via azide-alkyne click ligation chemistry both in vitro and in situ. In total, our results contribute to the growing list of proteasome activity tools to include five subunit-selective activity-based proteasome probes, four of which report on proteasome activities in living cells.

A suicide inhibitor of nematode trehalose-6-phosphate phosphatases

Protein-based drug discovery strategies have the distinct advantage of providing insights into the molecular mechanisms of chemical effectors. Currently, there are no known trehalose-6-phosphate phosphatase (TPP) inhibitors that possess reasonable inhibition constants and chemical scaffolds amenable to convenient modification. In the present study, we subjected recombinant TPPs to a two-tiered screening approach to evaluate several diverse compound groups with respect to their potential as TPP inhibitors. From a total of 5452 compounds tested, N-(phenylthio)phthalimide was identified as an inhibitor of nematode TPPs with apparent Ki values of 1.0 μM and 0.56 μM against the enzymes from the zoonotic roundworms Ancylostoma ceylanicum and Toxocara canis, respectively. Using site-directed mutagenesis, we demonstrate that this compound acts as a suicide inhibitor that conjugates a strictly conserved cysteine residue in the vicinity of the active site of nematode TPPs. The anthelmintic properties of N-(phenylthio)phthalimide were assessed in whole nematode assays using larvae of the ascaroids T. canis and T. cati, as well as the barber’s pole worm Haemonchus contortus. The compound was particularly effective against each of the ascaroids with an IC50 value of 9.3 μM in the survival assay of T. cati larvae, whereas no bioactivity was observed against H. contortus.

Decomposition of enzyme kinetics equations

The problem of the splitting of singularly perturbed differential systems is invesigated and the application to the problem of suicide substrate kinetics is given.

Numerical Analysis of Time-Dependent Inhibition by MDMA.

Methylenedioxymethamphetamine (MDMA) is a known drug of abuse and schedule 1 narcotic under the Controlled Substances Act. Previous pharmacokinetic work on MDMA used classic linearization techniques to conclude irreversible mechanism-based inhibition of CYP2D6. The current work challenges this outcome by assessing the possibility of two alternative reversible kinetic inhibition mechanisms known as the quasi-irreversible (QI) model and equilibrium model (EM). In addition, progress curve experiments were used to investigate the residual metabolism of MDMA by liver microsomes and CYP2D6 baculosomes over incubation periods up to 30 minutes. These experiments revealed activity in a terminal linear phase at the fractional rates with respect to initial turnover of 0.0354 ± 0.0089 in human liver microsomes and 0.0114 ± 0.0025 in baculosomes. Numerical model fits to percentage of remaining activity (PRA) data were consistent with progress curve modeling results, wherein an irreversible inhibition pathway was found unnecessary for good fit scoring. Both QI and EM kinetic mechanisms fit the PRA data well, although in CYP2D6 baculosomes the inclusion of an irreversible inactivation pathway did not allow for convergence to a reasonable fit. The kinetic complexity accessible to numerical modeling has been used to determine that MDMA is not an irreversible inactivator of CYP2D6, and instead follows what can be generally referred to as slowly reversible inhibition. SIGNIFICANCE STATEMENT: The work herein describes the usage of computational models to delineate between irreversible and slowly reversible time-dependent inhibition. Such models are used in the paper to analyze MDMA and classify it as a reversible time-dependent inhibitor.

Protease Inhibitors for the Treatment of HIV/AIDS: Recent Advances and Future Challenges.

Acquired Immunodeficiency Syndrome (AIDS) is a chronic disease characterized by multiple life-threatening illnesses caused by a retro-virus, Human Immunodeficiency Virus (HIV). HIV infection slowly destroys the immune system and increases the risk of various other infections and diseases. Although, there is no immediate cure for HIV infection/AIDS, several drugs targeting various cruxes of HIV infection are used to slow down the progress of the disease and to boost the immune system. One of the key therapeutic strategies is Highly Active Antiretroviral Therapy (HAART) or ' AIDS cocktail' in a general sense, which is a customized combination of anti-retroviral drugs designed to combat the HIV infection. Since HAART's inception in 1995, this treatment was found to be effective in improving the life expectancy of HIV patients over two decades. Among various classes of HAART treatment regimen, Protease Inhibitors (PIs) are known to be widely used as a major component and found to be effective in treating HIV infection/AIDS. For the past several years, a variety of protease inhibitors have been reported. This review outlines the drug design strategies of PIs, chemical and pharmacological characteristics of some mechanism-based inhibitors, summarizes the recent developments in small molecule based drug discovery with HIV protease as a drug target. Further discussed are the pharmacology, PI drug resistance on HIV PR, adverse effects of HIV PIs and challenges/impediments in the successful application of HIV PIs as an important class of drugs in HAART regimen for the effective treatment of AIDS.

A Mass Spectrometric Assay of METTL3/METTL14 Methyltransferase Activity.

A variety of covalent modifications of RNA have been identified and demonstrated to affect RNA processing, stability, and translation. Methylation of adenosine at the N6 position (m6A) in messenger RNA (mRNA) is currently the most well-studied RNA modification and is catalyzed by the RNA methyltransferase complex METTL3/METTL14. Once generated, m6A can modulate mRNA splicing, export, localization, degradation, and translation. Although potent and selective inhibitors exist for several members of the Type I S-adenosylmethionine (SAM)-dependent methyltransferase family, no inhibitors have been reported for METTL3/METTL14 to date. To facilitate drug discovery efforts, a sensitive and robust mass spectrometry-based assay for METTL3/METTL14 using self-assembled monolayer desorption/ionization (SAMDI) technology has been developed. The assay uses an 11-nucleotide single-stranded RNA compared to a previously reported 27-nucleotide substrate. IC50 values of mechanism-based inhibitors S-adenosylhomocysteine (SAH) and sinefungin (SFG) are comparable between the SAMDI and radiometric assays that use the same substrate. This work demonstrates that SAMDI technology is amenable to RNA substrates and can be used for high-throughput screening and compound characterization for RNA-modifying enzymes.

Identification and Optimization of Mechanism-Based Fluoroallylamine Inhibitors of Lysyl Oxidase-like 2/3.

Lysyl oxidase-like 2 (LOXL2) is a secreted enzyme that catalyzes the formation of cross-links in extracellular matrix proteins, namely, collagen and elastin, and is indicated in fibrotic diseases. Herein, we report the identification and subsequent optimization of a series of indole-based fluoroallylamine inhibitors of LOXL2. The result of this medicinal chemistry campaign is PXS-5120A (12k), a potent, irreversible inhibitor that is >300-fold selective for LOXL2 over LOX. PXS-5120A also shows potent inhibition of LOXL3, an emerging therapeutic target for lung fibrosis. Key to the development of this compound was the utilization of a compound oxidation assay. PXS-5120A was optimized to show negligible substrate activity in vitro for related amine oxidase family members, leading to metabolic stability. PXS-5120A, in a pro-drug form (PXS-5129A, 12o), displayed anti-fibrotic activity in models of liver and lung fibrosis, thus confirming LOXL2 as an important target in diseases where collagen cross-linking is implicated.

Validation of a 3-h Sampling Interval to Assess Variability in Cytochrome P450 3A Phenotype and the Impact of Induction and Mechanism-Based Inhibition Using Midazolam as a Probe Substrate.

Background: Drug probe phenotyping is used extensively in academic and industry research to evaluate cytochrome P450 (CYP) phenotype in order to account for sources of between- and within- subject variability in metabolic clearance. In terms of application, CYP3A is the most important drug metabolizing enzyme the most frequently studied. Currently, phenotyping studies for CYP3A involve the administration of midazolam and collection of timed blood samples up to 24-48 hours in order to determine an area under the plasma concentration time curve (AUC). The key challenge that limits the use of midazolam-based phenotyping for CYP3A in academic research settings and preclude the use of this approach in a clinical setting is the logistical burden of collecting frequent blood samples for up to 48 h post dose following the administration of a probe drug ± an interacting drug. Aim: The current study sought to validate if a reduced sampling interval could be used to accurately define both between-subject variability in CYP3A phenotype and the magnitude of changes in CYP3A activity due to either induction or mechanism-based inhibition. Methods: The area under the curve (AUC) for midazolam was assessed under baseline, induction (7 days rifampin, 300 mg daily) and, following a washout period of 4 days, mechanism based inhibition (3 days clarithromycin, 250 mg daily) conditions in a cohort of 30 health males. The capacity of normalized reduced sampling interval AUCs measured over 0 to 1, 0 to 2, 0 to 3, and 0 to 4 h to accurately define the AUC0-6 was evaluated with respect to precision (R2 for correlation), bias (slope of normalized correlation), agreement (Bland Altman analysis) and proportional bias (linear regression of Bland Altman parameters). Results: Robust concordance was observed between the AUC calculated from PK collection intervals of 0 to 3 and 0 to 6 h in terms of both the measurement of between-subject variability in midazolam AUC and changes in midazolam AUC due to induction and mechanism-based inhibition of CYP3A4. Conclusion: On this basis, it is proposed that a 3-h assessment of midazolam AUC (AUC0-3) represents a viable strategy to reduce the logistical burden associated with the assessment of CYP3A phenotype.

Aspergillus nidulans thermostable arginine deiminase-Dextran conjugates with enhanced molecular stability, proteolytic resistance, pharmacokinetic properties and anticancer activity

The potential anticancer activity of arginine deiminase (ADI) via deimination of l-arginine into citrulline has been extensively verified against various arginine-auxotrophic tumors, however, the higher antigenicity, structural instability and in vivo proteolysis are the major challenges that limit this enzyme from further clinical implementation. Since, this clinically applied enzyme was derived from Mycobacterium spp, thus, searching for ADI from eukaryotic microbes “especially thermophilic fungi” could have a novel biochemical, conformational and catalytic properties. Aspergillus nidulans ADI was purified with 5.3 folds, with molecular subunit structure 48 kDa and entire molecular mass 120 kDa, ensuring its homotrimeric identity. The peptide fingerprinting analysis revealing the domain Glu95-Gly96-Gly97 as the conserved active site of A. nidulans ADI, with higher proximity to Mycobacterium ADI clade IV. In an endeavor to fortify the structural stability and anticancer activity of A. nidulans ADI, the enzyme was chemically modified with dextran. The optimal activity of Dextran-ADI conjugates was determined at 0.08:20 M ratio of ADI: Dextran, with an overall increase to ADI molecular subunit mass to ˜100 kDa. ADI was conjugated with dextran via the ε-amino groups interaction of surface lysine residues of ADI. The resistance of Dextran-ADI conjugate to proteolysis had been increased by 2.5 folds to proteinase K and trypsin, suggesting the shielding of >50% of ADI surface proteolytic recognition sites. The native and Dextran-ADI conjugates have the same optimum reaction temperature (37 °C), reaction pH and pH stability (7.0–8.0) with dependency on K+ ions as a cofactor. Dextran-ADI conjugates exhibited a higher thermal stability by ˜ 2 folds for all the tested temperatures, ensuring the acquired structural and catalytic stability upon dextran conjugation. Dextran conjugation slightly protect the reactive amino and thiols groups of surface amino acids of ADI from amino acids suicide inhibitors. The affinity of ADI was increased by 5.3 folds to free L-arginine with a dramatic reduction in citrullination of peptidylarginine residues upon dextran conjugation. The anticancer activity of ADI to breast (MCF-7), liver (HepG-2) and colon (HCT8, HT29, DLD1 and LS174 T) cancer cell lines was increased by 1.7 folds with dextran conjugation in vitro. Pharmacokinetically, the half-life time of ADI was increased by 1.7 folds upon dextran conjugation, in vivo. From the biochemical and hematological parameters, ADIs had no signs of toxicity to the experimental animals. In addition to the dramatic reduction of L-arginine in serum, citrulline level was increased by 2.5 folds upon dextran conjugation of ADI. This is first report exploring thermostable ADI from thermophilic A. nidulans with robust structural stability, catalytic efficiency and proteolytic resistance.

Structural Insights into the Interaction of Cytochrome P450 3A4 with Suicide Substrates: Mibefradil, Azamulin and 6',7'-Dihydroxybergamottin.

Human cytochrome P450 3A4 (CYP3A4) is the most important drug-metabolizing enzyme. Some drugs and natural compounds can act as suicide (mechanism-based) inactivators of CYP3A4, leading to unanticipated drug-drug interactions, toxicity and therapeutic failures. Despite significant clinical and toxicological implications, the mechanism-based inactivation remains incompletely understood. This study provides the first direct insights into the interaction of CYP3A4 with three suicide substrates: mibefradil, an antihypertensive drug quickly withdrawn from the market; a semi-synthetic antibiotic azamulin; and a natural furanocoumarin, 6′,7′-dihydroxybergamottin. Novel structural findings help better understand the suicide substrate binding and inhibitory mechanism, and can be used to improve the predictability of the binding ability, metabolic sites and inhibitory/inactivation potential of newly developed drugs and other chemicals relevant to public health.

Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis.

Activated endocytosis of extracellular macromolecules and their intracellular trafficking to lysosomes is an essential metabolic mechanism in cancer cells during their rapid proliferation. Cancer cells reuse a vast amount of N-acetylglucosamine (GlcNAc) supplied from the GlcNAc salvage pathway for the accelerated synthesis of a pivotal uridine diphosphate (UDP)-GlcNAc. A method to inactivate key glycosidases in lysosomes could critically contribute to the development of potent anticancer therapy. Here we demonstrate that "nanosomes" made of core metals covered by an antiadhesive mixed self-assembled monolayer allow for avoiding nonspecific surface protein corona and targeted molecular delivery through activated endocytosis. Nanosomes carrying suicide substrates showed that lysosomal glycosidases such as β-hexosaminidase and β-galactosidase in cancer cells are promising targets for novel anticancer therapeutic nanomedicine that induce apoptotic cell death through lysosomal membrane permeabilization. The advantage of this method is evident because multivalent surface loading by antiadhesive nanosomes makes it possible to highlight "weak interactions" such as carbohydrate-lectin interactions independent of surface protein corona.

Antiadhesive Nanosomes Facilitate Targeting of the Lysosomal GlcNAc Salvage Pathway through Derailed Cancer Endocytosis

AbstractActivated endocytosis of extracellular macromolecules and their intracellular trafficking to lysosomes is an essential metabolic mechanism in cancer cells during their rapid proliferation. Cancer cells reuse a vast amount of N‐acetylglucosamine (GlcNAc) supplied from the GlcNAc salvage pathway for the accelerated synthesis of a pivotal uridine diphosphate (UDP)‐GlcNAc. A method to inactivate key glycosidases in lysosomes could critically contribute to the development of potent anticancer therapy. Here we demonstrate that “nanosomes” made of core metals covered by an antiadhesive mixed self‐assembled monolayer allow for avoiding nonspecific surface protein corona and targeted molecular delivery through activated endocytosis. Nanosomes carrying suicide substrates showed that lysosomal glycosidases such as β‐hexosaminidase and β‐galactosidase in cancer cells are promising targets for novel anticancer therapeutic nanomedicine that induce apoptotic cell death through lysosomal membrane permeabilization. The advantage of this method is evident because multivalent surface loading by antiadhesive nanosomes makes it possible to highlight “weak interactions” such as carbohydrate–lectin interactions independent of surface protein corona.

Structure Kinetics Relationships and Molecular Dynamics Show Crucial Role for Heterocycle Leaving Group in Irreversible Diacylglycerol Lipase Inhibitors

Drug discovery programs of covalent irreversible, mechanism-based enzyme inhibitors often focus on optimization of potency as determined by IC50-values in biochemical assays. These assays do not allow the characterization of the binding activity (Ki) and reactivity (kinact) as individual kinetic parameters of the covalent inhibitors. Here, we report the development of a kinetic substrate assay to study the influence of the acidity (pKa) of heterocyclic leaving group of triazole urea derivatives as diacylglycerol lipase (DAGL)-α inhibitors. Surprisingly, we found that the reactivity of the inhibitors did not correlate with the pKa of the leaving group, whereas the position of the nitrogen atoms in the heterocyclic core determined to a large extent the binding activity of the inhibitor. This finding was confirmed and clarified by molecular dynamics simulations on the covalently bound Michaelis–Menten complex. A deeper understanding of the binding properties of covalent serine hydrolase inhibitors is expected to aid in the discovery and development of more selective covalent inhibitors.

Targeting Mitochondrial Proline Dehydrogenase with a Suicide Inhibitor to Exploit Synthetic Lethal Interactions with p53 Upregulation and Glutaminase Inhibition.

Proline dehydrogenase (PRODH) is a p53-inducible inner mitochondrial membrane flavoprotein linked to electron transport for anaplerotic glutamate and ATP production, most critical for cancer cell survival under microenvironmental stress conditions. Proposing that PRODH is a unique mitochondrial cancer target, we structurally model and compare its cancer cell activity and consequences upon exposure to either a reversible (S-5-oxo: S-5-oxo-2-tetrahydrofurancarboxylic acid) or irreversible (N-PPG: N-propargylglycine) PRODH inhibitor. Unlike 5-oxo, the suicide inhibitor N-PPG induces early and selective decay of PRODH protein without triggering mitochondrial destruction, consistent with N-PPG activation of the mitochondrial unfolded protein response. Fly and breast tumor (MCF7)-xenografted mouse studies indicate that N-PPG doses sufficient to phenocopy PRODH knockout and induce its decay can be safely and effectively administered in vivo Among breast cancer cell lines and tumor samples, PRODH mRNA expression is subtype dependent and inversely correlated with glutaminase (GLS1) expression; combining inhibitors of PRODH (S-5-oxo and N-PPG) and GLS1 (CB-839) produces additive if not synergistic loss of cancer cell (ZR-75-1, MCF7, DU4475, and BT474) growth and viability. Although PRODH knockdown alone can induce cancer cell apoptosis, the anticancer potential of either reversible or irreversible PRODH inhibitors is strongly enhanced when p53 is simultaneously upregulated by an MDM2 antagonist (MI-63 and nutlin-3). However, maximum anticancer synergy is observed in vitro when the PRODH suicide inhibitor, N-PPG, is combined with both GLS1-inhibiting and a p53-upregulating MDM2 antagonist. These findings provide preclinical rationale for the development of N-PPG-like PRODH inhibitors as cancer therapeutics to exploit synthetic lethal interactions with p53 upregulation and GLS1 inhibition.

Bergamottin can be used to assess CYP3A-mediated intestinal first-pass metabolism without affecting P-glycoprotein-mediated efflux in rats

1. We investigated whether bergamottin would be useful for evaluating CYP3A-mediated intestinal metabolism in rats utilising its characteristics as a mechanism-based inhibitor of CYP3A.2. Buspirone and fexofenadine, probe substrates of CYP3A and P-glycoprotein (P-gp), respectively, were orally co-administered to rats with bergamottin (2.5 mg/kg) or orally administered 2 h after bergamottin pre-treatment. The effect of bergamottin pre-treatment on hepatic CYP3A specifically was investigated with intravenous administration of buspirone. The kobs of bergamottin for CYP3A was calculated based on the portal unbound Cmax.3. Co-administration of bergamottin significantly increased the AUC0–inf for buspirone and fexofenadine by 1.6-fold and 1.7-fold, respectively, indicating that bergamottin inhibited both CYP3A and P-gp.4. Bergamottin pre-treatment significantly elevated the AUC0–inf of oral buspirone by 3.7-fold but exerted no effect on the pharmacokinetics of intravenous buspirone, indicating that bergamottin pre-treatment selectively inhibited CYP3A-mediated intestinal metabolism without affecting the hepatic CYP3A. These findings were supported by the result that the kobs (0.00000118 min−1) of bergamottin for CYP3A was lower than the kdeg (0.0005 min−1) for CYP3A. Furthermore, bergamottin pre-treatment did not affect the pharmacokinetics of oral fexofenadine, suggesting that P-gp was not influenced.5. These profiles of bergamottin enable the convenient assessment of CYP3A-mediated intestinal metabolism.

Identification of O-acetylhomoserine sulfhydrylase, a putative enzyme responsible for methionine biosynthesis in Clostridioides difficile: Gene cloning and biochemical characterizations.

O-acetylhomoserine sulfhydrylase (OAHS) is a pyridoxal 5'-phosphate-dependent enzyme involved in microbial methionine biosynthesis. In this study, we report gene cloning, protein purification, and some biochemical characteristics of OAHS from Clostridioides difficile. The enzyme is a tetramer with molecular weight of 185 kDa. It possesses a high activity in the reaction of L-homocysteine synthesis, comparable to reported activities of OAHSes from other sources. OAHS activity is inhibited by metabolic end product L-methionine. L-Propargylglycine was found to be a suicide inhibitor of the enzyme. Substrate analogue Nγ -acetyl-L-2,4-diaminobutyric acid is a competitive inhibitor of OAHS with Ki = 0.04 mM. Analysis of C. difficile genome allows to suggest that the bacterium uses the way of direct sulfhydrylation for the synthesis of L-methionine. The data obtained may provide the basis for further study of the role of OAHS in the pathogenic bacterium and the development of potential inhibitors.

Population Structure, Molecular Epidemiology, and β-Lactamase Diversity among Stenotrophomonas maltophilia Isolates in the United States.

Stenotrophomonas maltophilia is a Gram-negative, nonfermenting, environmental bacillus that is an important cause of nosocomial infections, primarily associated with the respiratory tract in the immunocompromised population. Aiming to understand the population structure, microbiological characteristics and impact of allelic variation on β-lactamase structure and function, we collected 130 clinical isolates from across the United States. Identification of 90 different sequence types (STs), of which 63 are new allelic combinations, demonstrates the high diversity of this species. The majority of the isolates (45%) belong to genomic group 6. We also report excellent activity of the ceftazidime-avibactam and aztreonam combination, especially against strains recovered from blood and respiratory infections for which the susceptibility is higher than the susceptibility to trimethoprim-sulfamethoxazole, considered the "first-line" antibiotic to treat S. maltophilia Analysis of 73 blaL1 and 116 blaL2 genes identified 35 and 43 novel variants of L1 and L2 β-lactamases, respectively. Investigation of the derived amino acid sequences showed that substitutions are mostly conservative and scattered throughout the protein, preferentially affecting positions that do not compromise enzyme function but that may have an impact on substrate and inhibitor binding. Interestingly, we detected a probable association between a specific type of L1 and L2 and genomic group 6. Taken together, our results provide an overview of the molecular epidemiology of S. maltophilia clinical strains from the United States. In particular, the discovery of new L1 and L2 variants warrants further study to fully understand the relationship between them and the β-lactam resistance phenotype in this pathogen.IMPORTANCE Multiple antibiotic resistance mechanisms, including two β-lactamases, L1, a metallo-β-lactamase, and L2, a class A cephalosporinase, make S. maltophilia naturally multidrug resistant. Thus, infections caused by S. maltophilia pose a big therapeutic challenge. Our study aims to understand the microbiological and molecular characteristics of S. maltophilia isolates recovered from human sources. A highlight of the resistance profile of this collection is the excellent activity of the ceftazidime-avibactam and aztreonam combination. We hope this result prompts controlled and observational studies to add clinical data on the utility and safety of this therapy. We also identify 35 and 43 novel variants of L1 and L2, respectively, some of which harbor novel substitutions that could potentially affect substrate and/or inhibitor binding. We believe our results provide valuable knowledge to understand the epidemiology of this species and to advance mechanism-based inhibitor design to add to the limited arsenal of antibiotics active against this pathogen.

S-3-Carboxypropyl-l-cysteine specifically inhibits cystathionine γ-lyase–dependent hydrogen sulfide synthesis

Hydrogen sulfide (H2S) is a gaseous signaling molecule, which modulates a wide range of mammalian physiological processes. Cystathionine γ-lyase (CSE) catalyzes H2S synthesis and is a potential target for modulating H2S levels under pathophysiological conditions. CSE is inhibited by propargylglycine (PPG), a widely used mechanism-based inhibitor. In this study, we report that inhibition of H2S synthesis from cysteine, but not the canonical cystathionine cleavage reaction catalyzed by CSE in vitro, is sensitive to preincubation of the enzyme with PPG. In contrast, the efficacy of S-3-carboxpropyl-l-cysteine (CPC) a new inhibitor described herein, was not dependent on the order of substrate/inhibitor addition. We observed that CPC inhibited the γ-elimination reaction of cystathionine and H2S synthesis from cysteine by human CSE with Ki values of 50 ± 3 and 180 ± 15 μm, respectively. We noted that CPC spared the other enzymes involved either directly (cystathionine β-synthase and mercaptopyruvate sulfurtransferase) or indirectly (cysteine aminotransferase) in H2S biogenesis. CPC also targeted CSE in cultured cells, inhibiting transsulfuration flux by 80-90%, as monitored by the transfer of radiolabel from [35S]methionine to GSH. The 2.5 Å resolution crystal structure of human CSE in complex with the CPC-derived aminoacrylate intermediate provided a structural framework for the molecular basis of its inhibitory effect. In summary, our study reveals a previously unknown confounding effect of PPG, widely used to inhibit CSE-dependent H2S synthesis, and reports on an alternative inhibitor, CPC, which could be used as a scaffold to develop more potent H2S biogenesis inhibitors.

Passaging of an influenza A(H1N1)pdm09 virus in a difluoro sialic acid inhibitor selects for a novel, but unfit I106M neuraminidase mutant

An influenza A(H1N1)pdm09 and an influenza B virus were passaged in 3-fluoro(eq)-4-guanidino difluoro sialic acid (3Feq4Gu DFSA), an inhibitor of the influenza neuraminidase (NA) to determine whether resistant variants could be selected. 3Feq4Gu DFSA is a mechanism-based inhibitor, forming a covalent link to Y406 in the NA active site. Given its similarity to the natural substrate, sialic acid, we predicted resistant variants would be difficult to select. Yields of both viruses decreased with passaging, so that after 12 passages both viruses were only growing to low titers. Drug concentrations were decreased for another three passages. There was no difference in NA sensitivity in the MUNANA fluorescence-based assay, nor in plaque assays for the passaged virus stocks. All influenza B plaques were still wild type in all assays. There were isolated small diffuse plaques in the P15 pdm09 stock, which after purification had barely detectable NA or hemagglutinin (HA) activity. These had a novel non-active site I106M substitution in the NA gene, but unexpectedly no HA changes. The I106M may impact NA function through steric effects on the movement of the 150 and 430-loops. The I106M viruses had similar replication kinetics in MDCK cells as wild type viruses, but their ability to bind to and infect CHO–K1 cells expressing high levels of cell-bound mucin was compromised. The I106M substitution was unstable, with progeny rapidly reverting to wild type by three different mechanisms. Some had reverted to I106, some had V106, both with wild type NA and HA properties. A third group retained the I106M, but had a compensating R363K substitution, which regained almost wild type NA properties. These viruses now agglutinated chicken red blood cells (CRBCs) but unlike the I/V106, they rebound after elution at 37 °C. There were no mutations in the HA, but each phenotype correlated with the NA sequence. We propose that the activity in the I106M mutant is insufficient to remove carbohydrates from the virion HA and NA, sterically limiting HA access to CRBC receptors, thus resulting in poor HA binding.