Activity-Based Protein Profiling Reveals That Cephalosporins Selectively Active on Non-replicating Mycobacterium tuberculosis Bind Multiple Protein Families and Spare Peptidoglycan Transpeptidases

This work is licensed under a Creative Commons Attribution 4.0 International License.As β-lactams are reconsidered for the treatment of tuberculosis (TB), their targets are assumed to be peptidoglycan transpeptidases, as verified by adduct formation and kinetic inhibition of Mycobacterium tuberculosis (Mtb) transpeptidases by carbapenems active against replicating Mtb. Here, we investigated the targets of recently described cephalosporins that are selectively active against non-replicating (NR) Mtb. NR-active cephalosporins failed to inhibit recombinant Mtb transpeptidases. Accordingly, we used alkyne analogs of NR-active cephalosporins to pull down potential targets through unbiased activity-based protein profiling and identified over 30 protein binders. None was a transpeptidase. Several of the target candidates are plausibly related to Mtb’s survival in an NR state. However, biochemical tests and studies of loss of function mutants did not identify a unique target that accounts for the bactericidal activity of these beta-lactams against NR Mtb. Instead, NR-active cephalosporins appear to kill Mtb by collective action on multiple targets. These results highlight the ability of these β-lactams to target diverse classes of proteins.NIH U19AI111143Milstein Program in Chemical Biology and Translational MedicineWilliam Randolph Hearst TrustWelch Foundation (A-0015

Mécanisme d’inhibition des L,D-transpeptidases par les β-lactamines et les diazabicyclooctanes

Antibiotic resistance is a growing and global threat to human health that has led to an acute need for the development of new antibiotics. Elucidating the mechanism of inhibition of antibiotic targets is crucial for the development of more potent drugs. The essentiality of peptidoglycan and more than seventy years of successful use of β-lactams have made polymerization of this major cell wall component an attractive and validated target for drug development. Active-site serine Penicillin-Binding Proteins (PBPs) have long been considered as the only enzymes catalyzing the essential cross-linking step of peptidoglycan polymerization. The thesis explores inhibition of a distinct family of enzymes, the active-site cysteine L,D-transpeptidases (LDTs), that have a preponderant role in peptidoglycan synthesis in Mycobacterium tuberculosis. We show that the efficacy of LDT inhibition by β-lactams is primarily governed by the reactivity of the four-membered ring. We propose that acylation of LDTs by β-lactams proceeds through formation of an amine anion intermediate, followed by a subsequent irreversible step that is essential for the antibacterial activity of the drugs. A fluorescence spectroscopy approach enabling kinetic analyses of the acylation steps was developed to explore inactivation mechanisms and to evaluate the efficacy of new synthetic drugs. We also identify diazabicyclooctanes (DBOs) as new pharmacophores that inactivate LDTs by formation of a thio-carbamoyl-enzyme. We discuss several mechanism-based strategies for rational optimization of LDT inhibitors belonging to the β-lactam and DBO families.La résistance aux antibiotiques est une menace mondiale qui conduit à un besoin urgent de développement de nouveaux antibiotiques. L'étude du mécanisme d'inhibition des cibles des antibiotiques est cruciale pour le développement de nouvelles molécules. Le caractère essentiel du peptidoglycane, le composant majeur de la paroi bactérienne, ainsi que soixante-dix ans d'utilisation des β-lactamines ont fait de la polymérisation du peptidoglycane une cible attractive et validée pour les antibiotiques. Les protéines de liaison à la pénicilline (PLP) ont longtemps été considérées comme les seules enzymes catalysant l’étape essentielle de réticulation du peptidoglycane. La thèse explore l'inhibition d'une famille distincte d'enzymes, les L,D-transpeptidases (LDT), qui jouent un rôle prépondérant dans la réticulation chez Mycobacterium tuberculosis. Nous avons montré que l'efficacité d'inhibition des LDT par les β-lactamines est principalement régie par la réactivité du noyau β-lactame. Nous proposons que l'acylation des LDT par les β-lactamines implique la formation d'un intermédiaire, une amine anionique, suivie d'une étape irréversible qui est essentielle pour l'activité antibactérienne. Une approche par spectroscopie de fluorescence a été développée pour explorer les mécanismes d'inactivation et évaluer l'efficacité de nouvelles molécules. Nous avons également identifié une nouvelle famille de molécules, les diazabicyclooctanes (DBO), qui inactivent les LDT par la formation d'un thio-carbamoyl-enzyme. Nous discutons de plusieurs stratégies basées sur le mécanisme d’inactivation pour l’optimisation rationnelle d’inhibiteurs appartenant aux familles des β-lactamines et des DBO

Reversible inactivation of a peptidoglycan transpeptidase by a β-lactam antibiotic mediated by β-lactam-ring recyclization in the enzyme active site

International audienceβ-lactam antibiotics act as suicide substrates of transpeptidases responsible for the last cross-linking step of peptidoglycan synthesis in the bacterial cell wall. Nucleophilic attack of the β-lactam carbonyl by the catalytic residue (Ser or Cys) of transpeptidases results in the opening of the β-lactam ring and in the formation of a stable acyl-enzyme. The acylation reaction is considered as irreversible due to the strain of the β-lactam ring. In contradiction with this widely accepted but poorly demonstrated premise, we show here that the acylation of the L,D-transpeptidase Ldtfm from Enterococcus faecium by the β-lactam nitrocefin is reversible, leading to limited antibacterial activity. Experimentally, two independent methods based on spectrophotometry and mass spectrometry provided evidence that recyclization of the β-lactam ring within the active site of Ldtfm regenerates native nitrocefin. Ring strain is therefore not sufficient to account for irreversible acylation of peptidoglycan transpeptidases observed for most β-lactam antibiotics

Peptidoglycan cross-linking activity of L,D-transpeptidases from Clostridium difficile and inactivation of theses enzymes by β-lactams

International audienc

DIAZABICYCLOOCTANE FUNCTIONALIZATION FOR INHIBITION OF -LACTAMASES FROM ENTEROBACTERIA

International audienceSecond-generation-lactamase inhibitors containing a dia abic clooctane (DBO) scaffold restore the activit of-lactams against pathogenic bacteria, including those producing class A, C, and D en mes that are not susceptible to first-generation inhibitors containing a-lactam ring. Here, e report optimi ation of a s nthetic route to access tria ole-containing DBOs and biological evaluation of a series of 17 compounds for inhibition of five-lactamases representative of en mes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; = 4.7 10-21) as observed bet een the inhibition efficac of purified-lactamases and the potentiation of-lactam antibacterial activit indicating that DBO functionali ation did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displa ed reduced efficac likel due to the absence of h drogen bonding ith a conserved asparagine residue at position 132. This as partiall compensated b additional interactions involving certain tria ole substituents

Synthesis of carbapenems containing peptidoglycanmimetics and inhibition of the cross-linking activity of a transpeptidase of the L,D specificity

International audienceThe carbapenem class of β‐lactams has been optimized against Gram‐negative bacteria producing extended‐spectrum β‐lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d‐transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As β‐lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido‐carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems

Molecular Basis of the Interaction of the Human Protein Tyrosine Phosphatase Non-receptor Type 4 (PTPN4) with the Mitogen-activated Protein Kinase p38γ

International audienceThe human protein tyrosine phosphatase non-receptor type 4 (PTPN4) prevents cell death induction in neuroblastoma and glioblastoma cell lines in a PDZ·PDZ binding motifs-dependent manner, but the cellular partners of PTPN4 involved in cell protection are unknown. Here, we described the mitogen-activated protein kinase p38γ as a cellular partner of PTPN4. The main contribution to the p38γ·PTPN4 complex formation is the tight interaction between the C terminus of p38γ and the PDZ domain of PTPN4. We solved the crystal structure of the PDZ domain of PTPN4 bound to the p38γ C terminus. We identified the molecular basis of recognition of the C-terminal sequence of p38γ that displays the highest affinity among all endogenous partners of PTPN4. We showed that the p38γ C terminus is also an efficient inducer of cell death after its intracellular delivery. In addition to recruiting the kinase, the binding of the C-terminal sequence of p38γ to PTPN4 abolishes the catalytic autoinhibition of PTPN4 and thus activates the phosphatase, which can efficiently dephosphorylate the activation loop of p38γ. We presume that the p38γ·PTPN4 interaction promotes cellular signaling, preventing cell death induction

Click and release chemistry for activity‐based purification of β‐lactam targets

International audienceβ-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of β-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of β-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step

Negative impact of carbapenem methylation on the reactivity of β-lactams for cysteine acylation revealed by quantum calculations and kinetic analyses

International audienceThe Ldtfm L,D-transpeptidase mediates resistance to most β-lactam antibiotics in Enterococcus faecium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by β-lactams belonging to the carbapenem class but not by penams and cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding (KD values in the 50-80 mM range). Here, we analyze the reaction of a Cys sulfhydryl with various β-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the β-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for non-methylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of β-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the β-lactam reactivity with one exception as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation

Synthesis of Avibactam Derivatives and Activity on β-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 μg mL-1 for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria