ABSTRACT
Startling reports described the paradoxical triggering of the human mitogen-activated protein kinase pathway when a small-molecule inhibitor specifically inactivates the BRAF V600E protein kinase but not wt-BRAF. We performed a conceptual analysis of the general phenomenon "activation by inhibition" using bacterial and human HtrA proteases as models. Our data suggest a clear explanation that is based on the classic biochemical principles of allostery and cooperativity. Although substoichiometric occupancy of inhibitor binding sites results in partial inhibition, this effect is overrun by a concomitant activation of unliganded binding sites. Therefore, when an inhibitor of a cooperative enzyme does not reach saturating levels, a common scenario during drug administration, it may cause the contrary of the desired effect. The implications for drug development are discussed.
Subject(s)
Allosteric Site , Antineoplastic Agents/pharmacology , Heat-Shock Proteins/antagonists & inhibitors , High-Temperature Requirement A Serine Peptidase 1/antagonists & inhibitors , Periplasmic Proteins/antagonists & inhibitors , Protease Inhibitors/pharmacology , Allosteric Regulation , Antineoplastic Agents/chemistry , Escherichia coli , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/metabolism , High-Temperature Requirement A Serine Peptidase 1/chemistry , High-Temperature Requirement A Serine Peptidase 1/metabolism , Humans , Periplasmic Proteins/chemistry , Periplasmic Proteins/metabolism , Protease Inhibitors/chemistry , Protein Binding , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolismABSTRACT
Despite the availability of hundreds of antibiotic drugs, infectious diseases continue to remain one of the most notorious health issues. In addition, the disparity between the spread of multidrug-resistant pathogens and the development of novel classes of antibiotics exemplify an important unmet medical need that can only be addressed by identifying novel targets. Herein we demonstrate, by the development of the first inâ vivo active DegS inhibitors based on a pyrazolo[1,5-a]-1,3,5-triazine scaffold, that the serine protease DegS and the cell envelope stress-response pathway σE represent a target for generating antibiotics with a novel mode of action. Moreover, DegS inhibition is synergistic with well-established membrane-perturbing antibiotics, thereby opening promising avenues for rational antibiotic drug design.
Subject(s)
Anti-Bacterial Agents/pharmacology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli/drug effects , Serine Proteinase Inhibitors/pharmacology , Anti-Bacterial Agents/chemical synthesis , Anti-Bacterial Agents/chemistry , Dose-Response Relationship, Drug , Escherichia coli/metabolism , Escherichia coli Proteins/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Molecular Structure , Serine Proteinase Inhibitors/chemical synthesis , Serine Proteinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Covalent modifications of nonactive site lysine residues by small molecule probes has recently evolved into an important strategy for interrogating biological systems. Here, we report the discovery of a class of bioreactive compounds that covalently modify lysine residues in DegS, the rate limiting protease of the essential bacterial outer membrane stress response pathway. These modifications lead to an allosteric activation and allow the identification of novel residues involved in the allosteric activation circuit. These findings were validated by structural analyses via X-ray crystallography and cell-based reporter systems. We anticipate that our findings are not only relevant for a deeper understanding of the structural basis of allosteric activation in DegS and other HtrA serine proteases but also pinpoint an alternative use of covalent small molecules for probing essential biochemical mechanisms.