Rational Optimization of Drug-Target Residence Time: Insights from Inhibitor Binding to the Staphylococcus aureus FabI Enzyme–Product Complex

Abstract

Drug-target kinetics has recently emerged as an especially important facet of the drug discovery process. In particular, prolonged drug-target residence times may confer enhanced efficacy and selectivity in the open in vivo system. However, the lack of accurate kinetic and structural data for series of congeneric compounds hinders the rational design of inhibitors with decreased off-rates. Therefore, we chose the Staphylococcus aureus enoyl-ACP reductase (saFabI) - an important target for the development of new anti-staphylococcal drugs - as a model system to rationalize and optimize the drug-target residence time on a structural basis. Using our new, efficient and widely applicable mechanistically informed kinetic approach, we obtained a full characterization of saFabI inhibition by a series of 20 diphenyl ethers complemented by a collection of 9 saFabI-inhibitor crystal structures. We identified a strong correlation between the affinities of the investigated saFabI diphenyl ether inhibitors and their corresponding residence times, which can be rationalized on a structural basis. Due to its favorable interactions with the enzyme, the residence time of our most potent compound exceeds 10 hours. In addition, we found that affinity and residence time in this system can be significantly enhanced by modifications predictable by a careful consideration of catalysis. Our study provides a blueprint for investigating and prolonging drug-target kinetics and may aid in the rational design of long-residence-time inhibitors targeting the essential saFabI enzyme.

Structural Basis for the Recognition of Mycolic Acid Precursors by KasA, a Condensing Enzyme and Drug Target from Mycobacterium Tuberculosis

“…This is nicely illustrated by recent studies concerning the induced fit binding of peptide deformylase and enoyl-ACP reductase inhibitors (20,49,61). For instance, the picomolar affinity of diphenyl ether enoyl-ACP reductase inhibitors can be related to their capability to mimic the transition state of the catalyzed reaction and to trigger the correlated ordering of a flexible loop (20,61). In the present study, we reveal snapshots of the dynamic process that is involved in the recognition of very long chain fatty acyl substrates by the drug target KasA.…”

“…In light of the fact that the free energy of the transition state along the reaction coordinate has to be lowered efficiently, it is not very surprising that the dynamics of substrate binding and catalysis are also reflected in the inhibition of enzymes (49,59,60). This is nicely illustrated by recent studies concerning the induced fit binding of peptide deformylase and enoyl-ACP reductase inhibitors (20,49,61). For instance, the picomolar affinity of diphenyl ether enoyl-ACP reductase inhibitors can be related to their capability to mimic the transition state of the catalyzed reaction and to trigger the correlated ordering of a flexible loop (20,61).…”

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

“…The k obs versus [triclosan] plot fit best to a straight line indicating reversible, slow binding of triclosan to FabI(M99T), with an apparent on rate (k 3 ) of 1.7 ϫ 10 4 M Ϫ1 s Ϫ1 . This "slow binding" behavior of triclosan is characteristic of its interactions with many FabIs (7,12,39), and the importance of slow off rates to the in vivo drug efficacy has recently become clear (12,39). However, the basis for the lower MIC in our closed system was attributed to the higher affinity of triclosan for the FabI(M99T)⅐NAD ϩ complex.…”

Clinical Relevance of Type II Fatty Acid Synthesis Bypass in Staphylococcus aureus

“…A class of new-generation antimicrobials in intensive development uses the type II fatty acid synthesis (FASII) pathway as an antibacterial target (2-5) to treat S. aureus infections (6)(7)(8). A widely used biocide, triclosan (5-chloro-2-[2,4-dichlorophenoxy]phenol; commercialized as Irgasan or Microban), is a prototype for further anti-FASII development (4,9,10).…”