Development and binding characteristics of phosphonate inhibitors of SplA protease from Staphylococcus aureus

Abstract

Staphylococcus aureus is responsible for a variety of human infections, including lifethreatening, systemic conditions. Secreted proteome, including a range of proteases, constitutes the major virulence factor of the bacterium. However, the functions of individual enzymes, in particular SplA protease, remain poorly characterized. Here, we report development of specific inhibitors of SplA protease. The design, synthesis, and activity of a series of a-aminoalkylphosphonate diaryl esters and their peptidyl derivatives are described. Potent inhibitors of SplA are reported, which may facilitate future investigation of physiological function of the protease. The binding modes of the high-affinity compounds Cbz-Phe P -(OC 6 H 4 24-SO 2 CH 3 ) 2 and Suc-Val-Pro-Phe P -(OC 6 H 5 ) 2 are revealed by high-resolution crystal structures of complexes with the protease. Surprisingly, the binding mode of both compounds deviates from previously characterized canonical interaction of a-aminoalkylphosphonate peptidyl derivatives and family S1 serine proteases.

A Fluorescent‐Labeled Phosphono Bisbenzguanidine As an Activity‐Based Probe for Matriptase

“…The calculations yieldedaplausible binding mode as shown in Figure 2. In accordance with previous reports, [45][46][47] one of the (partially) negatively charged oxygen atomso ft he phosphonate is proposed to form ionic interactions with the protonated e 2 -nitrogen of His57, whereas the other is orientated towards the oxyanion hole. The "right" benzguanidinium moiety,a sa na rginine mimetic, expectedly interacts with Asp189 in the deep S1 pocket by charge-assisted hydrogen bonding.…”

“…In order to propose possible binding modes of probe 18 , molecular docking calculations were performed, using the crystal structure of a complex of matriptase with a bis‐benzamidine inhibitor (PDB‐ID: 4JZI) . The interaction of serine proteases with diphenyl phosphonate inhibitors includes the formation of a serine phosphono diester through the nucleophilic attack of the active site residue Ser195 at the phosphorus and the release of one phenoxy group, followed by a hydrolytic “aging” into a phosphono monoester . Such a monoester complex with phosphorus in a tetrahedral configuration was generated by covalent docking as follows.…”

“…[4] The interaction of serine proteases with diphenyl phosphonate inhibitors includes the formation of as erine phosphono diester through the nucleophilic attack of the active site residue Ser195a tt he phosphorus and the release of one phenoxyg roup, followed by ah ydrolytic "aging"i nto ap hosphono monoester. [45][46][47] Such am onoester complexw ith phosphorusi natetrahedral configurationw as generated by covalent docking as follows. Both phenoxy groups were removedf rom the inhibitor andt he single bond between the oxygen of Ser195a nd the phosphorus was manually built.…”

ClpP Protease, a Promising Antimicrobial Target

“…Recently, based on that co-crystallized structure, a new class of ClpP inhibitors with an α-amino diarylphosphonate warhead was reported by our group [108]. This family of compounds is a classic example of irreversible inhibitors of serine proteases [109,110,111,112,113,114]. Their mechanism of action consists of a nucleophilic attack of the catalytic serine to the phosphonate, which undergoes release of a phenolate group to recover the tetrahedral geometry from the pentacoordinate transition state (Figure 8B).…”

Recent Developments in Peptidyl Diaryl Phoshonates as Inhibitors and Activity-Based Probes for Serine Proteases

“…4-Methoxy substitution was found optimal, however, this compound was also potent towards subtilisin [27]. For SpIA protease, N-protected hydrophobic tripeptide derivatives (Val-Pro-Leu and Val-Pro-Phe, Table 1, Entry 7 ) typically appeared more potent than nonextended α-aminoalkylphosphonates [26]. Selected inhibitors served for cocrystallization with this enzyme and provided high resolution structures of the ligand–protein complexes.…”