Probing of the cis-5-phenyl proline scaffold as a platform for the synthesis of mechanism-based inhibitors of the Staphylococcus aureus sortase SrtA isoform

https://doi.org/10.1016/j.bmc.2009.02.008Get rights and content

Abstract

cis-5-Phenyl prolinates with electrophilic substituents at the fourth position of a pyrrolidine ring were synthesized by 1,3-dipolar cycloaddition of arylimino esters with divinyl sulfone and acrylonitrile. 4-Vinylsulfonyl 5-phenyl prolinates inhibit Staphylococcus aureus sortase SrtA irreversibly by modification of the enzyme Cys184 and could be used as hits for the development of antibacterials and antivirulence agents.

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) has been nominated by the Antimicrobial Availability Task Force of the Infectious Diseases Society of America as one of six high-priority problematic pathogens.1 This nomination reflects the high incidence of MRSA infections, substantial morbidity, and peculiar virulence factors circumventing usual antimicrobial therapy. Other concerns are caused by emerging resistance of S. aureus strains to modern therapies and a lack of novel drug candidates, especially those with new mechanism of action. S. aureus, like other Gram-positive organisms, utilizes surface proteins for adhesion to host cells and invasion of tissues. The vast majority of surface proteins involved in these aspects of staphylococcal disease are substrates of sortases — cysteine transpeptidases which link surface proteins to peptidoglycan, thus incorporating them into the envelope and leading to their display on the microbial surface.2 S. aureus strains in which the srtA gene has been deleted display no defect in survival and growth but exhibit reduced pathogenicity and virulence, due to a failure to display MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) on the bacterial surface. The same effect has been demonstrated for wild Gram-positive organisms treated with sortase inhibitors, implicating sortase enzymes as important targets for the treatment of staphylococcal disease.2 Different types of sortase inhibitors have been recently reviewed,3 and the most active and promising small-molecule inhibitors4, 5, 6, 7, 8, 9, 10, 11, 12 are presented in Figure 1. Three types of small molecules, aryl (β-amino)ethyl ketones,7 trans-3-(furan-2-yl) acrylic acid amides8 and aaptamines,12 have been reported since the publication of the review. This work describes our studies towards the design and synthesis of novel types of S. aureus SrtA inhibitors, and an understanding of their mechanisms of action.

S. aureus sortase SrtA is a 206 amino acid cysteine transpeptidase with an N-terminal transmembrane anchor. His120, Cys184 and Arg197 conserved side chains constitute the enzyme active site.2, 3 Of the reported small molecule inhibitors of SrtA, only a few have been subjected to detailed kinetic and mechanistic studies. Commercial vinyl sulfones4 and aryl (β-amino)ethyl ketones7 irreversibly modify Cys184, preventing acylation of the Thr-Gly carbonyl, and thus cleavage of the sorting signal. Diarylacrylonitriles demonstrate a competitive character of inhibition,5 although the strong Michael acceptor moiety of these molecules does not exclude interaction with different nucleophilic species in vivo. Synthetic trans-3-hetaryl acrylic acid amides SrtA inhibitors have been developed from in silico virtual screening of commercial compound libraries and docking model interactions of inhibitors with amino acid residues in SrtA active site have been identified recently.8 All other small-molecule inhibitors of SrtA have been isolated from natural plant extracts, and only indirect data of their mechanism of interaction with the enzyme are available.3 Although X-ray structures of both S. aureus SrtA with its substrate complex13 and Bacillus anthracis SrtB with and without inhibitor7 have been solved, this information is insufficient to allow for construction of a pharmaceutically suitable inhibitor and additional studies of sortase–small molecules interactions are desired. We have employed conformationally constrained derivatives of cis-5-phenyl prolines with directed functional groups to evaluate the binding modes of these compounds with S. aureus SrtA (Fig. 2).

Section snippets

Chemistry

Only two types of synthetic compounds were known to inhibit SrtA at the beginning of this project — vinyl sulfones4 and diarylacrylonitriles.5 The utility of vinyl sulfones for different medicinal chemistry applications has been recently reviewed.14 As effective Michael acceptors, some vinyl sulfones are in clinical studies as cysteine protease inhibitors.15 Similarly, the nitrile group has been shown to be a key structural element for effective SrtA inhibition.5 We planned to attach vinyl

Inhibition studies with SrtAΔN24

As a first test, the compounds were assayed for the ability to inhibit SrtA activity in an HPLC-based assay at three separate concentrations: 39.1 μM, 625 μM, and 5 mM. Compounds 2ac displayed complete inhibition at a concentration of 5 mM, while the remainder showed more modest inhibition. However, only the vinyl sulfone compounds were potent inhibitors at mid-micromolar concentrations. The carbonitriles 46 and 9 failed to completely inhibit SrtA even at a concentration of 5 mM and were not

Discussion

Inspecting known S. aureus SrtA inhibitors structures (Fig. 1), it is interesting to note that many are two-dimensional molecules with limited possibility for further modification. In this regard it seems attractive to investigate the three-dimensional space of enzyme–ligand interactions within the SrtA active site and to explore a set of related structural analogs. We selected cis-5-phenyl proline scaffold for construction of potential inhibitors (Fig. 2) since efficient straightforward

Conclusions

In conclusion we have developed an effective synthetic approach to a diverse set of cis-5-phenyl prolinates functionalized by electrophilic groups. We have also tested these synthesized compounds on the in vitro inhibitory activity of the S. aureus sortase SrtA transpeptidase. Racemic vinyl sulfonyl 5-phenyl prolinates 2 inhibit S. aureus sortase SrtA irreversibly by modification of the enzyme Cys184. Modifications of most active compounds including asymmetric synthetic approaches are under

Chemistry

Reagents were obtained from Alfa Aesar and used without further purification unless otherwise stated. Solvents were dried using standard procedures. Reactions were monitored by thin layer chromatography (TLC) on precoated silica gel plates (Sorbfil) with a UV indicator. Column chromatography was performed with Alfa Aesar Silica Gel 60 (0.040–0.063 mm). Melting points were determined in open capillary and are uncorrected. 1H NMR and 13C NMR spectra were recorded with a Bruker Avance 400 MHz

Expression and purification of 6-His tagged SrtAΔN24

N-Terminally His6-tagged SrtA lacking the amino-terminal 24 amino acids was expressed in BL21(DE3) cells containing the plasmid pET15bSrtAΔN24. Cells were grown in Luria Broth containing 100 μg/ml of ampicillin at 37 °C until the OD600 reached 0.5–0.6. Protein expression was induced by the addition of 1 mM isopropyl β-d-thiogalactopyranoside (IPTG), and the cells were grown for an additional 3 h at 37 °C then harvested by centrifugation at 3000g for 10 min. Cells were resuspended in 150 mM NaCl, 50 mM

Solid-phase synthesis of Abz-LPETG-Dap(DNP)-NH2

The peptide Abz-LPETG-Dap(DNP)-NH2 was synthesized by the Fmoc/piperidine strategy on PAL resin on a 0.25 mmol scale. Cleavage from the resin was achieved via incubation with a 95:2.5:2.5 TFA/water/triisopropylsilane (TIPS) mixture for 2.5 h. The peptide was precipitated using cold diethyl ether following the removal of excess TFA via rotary evaporation. After filtration, the precipitate was dissolved in a 50:50 water/acetonitrile mixture and lyophilized to yield crude peptide. Crude peptide was

Steady-state activity assays

Purified recombinant SrtAΔN24 at 1 μM was incubated with 2 mM Abz-LPETG-Dap(DNP)-NH2, 2 mM NH2-Gly5-OH, and increasing amounts of inhibitor at 37 °C in 2% DMSO, 0.1% CHAPS, 150 mM NaCl, 5 mM mM CaCl2 and 300 mM Tris (pH 7.5), in a total volume of 100 μL. To control for the possibility of time-dependent inactivation, the enzyme and inhibitor were pre-incubated in buffer at 37 °C for 30 min. Reactions were initiated by addition of substrate mix (Abz-LPETG-Dap(DNP)-NH2 and NH2-Gly5-OH), and allowed to

Acknowledgments

This research was kindly supported by a National Institutes of Health Allergy and Infectious Disease research Grant AI46611 to D.G.M. K.V.K. acknowledges a partial support from the Russian Foundation for Basic Research (Grant 08-04-01800a).

References and notes (29)

  • A.W. Maresso et al.

    J. Biol. Chem.

    (2007)
  • B.C. Chenna et al.

    Bioorg. Med. Chem. Lett.

    (2008)
  • K.B. Oh et al.

    Bioorg. Med. Chem. Lett.

    (2005)
  • K.H. Jang et al.

    Bioorg. Med. Chem. Lett.

    (2007)
  • Y. Zong et al.

    J. Biol. Chem.

    (2004)
  • O. Tsuge et al.
  • R.M. Oballa et al.

    Bioorg. Med. Chem. Lett.

    (2007)
  • G.H. Talbot et al.

    Clin. Infect. Dis.

    (2006)
  • L.A. Marraffini et al.

    Microbiol. Mol. Biol. Rev.

    (2006)
  • N. Suree et al.

    Mini-Rev. Med. Chem.

    (2007)
  • B.A. Frankel et al.

    J. Am. Chem. Soc.

    (2004)
  • K.B. Oh et al.

    J. Med. Chem.

    (2004)
  • K.B. Oh et al.

    Appl. Microbiol. Biotechnol.

    (2006)
  • S.S. Kang et al.

    Biol. Pharm. Bull.

    (2006)
  • Cited by (0)

    View full text