Evaluation of small molecule SecA inhibitors against methicillin-resistant Staphylococcus aureus

Abstract

Due to the emergence and rapid spread of drug resistance in bacteria, there is an urgent need for the development of novel antimicrobials. SecA, a key component of the general bacterial secretion system required for viability and virulence, is an attractive antimicrobial target. Earlier we reported that systematical dissection of a SecA inhibitor, Rose Bengal (RB), led to the development of novel small molecule SecA inhibitors active against Escherichia coli and Bacillus subtilis. In this study, two potent RB analogs were further evaluated for activities against methicillin-resistant Staphylococcus aureus (MRSA) strains and for their mechanism of actions. These analogs showed inhibition on the ATPase activities of S. aureus SecA1 (SaSecA1) and SecA2 (SaSecA2), and inhibition of SaSecA1-dependent protein-conducting channel. Moreover, these inhibitors reduce the secretion of three toxins from S. aureus and exert potent bacteriostatic effects against three MRSA strains. Our best inhibitor SCA-50 showed potent concentration-dependent bactericidal activity against MRSA Mu50 strain and very importantly, 2-60 fold more potent inhibitory effect on MRSA Mu50 than all the commonly used antibiotics including vancomycin, which is considered the last resort option in treating MRSA-related infections. Protein pull down experiments further confirmed SaSecA1 as a target. Deletion or overexpression of NorA and MepA efflux pumps had minimal effect on the antimicrobial activities against S. aureus, indicating that the effects of SecA inhibitors were not affected by the presence of these efflux pumps. Our studies show that these small molecule analogs target SecA functions, have potent antimicrobial activities, reduce the secretion of toxins, and have the ability to overcome the effect efflux pumps, which are responsible for multi-drug resistance. Thus, targeting SecA is an attractive antimicrobial strategy against MRSA.

Keywords: Efflux; MRSA; Rose Bengal analogs; SecA inhibitors; SecA-dependent secretion; SecA-liposomes.

Figure 1. Inhibition on the ATPase activities of SaSecA1 and SaSecA2

(a) Structures of RB (MW, 1017 daltons) and RB analogs, SCA-41 (MW, 282 daltons) and SCA-50 (MW 298 daltons). (b) Inhibition of the intrinsic ATPase activity of soluble SaSecA1 (n = 6). (c) Inhibition of the intrinsic ATPase activity of SaSecA2 (n = 6). The ATPase activity without inhibitor was defined as 100%.

Figure 2. Inhibition of the ion-channel activity of SaSecA1

SaSecA1-liposomes ion-channel activity was determined in oocytes with or without inhibitors (n = 20–30). The ion-channel activity without inhibitors was defined as 100%.

Figure 3. Non-competitive inhibition of the membrane ATPase activity of SaSecA1

SaSecA1-liposomes ion-channel activity was determined in the oocytes with different concentration of ATP and SCA-50 (n = 9–10). (a) Inhibition of RB. (b) Inhibition of SCA-50.

Figure 4. Inhibition of in vitro translocation activity of SaSecA1

Liposomes-SaSecA1-dependent proOmpA translocation activity was determined at 30 °C with or without SCA-50 (n = 3). The in vitro translocation activity of SaSecA1 without inhibitor was defined as 100%.

Figure 5. Validation SaSecA1 as a drug target with pull down assay

(a) Structure of SCA-254. (b) Whole cell lysate of S. aureus ATCC 6538 was mixed with or without SCA-254 (biotinylated SCA-50 analog) for 1 hr, then Streptavidin magnetic beads were used to pull out proteins interact with SCA-254. The interaction between SaSecA1 and SCA-254 was examined by Western blot with SecA antibody.

Figure 6. Inhibition of the secretion of S. aureus toxins

SCA-50 or same volume of DMSO as control was added to of S. aureus Mu50 at time point 0 hr as indicated. (a) Growth curve. (b) Western blot analysis the amount of toxins in the supernatant.

Figure 7. Bactericidal effects of SCA-50

Bactericidal effects were determined by counting CFU after 1 hr treatment with different concentration of SCA-50.

Figure 8. Photooxidation does not contribute to the bactericidal effects of SCA-50

Early log phase cells of S. aureus ATCC 6538 were treated with 40 µM of inhibitor for 2 hr with or without exposure to light. Control was absence of inhibitor, and bactericidal effects were determined by the reduction of CFU comparing inhibitor treatment with control.

Figure 9

Molecular modeling of SCA-50’s binding site in E. Coli SecA. Top panel shows SCA-50 partially blocks the ATP entrance in chain B. Bottom panel shows the amino acid residues in the binding pocket.