Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram-Negative Bacteria

Abstract

With the widespread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. The protein translocase SecA is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we developed three structural classes of SecA inhibitors that have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain an understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane of Gram-negative bacteria. We also found that the expression of efflux pumps, which are responsible for multidrug resistance (MDR), have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA is indeed the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.

Keywords: Gram-negative bacteria; SecA translocase; antibiotics; membrane permeability; multidrug resistance.

Figure 1

Three structural classes of small molecule SecA inhibitors

Figure 2. Bactericidal effects of SCA-107

E. coli NR698 and MC4100 with or without PMBN (25 μg/mL) were treated with SCA-107 at the concentration indicated at 37 °C for 2 hours. Then CFU were determined.

Figure 3. Non-competitive Inhibition of channel activity of SecA-liposomes in oocytes

(A–C) All 3 classes of SecA inhibitors with EcSecA show non-competitive inhibition with ATP; the data of SCA-107 is from ref. ^[9], presented here for comparisons with 3 classes of inhibitors, and with SecA homologs (D) PaSecA and (E) StSecA

Figure 4. Target pulled-down of SecA from E. coli lysates by Inhibitors conjugates

E. coli MC4100 whole cell lysates (33 μg) were treated with SecA inhibitor conjugates as indicated, and the pull- down bead samples were washed, run on SDS gels, and analyzed: (A) Coomassie Blue staining. Arrow indicates SecA band. (B) Immunoblots by EcSecA antibodies.

Figure 5. Altered trypsin sensitivity tests by SecA inhibitors

(A) E. coli MC4100 cell lysates (200 μg) were incubated with or without 2.5 mM SCA-15 on ice for 2 hrs. The mixtures were digested with trypsin (50 μg) at 25 °C for 20 min. The digestion was stopped by adding SDS sample buffer and boiling for 20 min. and run on 10–20% gradient SDS-PAGE gel. (B) Immunoblot by EcSecA antibodies. E. coli MC4100 whole cell lysates with or without addition of purified EcSecA protein were similarly incubated with or without SCA-15 or SCA-21, and treated with trypsin as in (A). Arrows indicate the bands altered by treatment with the inhibitors.