Design, Synthesis and Evaluation of Triazole-Pyrimidine Analogues as SecA Inhibitors

Abstract

SecA, a key component of the bacterial Sec-dependent secretion pathway, is an attractive target for the development of new antimicrobial agents. Through a combination of virtual screening and experimental exploration of the surrounding chemical space, we identified a hit bistriazole SecA inhibitor, SCA-21, and studied a series of analogues by systematic dissections of the core scaffold. Evaluation of these analogues allowed us to establish an initial structure-activity relationship in SecA inhibition. The best compounds in this group are potent inhibitors of SecA-dependent protein-conducting channel activity and protein translocation activity at low- to sub-micromolar concentrations. They also have minimal inhibitory concentration (MIC) values against various strains of bacteria that correlate well with the SecA and protein translocation inhibition data. These compounds are effective against methicillin-resistant Staphylococcus aureus strains with various levels of efflux pump activity, indicating the capacity of SecA inhibitors to null the effect of multidrug resistance. Results from studies of drug-affinity-responsive target stability and protein pull-down assays are consistent with SecA as a target for these compounds.

Keywords: SecA inhibitors; antimicrobials; protein secretion; pyrimidines; triazoles.

Figure 1

Optimization of the hit compound SCA-21

Figure 2. Screening for inhibition of the ATPase activity of EcSecAN68

The initial screening assays were carried out at 50 μM for all compounds.

Figure 3. Validation SaSecA1 as a drug target with pull down assay. (a)

Structure of SCA-256. (b) Whole cell lysate of S. aureus ATCC 6538 was mixed with SCA-256 (biotinylated 7b analog) for 1 hr, then Streptavidin magnetic beads were used to pull out proteins interact with SCA-256. The interaction between SaSecA1 and SCA-256 was examined by Western blot with the cross-reacting EcSecA antibody (1:5000 dilution). Synthesis of SCA-256 is described in the Supplemental Information section.

Figure 4. Ion-channel activity against EcSecA and SaSecA1

Ion-channel activity of SecA-liposomes was determined by adding reconstitute SecA-liposome complex into oocytes and recording the current inducted by proOmpA. EcSecA: E. coli SecA; SaSecA1: Staphlocucus aureus SecA1.

Figure 5. Molecular modeling of 7b (SCA-107) binding site in E. coli SecA. 6A

Docking was done in SYBYL 2.0 using PDB (2FSG) with SecA dimers A and B. 7b binds to the interface of the A and B monomers, closer to A (6A), partially blocking the entrance to the ATP site. 6B: SCA-107 surrounded by amino acid residues.

Figure 6. Bactericidal effect of 7b (SCA-107) against S. aureus Mu50

Bactericidal effects were determined by counting CFU after 2 hr treatment with different concentration of 7b.

Scheme 1

a) THF, 0 ºC ~rt, overnight; b) 5% NaOH, reflux 5 h, 65–87% overall yields

Scheme 2

a) K₂CO₃, acetone, rt, 2–3h, 35%–80%.

Scheme 3

a) CS₂, H₂O/EtOH, reflux 5 h, 65%; b) K₂CO₃, acetone, rt, 2– 3 h, 80%.

Scheme 4

a) K₂CO₃, acetone, rt, 2–3 h, 35%–80%.

Scheme 5

a) CS₂, NH₂NH₂, KOH/EtOH, 82%; b) DMF, 60 ºC, 4 h, 76%; c) NaBH₄ (1 eq), EtOH, rt, 2 h, 82%; d) NaBH₄ (10 eq), EtOH, rt, 2 h, 40%.