Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase

Figures & data

Scheme 1.  Reaction of synthesis of the boron-containing compound.

Scheme 2.  One step amide and imide formation at room temperature.

Figure 1.  Binding site of: (a) d-glutamic acid and (b) l-glutamic acid on the RacE1 isoform with two principal amino acid residues, (c) d-glutamic acid and (d) l-glutamic acid on the RacE2 isoform with two principal amino acid residues. Carbon atoms are colored cyan; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms of the protein, yellow for enzyme active site.

Figure 2.  Binding site of: (a) l-GM, (b) d-DFB, (c) l-DFB on the RacE1 isoform, with two principal amino acid residues. Carbon atoms are colored cyan; oxygen atoms, red; nitrogen atoms, blue; sulfur atoms, yellow for enzyme active site.

Figure 3.  Ligand on racemase enzyme allosteric site of: (a) l-GM, (b) d-DFB, (c) l-DFB on the RacE1 isoform, with some amino acid residues. Carbon atoms are colored cyan; oxygen atoms, red; nitrogen atoms, blue.

Table 1.  Free energies values and K_d of the natural substrates of l and d-glutamic acid and all three test compounds in relation to RacE1 and RacE2, according to docking studies.

Compound	RacE1		RacE2
	ΔG (Kcal/mol)	Kd (M)	ΔG (Kcal/mol)	Kd (M)
l-Glu	−7.4	3.77 × 10^-6	−8.21	9.521 × 10^-7
d-Glu	−8	1.38 × 10^-6	−8.94	2.799 × 10^-7
l-GM	−7.72	1.25 × 10^-5	−9.02	1.46 × 10^-6
d-DFB	−11.86	2.04 × 10^–12	−11.47	3.97 × 10^–12
l-DFB	−12.33	9.81 × 10^–13	−11.05	8.0 × 10^–12

Table 2.  MIC₅₀ and MIC₉₀ for the Bacillus spp. strains tested, by the microdilution technique in Mueller Hilton broth.

Antimicrobial agent	MIC (μg/mL)
	B. cereus		B. subtilis		B. anthracis
	50%	90%	50%	90%	50%	90%
TTC	8	16	0.25	0.5	0.25	0.5
d-DFB	64	128	32	64	32	64
l-DFB	32	64	16	32	32	64
l-GM	>256	>256	>256	>256	>256	>256

Table 3.  Determination of IC₅₀ of compound on Bacillus spp.

Cell extracts* (racemase enzyme)	IC50 μg/mL
	d-DFB	l-DFB	LGM
B. cereus	<64	<32	>500
B. subtilis	<32	<16	>500
B. anthracis	32	32	>500

*Cellular extracts containing the racemase enzyme were obtained according to the materials and methods section. The specific activity for each extract was: 198 mU/mg for B. cereus (RacBc), 192.6 mU/mg for B. subtilis (RacBs), and 207.6 mU/mg for B. antrhacis (RacBa). The IC₅₀ was determined using different concentrations for each inhibitor. To inhibit the activity of RacBc we used 64 μg/mL d-DFB, 32 μg/mL l-DFB, and 500 μg/mL LGM. For RacBs we used 32 μg/mL d-DFB, 16 μg/mL l-DFB, and 500 μg/mL LGM. For RacBa we used 32 μg/mL d-DFB, 32 μg/mL l-DFB, and 500 μg/mL LGM.