Bioisosteric replacement of pyridoxal-5'-phosphate to pyridoxal-5'-tetrazole targeting Bacillus subtilis GabR

Abstract

Antimicrobial resistance is a significant cause of mortality globally due to infections, a trend that is expected to continue to rise. As existing treatments fail and new drug discovery slows, the urgency to develop novel antimicrobial therapeutics grows stronger. One promising strategy involves targeting bacterial systems exclusive to pathogens, such as the transcription regulator protein GabR. Expressed in diverse bacteria including Escherichia coli, Bordetella pertussis, and Klebsiella pneumoniae, GabR has no homolog in eukaryotes, making it an ideal therapeutic target. Bacillus subtilis GabR (bsGabR), the most studied variant, regulates its own transcription and activates genes for GABA aminotransferase (GabT) and succinic semialdehyde dehydrogenase (GabD). This intricate regulatory system presents a compelling antimicrobial target with the potential for agonistic intervention to disrupt bacterial gene expression and induce cellular dysfunction, especially in bacterial stress responses. To explore manipulation of this system and the potential of this protein as an antimicrobial target, an in-depth understanding of the unique PLP-dependent transcription regulation is critical. Herein, we report the successful structural modification of the cofactor PLP and demonstrate the biochemical reactivity of the PLP analog pyridoxal-5'-tetrazole (PLT). Through both spectrophotometric and X-ray crystallographic analyses, we explore the interaction between bsGabR and PLT, together with a synthesized GABA derivative (S)-4-amino-5-phenoxypentanoate (4-phenoxymethyl-GABA or 4PMG). Most notably, we present a crystal structure of the condensed, external aldimine complex within bsGabR. While PLT alone is not a drug candidate, it can act as a probe to study the detailed mechanism of GabR-mediated function. PLT employs a tetrazole moiety as a bioisosteric replacement for phosphate in PLP. In addition, the PLP-4PMG adduct observed in the structure may serve as a novel chemical scaffold for subsequent structure-based antimicrobial design.

Keywords: GabR; antibiotic; bioisosterism; external aldimine; internal aldimine; pyridoxal‐5'‐phosphate; tetrazole.

FIGURE 1

Pharmacophore modification of pyridoxal‐5′‐phosphate (PLP) to pyridoxal‐5′‐tetrazole (PLT). Classical representation of the formation of the internal aldimine via nucleophilic attack of the bsGabR Eb/O K312 residue and subsequent external aldimine formation via nucleophilic attack of the 4‐phenoxymethyl‐GABA (4PMG) γ‐amino group and elimination of the K312 amine.

SCHEME 1

Synthesis of pyridoxal‐5′‐tetrazole 1 (PLT). Reagents and conditions: (a) 2,2‐DMP, PTSA, rt., 22 h; (b) MnO₂, PhMe, 50°C, 26 h; (c) diethyl cyanomethylphosphonate, 5.0 M K₂CO₃, rt., 45 min; (d) NaN₃, ZnBr₂, 4:1 H₂O:i‐PrOH, reflux, 46 h; (e) 1.0 M HCl, reflux, 3 h; (f) MnO₂, 1.0 M NaOH, H₂O, rt., 18 h.

FIGURE 2

Spectrophotometric characterization of 100 μM pyridoxal‐5′‐phosphate (PLP; red‐dotted line) and pyridoxal‐5′‐tetrazole (PLT; purple‐dotted line) cofactors. Characterization of 20 μM apo‐bsGabR Eb/O domain (black solid line) in the presence of 100 μM PLP (orange solid line) or PLT (turquoise solid line) following a 60 min incubation. Spectral changes of bsGabR and PLT over time (100 μM PLT, at 0, 10, 20, 30, and 60 min) (inset).

FIGURE 3

Model of apo‐bsGabR Eb/O domain binding pocket surrounded by refined 2Fo‐Fc at 1.1σ.

FIGURE 4

Spectrophotometric characterization of the bsGabR Eb/O‐PLT internal aldimine, bsGabR Eb/O‐PLT‐4PMG external aldimine, and PLT‐4PMG reaction free of bsGabR Eb/O domain. Spectral changes over time (at 0.5, 5, 10, and 40 min) and complete difference spectrum of the internal aldimine (A, B). Spectral changes over time (at 0.5, 2, 4, 8, and 30 min) and complete difference spectrum of the external aldimine (C, D). Spectral changes over time (at 0.5, 4, 16, 32, and 64 min) of PLT‐4PMG mixture absent the bsGabR Eb/O domain (E, F). Reaction scheme for the proposed formation of the internal aldimine intermediate between bsGabR and PLT (G). Reaction scheme for the proposed condensation of the external aldimine intermediate PLT‐4PMG facilitated by bsGabR (H).

FIGURE 5

Polder map of PLT‐4PMG external aldimine complex (3.0σ). Key hydrogen bonding and hydrophobic interactions are presented as dashed lines.

FIGURE 6

Characteristic structural determinants consistent with the formation of PLT‐4PMG external aldimine. (a) Internal aldimine (PDB Code 4N0B) superimposed onto the PLP‐GABA external aldimine structure (PDB Code 5T4J). (b) PLP‐GABA external aldimine superimposed onto the PLP‐4PMG external aldimine structures (PDB Code 6UXZ). (c) PLP‐4PMG external aldimine superimposed to PLT‐4PMG external aldimine structure. Key structural determinants Y281, K312, and R319 are highlighted in pink. Carboxylate binding residues are labeled with * and highlighted in pink. Key hydrogen bonding distances are indicated as dashed lines. (d) The biological assembly of a bsGabR Eb/o domain dimer is shown as a ribbon diagram. The PLP‐4PMG external aldimine is shown as spheres.

FIGURE 7

Arginine 319 alternate conformation influenced by presence of the tetrazole or phosphate moiety of PLT or PLP, respectively. R319 surrounding polder map of R319‐PLT interaction (3.75σ) or R319‐PLP interaction (4.0σ).