Design and evaluation of novel analogs of 2-amino-4-boronobutanoic acid (ABBA) as inhibitors of human gamma-glutamyl transpeptidase

Abstract

Inhibitors of gamma-glutamyl transpeptidase (GGT1, aka gamma-glutamyl transferase) are needed for the treatment of cancer, cardiovascular illness and other diseases. Compounds that inhibit GGT1 have been evaluated in the clinic, but no inhibitor has successfully demonstrated specific and systemic GGT1 inhibition. All have severe side effects. L-2-amino-4‑boronobutanoic acid (l-ABBA), a glutamate analog, is the most potent GGT1 inhibitor in vitro. In this study, we have solved the crystal structure of human GGT1 (hGGT1) with ABBA bound in the active site. The structure was interrogated to identify interactions between the enzyme and the inhibitor. Based on these data, a series of novel ABBA analogs were designed and synthesized. Their inhibitory activity against the hydrolysis and transpeptidation activities of hGGT1 were determined. The lead compounds were crystalized with hGGT1 and the structures solved. The kinetic data and structures of the complexes provide new insights into the critical role of protein structure dynamics in developing compounds for inhibition of hGGT1.

Keywords: Computer modeling; Drug development; Enzyme inhibitor; Enzyme structure; Gamma-glutamyl transferase; Gamma-glutamyl transpeptidase; Molecular docking; Molecular modeling; Protein crystallization; Structure-based drug design.

Fig. 1.

Hydrolysis and transpeptidation reactions catalyzed by hGGT1. Yellow highlight denotes atoms of the enzyme. Non-highlighted atoms are the substrate, glutathione. Figure from our prior publication.

Fig. 2.

Structures of GGT1 inhibitors.

Fig. 3.. ABBA in the active site of hGGT1.

(A) Interactions of L/d-ABBA with nearby residues in the glutamyl binding pocket of hGGT1 observed in crystal structures (7LD9). (B) Surface image showing the open pocket adjacent to the nitrogen of d-ABBA. The image is colored according to hydrophobicity of the residues: from blue for the most hydrophilic, to white, to orange red for the most hydrophobic. Enzyme atoms are colored yellow, ABBA atoms are colored orange, oxygens are red, nitrogens are blue, and boron is green. Resolution is 1.42 Å.

Fig. 4.. Interaction diagram of d-ABBA bound to hGGT1.

Hydrogen bonds are shown with arrows, and ionic interactions are shown with colored lines. Note the covalent O-B bond formed between Thr381 and the ligand, denoted by the two lines connecting the Thr381 label to the boron atom.

Fig. 5.

ABBA analogs in the active site of hGGT1. (A) Interactions of Compounds 3 with hGGT1 (7LA5). Resolution is 2.07 Å. (B) Interaction of Compound 14 with hGGT1 (7LBC). Resolution is 2.28 Å. Enzyme atoms are colored yellow, ABBA atoms are colored orange, oxygens are red, nitrogens are blue, and boron is green.

Fig. 6.. Overlapped crystal structures of l-ABBA-bound hGGT1 (orange) and Compound 3-bound hGGT1 (blue).

The yellow highlighted residue is Phe433. Note its “open” and “closed” conformations differ depending on which ligand is bound.

Fig. 7.. Ligand interaction diagram of Compound 14-bound hGGT1.

Note the hydrogen bond (arrow) and salt bridge (line) formed between the terminal amine and Asn401 and Asp423, respectively.

Scheme 1.

Synthesis of l-ABBA. Reagents and conditions: (a) 2.5 equiv of Ipc₂BH, tetrahydrofuran (THF), under N₂, at room temperature for 24 h, used directly for next step without purification; (b) Excess of acetaldehyde, 45 °C overnight; (c) HCl (6 M), boiling 3 h. Overall isolated yield 77 %. The same procedure was used to synthesize d-ABBA starting with the enantiomer of Compound 1.

Scheme 2.

Synthesis of analogs of d-ABBA. (a) SOCl₂, CH₃OH, 4 °C; (b) ArCHO (in 3, 4, 5, 7, 8, 9), or methyl ester of aldehyde (in 6), or N-Boc protected aldehyde (in10–13), NaBH₄; (c) HPLC purification on a C18 column; (d) HCl (6 M), boiling. (*) Compound 3 was hydrophilic and was therefore purified on a silica column rather than by HPLC. Compounds 10–13 were synthetic intermediates and not evaluated as inhibitors of hGGT1.