Structural Characterization of Mycobacterium abscessus Phosphopantetheine Adenylyl Transferase Ligand Interactions: Implications for Fragment-Based Drug Design

Abstract

Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (K_d 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.

Keywords: CoaD/ PPAT; Coenzyme A pathway; Mycobacterium abscessus; Mycobacterium tuberculosis; antibiotics; drug discovery; fragment-based.

FIGURE 1

(A) Crystal structure of M. abscessus PPAT hexamer (grey) in complex with ATP (PDB code 7YWM). The detailed interaction map is also shown with ATP in salmon stick model and interacting residues in grey line representation. Polar hydrogen bond contacts are shown in black dotted lines and active site Mg²⁺ as green sphere. (B) Biosynthetic pathway of coenzyme A in bacteria. The step catalyzed by PPAT enzyme is highlighted in black box. Corresponding E. coli gene names are given in brackets. *These two steps are catalyzed by a single polypeptide in bacteria. (C) Mab PPAT in complex with 4′-phosphopantetheine (PhP) (PDB code 7YY0) showing interacting residues in grey and PhP in blue stick model. (D) Crystal structure of M. abscessus PPAT in ternary complex with substrates, 4′-phosphopantetheine (PhP) and non-hydrolysable ATP analogue AMPCPP, solved at 1.62 Å (PDB code 7YY1). PhP is shown in green, AMPCPP in blue stick models. Mab PPAT active site is shown as a space-filling model coloured according to surface electrostatics. Structural superposition with Mab PPAT:dpCoA (yellow stick model) shows close agreement in the positions of substrates and product.

FIGURE 2

(A) Structural superposition of apo Mab PPAT in complex with feed-back inhibitor CoA (grey), PDB code 7YXZ and product dpCoA (green), PDB code 5O08, showing conformational differences in the enzyme and ligand binding mode. (B) The pantetheine moiety of CoA adopts a similar conformation to that of dpCoA. However, the CoA adenosine moiety is seen very flexible and largely solvent exposed in comparison to dpCoA in the Mab PPAT active site. (C) Thermodynamic (ITC) profile of CoA interaction with Mab PPAT.

FIGURE 3

(A) Structural superposition of six representative M. abscessus PPAT crystal structures bound to fragments 1(purple), 2(white), 3(green), 4(blue), 5(gold) and 6(turquoise), showing fragments occupying four distinct regions in the PPAT active site. Mab PPAT is shown as surface electrostatic representation and grey cartoon model. (B) Fragment linking scheme leading to compound 20, originally designed to target M. tuberculosis PPAT and the corresponding biophysical data (thermal shift (ΔT_m) and binding affinity (K_d) for M. abscessus PPAT ortholog, determined in this study (C) Superposition of compound 20 bound Mab PPAT (salmon) PDB code 7YYZ and Mtb PPAT (green), PDB code 6QMH structures showing close similarities in overall binding mode at the enzyme adenyl pocket.

FIGURE 4

(A) Active site diagram showing binding interactions of compound 20 (salmon stick model) with Mab PPAT (grey). The interacting residues are shown as green stick representation. Polar hydrogen bond contacts are shown in black dotted lines and active site water molecules as red spheres. (B) Thermodynamic (ITC) profiles of compound 20 interaction with Mab PPAT. Surface electrostatic representation of M. abscessus PPAT illustrating (C) apo Mab PPAT (PDB code 5O06) having an open and predominantly negatively charged solvent channel and (D) Compound 20 (green spherical representation) bound form with closed conformation of the central solvent channel (PDB code 7YYZ). The key amino acid residues mediating the conformational switch are shown as grey stick model.