Acetazolamide-based fungal chitinase inhibitors

Abstract

Chitin is an essential structural component of the fungal cell wall. Chitinases are thought to be important for fungal cell wall remodelling, and inhibition of these enzymes has been proposed as a potential strategy for development of novel anti-fungals. The fungal pathogen Aspergillus fumigatus possesses two distinct multi-gene chitinase families. Here we explore acetazolamide as a chemical scaffold for the inhibition of an A. fumigatus 'plant-type' chitinase. A co-crystal structure of AfChiA1 with acetazolamide was used to guide synthesis and screening of acetazolamide analogues that yielded SAR in agreement with these structural data. Although acetazolamide and its analogues are weak inhibitors of the enzyme, they have a high ligand efficiency and as such are interesting leads for future inhibitor development.

Graphical abstract

Figure 1

IC₅₀ curves determined in triplicate, fitted to a four-parameter logistic dose–response curve (minimum, Hill slope, inflection point and maximum) against AfChiA1 for allosamidin (IC₅₀ = 128 μM, Hill slope 0.9), acetazolamide (IC₅₀ = 164 μM, Hill slope 1.1) and 8-chlorotheophylline (IC₅₀ = 410 μM, Hill slope 1.0) using 4-methylumbelliferyl β-d-N,N′,N″-triacetylchitotrioside (4MU-NAG₃) as a substrate.

Figure 2

(A) Structure-based sequence alignment of A. fumigatus ChiA1 and S. cerevisiae CTS1. AfChiA1 secondary structure elements are indicated above the sequence and labelled. Residue numbers are given for AfChiA1. The ScCTS1 sequence is shaded by sequence similarity between the two enzymes shown (black = identical, grey = chemically similar residues), while AfChiA1 is shaded by sequence conservation among A. fumigatus ChiA enzymes (purple = 100% identity, then a gradient from blue (mode identical) to white (less identical)). Residues lining the AfChiA1 active site are highlighted by green filled circles. (B) Acetazolamide (slate) binding to the active site of AfChiA1. The protein is shown as a grey cartoon with the side chains of active site residues shown as sticks and labelled. Unbiased (i.e., calculated before the addition of the ligand to the model) σ_A-weighted F_o − F_c density for acetazolamide contoured at 3.0σ is shown in cyan. Possible hydrogen bonds are indicated as black dotted lines, a water participating in indirect hydrogen bonding between ligand and protein is shown as a red sphere. (C) The active site cavity of AfChiA1 (with bound acetazolamide) coloured by similarity among AfChiA proteins as described for panel A. Y125, the only non-conserved residue of the acetazolamide-binding site, is labelled.

Scheme 1

Synthesis of acetazolomide analogues.