Drug design from the cryptic inhibitor envelope

Abstract

Conformational dynamics plays an important role in enzyme catalysis, allosteric regulation of protein functions and assembly of macromolecular complexes. Despite these well-established roles, such information has yet to be exploited for drug design. Here we show by nuclear magnetic resonance spectroscopy that inhibitors of LpxC--an essential enzyme of the lipid A biosynthetic pathway in Gram-negative bacteria and a validated novel antibiotic target--access alternative, minor population states in solution in addition to the ligand conformation observed in crystal structures. These conformations collectively delineate an inhibitor envelope that is invisible to crystallography, but is dynamically accessible by small molecules in solution. Drug design exploiting such a hidden inhibitor envelope has led to the development of potent antibiotics with inhibition constants in the single-digit picomolar range. The principle of the cryptic inhibitor envelope approach may be broadly applicable to other lead optimization campaigns to yield improved therapeutics.

Figure 1. Dynamic access of minor conformational states of LpxC inhibitors containing the threonyl head group.

(a) Crystal structure of the AaLpxC/LPC-011 complex, showing a single trans χ¹ rotamer of the threonyl side chain of the inhibitor. AaLpxC is shown in the cartoon model and catalytically important residues in the stick model. LPC-011 is shown in the stick model, with the purple mesh representing the inhibitor omit map (2mFo-DFc) contoured at 1.0σ. (b) NMR measurements of scalar couplings (³J_NCγ2 and ³J_C'Cγ2) of the threonyl-head-group-containing LpxC inhibitors CHIR-090 (orange) and LPC-011 (blue) reveal a dynamic distribution of all three rotameric χ¹ states. (c) Combining the two most-populated ligand states creates a dynamically accessible inhibitor envelope around the Cβ atom of the threonyl head group. The binding pockets near F180 and H253/K227 are coloured in yellow and grey, respectively, and a third binding pocket accessible to solvent is denoted by an open dashed circle in blue. (d) The Cβ-triply substituted compound LPC-040 occupies all three pockets within the inhibitor envelope. PaLpxC is shown in the cartoon model, with catalytically important residues shown in the stick model. Residue numbering reflects the corresponding residues in AaLpxC, with PaLpxC residue numbers shown in parentheses. LPC-040 is shown in the stick model, with the purple mesh representing the inhibitor omit map (2mFo-DFc) contoured at 1.0σ. (e) Inhibition constants (K_i*) of LpxC inhibitors. Chemical substitutions at the Cβ-position of the inhibitors and their observed (LPC-011 and LPC-040) and predicted (LPC-037) binding modes within the inhibitor envelope are labelled.

Figure 2. Expanded inhibitor envelope enables the design of a potent inhibitor, LPC-058.

(a) Crystal structure of AaLpxC in complex with LPC-023, an isoleucine derivative, reveals a gauche+ χ² rotamer conformation of the inhibitor. AaLpxC is shown in the cartoon model, with the catalytically important residues in the stick model. LPC-023 is shown in the stick model, with the purple mesh representing the inhibitor omit map (2mFo-DFc) contoured at 1.0σ. (b) Combined measurements of the Cδ1 chemical shift and the ³J_CαCδ1 coupling of LPC-023 in the protein-bound complex reveal a dynamic equilibrium between gauche+ and trans χ² rotameric states, with the gauche+ state being the predominant conformation (∼75% population) and the trans state being the minor conformation (∼25%). (c) Design and structural validation of LPC-058 that optimally occupies the inhibitor envelope. PaLpxC is shown in the cartoon model, with the catalytically important residues in the stick model. Residue numbering reflects the corresponding residues of AaLpxC, with PaLpxC numbers shown in parentheses. LPC-058 is shown in the stick model, with the purple mesh representing the inhibitor omit map (2mFo-DFc) contoured at 1.1σ.

Figure 3. LPC-058 is a superior inhibitor compared with the parent compounds LPC-011 and CHIR-090.

(a) Inhibition constants of CHIR-090, LPC-011, LPC-058 and LPC-083. The head group of each compound and its conformation within the inhibitor envelope is denoted. (b) LPC-058 is a potent antibiotic and displays enhanced activity over LPC-011 and CHIR-090 against a diverse array of Gram-negative pathogens. MIC enhancement of >4-fold over LPC-011 and ≥32-fold over CHIR-090 is labelled. Tested bacterial species include E. coli (Ec), P. aeruginosa (Pa), Salmonella typhimurium (St), Vibrio cholerae (Vc), Klebsiella pneumoniae (Kp), Enterobacter cloacae (Ent), Morganella morganii (Mm), Proteus mirabilis (Pm), Chlamydia trachomatis (Ct) and Acinetobacter baumannii (Ab).