Small molecule cores demonstrate non-competitive inhibition of lactate dehydrogenase

Abstract

Lactate dehydrogenase (LDH) has recently garnered attention as an attractive target for cancer therapies, owing to the enzyme's critical role in cellular metabolism. Current inhibition strategies, employing substrate or cofactor analogues, are insufficiently specific for use as pharmaceutical agents. The possibility of allosteric inhibition of LDH was postulated on the basis of theoretical docking studies of a small molecule inhibitor to LDH. The present study examined structural analogues of this proposed inhibitor to gauge its potency and attempt to elucidate the molecular mechanism of action. These analogues display encouraging in vitro inhibition of porcine heart LDH, including micromolar K_i values and a maximum inhibition of up to 50% in the steady state. Furthermore, Michaelis-Menten kinetics and fluorescence data both suggest the simple, acetaminophen derivatives are non-competitive in binding to the enzyme. Kinetic comparisons of a panel of increasingly decorated structural analogues imply that the binding is specific, and the small molecule core provides a privileged scaffold for further pharmaceutical development of a novel, allosteric drug.

Fig. 1. Conserved active site architecture of porcine and human LDH. Pig heart LDH, bound oxamate and NADH cofactor shown in greens (PDB ID: 6CEP), human heart LDH and ligands (PDB ID: ; 1I0Z) shown in blues, hydrogen bonding interactions shown in gray, hydride transfer in red. Image created with PyMol (Shroedinger, Version 1.6.8.0).

Fig. 2. EC₅₀ curves for molecules included in JPCL 2016 docking study, along with their structures and docking scores. Relative activity is measured against uninhibited enzyme activity.

Fig. 3. Substrate and cofactor competition assays. (A) and (C), Michaelis–Menten fits and (B) and (D) double reciprocal Lineweaver–Burk plots for substrate competition of 3-AP and DHB, respectively in the presence of 5 nN phLDH. (E) Cofactor competition of 3-AP vs. NADH in the presence of 5 nN phLDH. (F) and (G) Michaelis–Menten fits and Lineweaver–Burk plot for 3-AP in the presence of 3 μN phLDH.

Fig. 4. Single turnover kinetic traces and hydride transfer rate of phLDH.

Fig. 5. Fluorescence spectra and relative intensities in presence of oxamate and inhibitors. Sample concentrations 3 μN phLDH, 6 μM NADH, 5 μM sodium oxamate.