Allosteric Regulation of the Mobile Loop in Lactate Dehydrogenase from Plasmodium Falciparum

Abstract

Malaria is still considered one of the deadliest parasitic diseases. Plasmodium parasites that cause malaria, particularly P. falciparum, have become resistant to many commonly used treatments. To combat this illness, we propose developing novel therapeutics that selectively target the mobile loop region of lactate dehydrogenase (LDH), an enzyme that the parasite depends on for survival. In general, LDH catalyzes the interconversion of pyruvate to lactate and NADH to NAD+. The mobile loop of LDH from P. falciparum (pfLDH) is located proximal to the active site and contains five additional residues compared to mammalian LDH (Figure 1). This significant structural change makes the mobile loop region of pfLDH an attractive target for the development of therapeutics, as it contains a catalytically necessary arginine residue that interacts with the substrate upon loop closure. While inhibitors of LDH, both small molecule and peptide‐based, have been previously identified and developed, it is unclear if the mobile loop region of LDH serves as a viable target for drug discovery.Preliminary data recently obtained demonstrates that a previously developed DNA aptamer (2008s), designed to bind to the mobile loop region of pfLDH, inhibits the enzyme selectively over mammalian LDH. The developers of 2008s reported that the aptamer does not affect the kinetics of the enzyme. However, our steady‐state kinetics measurements show that the kcat or turnover number of pfLDH is initially decreased by half while the Km or Michaelis constant remains about the same at the highest aptamer concentration studied (100 nM). The decrease in Kcat with constant Km is characteristic of an uncompetitive inhibitor that does not compete with the substrate for the active site but decreases the overall rate of the enzyme reaction. The kinetic traces for these assays also show that activity fully recovers after approximately 20‐30 seconds, compared to assays performed in the absence of aptamer. Together, these preliminary data suggest that 2008s binds selectively to the pfLDH and inhibit the ternary complex initially; however, once the enzyme begins to turn over, the aptamer dissociates from the enzyme.Our results provide proof of concept that molecules designed to bind to the mobile loop of pfLDH can inhibit the enzyme. MD simulations using GROMACS will be utilized to explore the conformation space of the pfLDH loop motion and compare it to that of hhLDH. Regarding pfLDH, sampling the conformational space of the loop motion will allow for a better understanding of how aptamer binding impacts its dynamics and may also elucidate a druggable site on or near the mobile loop. Additional kinetics studies will be performed at higher aptamer concentrations to determine if more pronounced inhibitory effects are achievable. The development of allosteric inhibitors that bind to and limit the dynamics of the mobile loop region of pfLDH selectively could lead to a paradigm shift concerning the design of novel therapeutics and treatments for malaria and other diseases.