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 inhibition of porcine heart LDH, including micromolar 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.

Inline SAXS-coupled chromatography under extreme hydrostatic pressure

“…21 At ambient pressure (4°C), LDH elutes as a broadened peak preceded by a small shoulder of aggregates or higher oligomers ( Figure 6A ). The estimated molecular weight is constant across the peak and closely matches the known molecular weight of the LDH homotetramer (149.61 kDa) 22…”

“…21 At ambient pressure (4ºC), LDH elutes as a broadened peak preceded by a small shoulder of aggregates or higher oligomers (Figure 6A). The estimated molecular weight is constant across the peak and closely matches the known molecular weight of the LDH homotetramer (149.61 kDa) 22 At 100 MPa (4ºC), an additional peak appears in the elution profile (Figure 6B) and the estimated molecular weight falls appreciably, though not fully to the expected dimer value (74.8 kDa). Toward the end of the second peak, the estimated molecular weight, though very noisy, settles near the monomer value (37.4 kDa).…”

“…Toward the end of the second peak, the estimated molecular weight, though very noisy, settles near the monomer value (37.4 kDa). As in the previous case, the known crystal structure of porcine heart LDH (PDB ID: 6CEP) 22 was used to compute the volume fraction of tetramer and dimer at each point along the elution profiles. LDH is commonly referred to as a dimer of dimers where the proposed solution-state dimer conserves a strong intermolecular interaction, an alpha-helical barrel between the monomeric units.…”

Structural Evidence for Isoform-Selective Allosteric Inhibition of Lactate Dehydrogenase A

“…Indeed, allosteric activation by fructose-1,6-bisphosphate (FBP) has been described for the lactate dehydrogenase of Bifidobacterium longum, and the option of allosteric LDH inhibition has been postulated based on studies recording dynamic motions within LDH proteins. , Additional studies demonstrated the disruption of rate-promoting vibrations (RPVs) upon binding of 2-chloro- N -(3,5-dihydroxyphenyl)­acetamide (CPA; selected by docking studies) to a predicted pocket in direct proximity of the active site in human heart LDH (hLDHB) . Building on that, Andrews and Dyer have recently identified partial allosteric inhibitors of porcine heart LDH and reported a noncompetitive inhibitory activity for 3-acetamidophenol (3-AP) in the micromolar range in vitro (IC 50 = 78 ± 21 μM) …”

“…37 Building on that, Andrews and Dyer have recently identified partial allosteric inhibitors of porcine heart LDH and reported a noncompetitive inhibitory activity for 3-acetamidophenol (3-AP) in the micromolar range in vitro (IC 50 = 78 ± 21 μM). 38 We herein present two isoform-selective LDHA inhibitors with sub-micromolar activities in vitro and demonstrate both target engagement and inhibitor binding to a novel allosteric site with the help of protein−ligand crystal structures. Given that no crystal structures of human LDHA or homologous proteins deposited to date exhibit this binding-competent allosteric pocket in an open conformation (Supporting Information, Figure 1), it is tempting to speculate that binding of 3 and 7 may well result from ligand-dependent conformational selection.…”