Oligomeric interfaces as a tool in drug discovery: Specific interference with activity of malate dehydrogenase of Plasmodium falciparum in vitro

Abstract

Malaria remains a major threat to human health, as strains resistant to current therapeutics are discovered. Efforts in finding new drug targets are hampered by the lack of sufficiently specific tools to provide target validation prior to initiating expensive drug discovery projects. Thus, new approaches that can rapidly enable drug target validation are of significant interest.In this manuscript we present the crystal structure of malate dehydrogenase from Plasmodium falciparum (PfMDH) at 2.4 Å resolution and structure-based mutagenic experiments interfering with the inter-oligomeric interactions of the enzyme. We report decreased thermal stability, significantly decreased specific activity and kinetic parameters of PfMDH mutants upon mutagenic disruption of either oligomeric interface. In contrast, stabilization of one of the interfaces resulted in increased thermal stability, increased substrate/cofactor affinity and hyperactivity of the enzyme towards malate production at sub-millimolar substrate concentrations. Furthermore, the presented data show that our designed PfMDH mutant could be used as specific inhibitor of the wild type PfMDH activity, as mutated PfMDH copies were shown to be able to self-incorporate into the native assembly upon introduction in vitro, yielding deactivated mutant:wild-type species. These data provide an insight into the role of oligomeric assembly in regulation of PfMDH activity and reveal that recombinant mutants could be used as probe tool for specific modification of the wild type PfMDH activity, thus offering the potential to validate its druggability in vivo without recourse to complex genetics or initial tool compounds. Such tool compounds often lack specificity between host or pathogen proteins (or are toxic in in vivo trials) and result in difficulties in assessing cause and effect—particularly in cases when the enzymes of interest possess close homologs within the human host. Furthermore, our oligomeric interference approach could be used in the future in order to assess druggability of other challenging human pathogen drug targets.

Oligomeric protein interference validates druggability of aspartate interconversion in Plasmodium falciparum

“…In order to validate the druggability of the targeted malate‐aspartate pathway, structure‐based mutagenesis experiments of the two involved genes were performed. We have recently demonstrated that a surface mutation in Pf MDH, where valine 190 was mutated into tryptophan, disrupted the A‐C interface as anticipated, causing significant activity loss (Lunev et al, ). Our previous data suggested that the introduction of the V190W mutation at the oligomeric interface (A‐C) not only caused the splitting of the tetramer into a pair of dimers but it also made the re‐formation of the tetramer highly unlikely due to the introduction of molecular clashes.…”

“…Pf MDH possesses a tetrameric conformation where each monomer is comprised of 326 residues and is composed of two major domains: an N‐terminal cofactor‐binding domain containing a parallel structure of six beta‐sheets (Rossmann‐fold) and C‐terminal substrate‐binding domain. Our previous results suggested that a correctly formed tetrameric assembly of Pf MDH is essential for activity (Lunev et al, ). Indeed, the introduction of a tryptophan residue at one of the interfaces facilitating oligomerization (V190W) results in disruption of the tetramer, breaking it down into two dimers, and a significant reduction in activity.…”

“…As shown by subsequent activity assays, the isolated wild‐type:V190W chimera possessed no detectable activity in either direction, while recombinant wild‐type Pf MDH displayed both reductive and oxidative activity. These data demonstrate that recombinant mutants can be used as specific modifiers of wild‐type Pf MDH activity in vitro, offering the potential to validate it as a drug target, without recourse to complex genetics or initial tool compounds that may display significant off‐target effects (Lunev et al, ). However, neither Pf AspAT nor Pf MDH has as yet been validated as a drug target in vivo.…”

“…The structure Pf MDH has also been recently solved, and we have provided an insight into the role of oligomeric assembly in the regulation of Pf MDH activity (Lunev et al, ). Pf MDH possesses a tetrameric conformation where each monomer is comprised of 326 residues and is composed of two major domains: an N‐terminal cofactor‐binding domain containing a parallel structure of six beta‐sheets (Rossmann‐fold) and C‐terminal substrate‐binding domain.…”

“…Indeed, the introduction of a tryptophan residue at one of the interfaces facilitating oligomerization (V190W) results in disruption of the tetramer, breaking it down into two dimers, and a significant reduction in activity. Co‐purification and western blot experiments with mixed lysates of recombinantly expressed wild‐type Pf MDH (Strep‐tagged) and Pf MDH‐V190W (His 6 ‐tagged) mutant, with a predicted molecular mass of 35.28 and 36.74 kDa for each monomer, respectively, demonstrated that Pf MDH‐V190W was able to insert itself into a pre‐formed wild‐type Pf MDH assembly (Lunev et al, ). As shown by subsequent activity assays, the isolated wild‐type:V190W chimera possessed no detectable activity in either direction, while recombinant wild‐type Pf MDH displayed both reductive and oxidative activity.…”