Low Activity Oligomers of Porphobilinogen Synthase as Antibiotic Targets

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Abstract: Allosteric regulation through the interconversion of functionally distinct non-additive quaternary structure assemblies has recently been described for the essential enzyme porphobilinogen synthase (PBGS). The oligomeric rearrangements seen with PBGS are different from the MWC or Koshland models for allostery; thus, the term “morpheein” has been introduced to describe this structural basis for allostery. Small molecules that can stabilize either an active or inactive quaternary structure assembly, herein called morphlocks, are proposed to function as therapeutic agents. Stabilization of an inactive quaternary structure assembly of porphobilinogen synthase (PBGS) is proposed as the basis for a new class of antibiotic agents. Although PBGS, which catalyzes the first common step in the biosynthesis of the tetrapyrrole cofactors (e.g. heme), is essential in both humans and most human pathogens, there is extensive phylogenetic variation in the putative morphlock binding sites. This allows us to target PBGS for drug development. There are four Specific Aims. Aim 1 is the application of in silico methods (protein structure modeling and docking) to the discovery of morphlocks that will trap inactive quaternary structure assemblies of PBGS from the human pathogens Pseudomonas aeruginosa, Vibrio cholera, Toxoplasma gondii, and Plasmodium falciparum. Post-docking criteria will be used to select compounds for purchase. Aim 2 is the in vitro testing of the purchased compounds as inhibitors of PBGS from the targeted pathogens. Using native gel shift analysis and extensive kinetic criteria, purchased compounds will be carefully and critically evaluated to select inhibitors that function through the oligomer stabilization mechanism and with species selectivity. Aim 3 is the in vivo testing of chosen morphlocks using a human cell culture system and standard microbiological assays. In Aim 4 we will use X-ray crystallography to evaluate the structures of the morphlock-PBGS complexes. To further probe the structure/function of select morphlocks, we will synthesize and evaluate structural analogs. The proposed methods have been used to discover the first morphlock, morphlock-1, which inhibits a plant PBGS in a species selective fashion by trapping the inactive hexameric assembly. Should the proposed morpheein mechanism for allosteric regulation be applicable to other medically important proteins, the proposed work will serve as a roadmap for harnessing this allosteric mechanism for drug discovery.