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The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

The sulfonamide class of antibiotics has been in continuous use for over 70 years. They are thought to act by directly inhibiting dihydropteroate synthase (DHPS), and also acting as prodrugs that sequester pterin pools by forming dead end pterin-sulfonamide conjugates. In this study, eight pterin-sulfonamide conjugates were synthesized using a novel synthetic strategy and their biochemical and microbiological properties were investigated. The conjugates were shown to competitively inhibit DHPS, and inhibition was enhanced by the presence of pyrophosphate that is crucial to catalysis and is known to promote an ordering of the DHPS active site. The co-crystal structure of Yersinia pestis DHPS bound to one of the more potent conjugates revealed a mode of binding that is similar to that of the enzymatic product analog pteroic acid. The antimicrobial activities of the pterin-sulfonamide conjugates were measured against Escherichia coli in the presence and absence of folate precursors and dependent metabolites. These results show that the conjugates have appreciable antibacterial activity and act by an on target, anti-folate pathway mechanism rather than as simple dead end products.