Structure-Based Design of AG-946, a Pyruvate Kinase Activator.

Pyruvate kinase (PK) is the enzyme that catalyzes the conversion of phosphoenolpyruvate and adenosine diphosphate to pyruvate and adenosine triphosphate in glycolysis and plays a crucial role in regulating cell metabolism. We describe the structure-based design of AG-946, an activator of PK isoforms, including red blood cell-specific forms of PK (PKR). This was designed to have a pseudo-C2-symmetry matching its allosteric binding site on the PK enzyme, which increased its potency toward PKR while reducing activity against off-targets observed from the original scaffold.

Identification and characterization of an allosteric inhibitory site on dihydropteroate synthase.

The declining effectiveness of current antibiotics due to the emergence of resistant bacterial strains dictates a pressing need for novel classes of antimicrobial therapies, preferably against molecular sites other than those in which resistance mutations have developed. Dihydropteroate synthase (DHPS) catalyzes a crucial step in the bacterial pathway of folic acid synthesis, a pathway that is absent in higher vertebrates. As the target of the sulfonamide class of drugs that were highly effective until resistance mutations arose, DHPS is known to be a valuable bacterial Achilles heel that is being further exploited for antibiotic development.

Replacing sulfa drugs with novel DHPS inhibitors.

More research effort needs to be invested in antimicrobial drug development to address the increasing threat of multidrug-resistant organisms. The enzyme DHPS has been a validated drug target for over 70 years as the target for the highly successful sulfa drugs. The use of sulfa drugs has been compromised by the widespread presence of resistant organisms and the adverse side effects associated with their use.

Structure-based design of novel pyrimido[4,5-c]pyridazine derivatives as dihydropteroate synthase inhibitors with increased affinity.

Dihydropteroate synthase (DHPS) is the validated drug target for sulfonamide antimicrobial therapy. However, due to widespread drug resistance and poor tolerance, the use of sulfonamide antibiotics is now limited. The pterin binding pocket in DHPS is highly conserved and is distinct from the sulfonamide binding site.

Development of a pterin-based fluorescent probe for screening dihydropteroate synthase.

Dihydropteroate synthase (DHPS) is the classical target of the sulfonamide class of antimicrobial agents, whose use has been limited by widespread resistance and pharmacological side effects. We have initiated a structure-based drug design approach for the development of novel DHPS inhibitors that bind to the highly conserved and structured pterin subsite rather than to the adjacent p-aminobenzoic acid binding pocket that is targeted by the sulfonamide class of antibiotics. To facilitate these studies, a robust pterin site-specific fluorescence polarization (FP) assay has been developed and is discussed herein.

Multiple independent binding sites for small-molecule inhibitors on the oncoprotein c-Myc.

Deregulation of the c-Myc transcription factor is involved in many types of cancer, making this oncoprotein an attractive target for drug discovery. One approach to its inhibition has been to disrupt the dimeric complex formed between its basic helix-loop-helix leucine zipper (bHLHZip) domain and a similar domain on its dimerization partner, Max. As monomers, bHLHZip proteins are intrinsically disordered (ID).

Discovery of novel Myc-Max heterodimer disruptors with a three-dimensional pharmacophore model.

A three-dimensional pharmacophore model was generated utilizing a set of known inhibitors of c-Myc-Max heterodimer formation. The model successfully identified a set of structurally diverse compounds with potential inhibitory activity against c-Myc. Nine compounds were tested in vitro, and four displayed affinities in the micromolar range and growth inhibitory activity against c-Myc-overexpressing cells.

Small-molecule perturbation of competing interactions between c-Myc and Max.

The oncogenic transcription factor c-Myc undergoes coupled binding and folding of its basic-helix-loop-helix-leucine zipper domain (bHLHZip) upon heterodimerization with its partner protein Max. The latter exists in two isoforms: p21, which homodimerizes poorly, and p22, which homodimerizes well. We show that the effect of 10058-F4 (a small-molecule that binds disordered c-Myc monomers and disrupts the c-Myc-Max complex) on both c-Myc-Max heterodimerization and DNA binding is dependent on the nature of the Max isoform.

Structural rationale for the coupled binding and unfolding of the c-Myc oncoprotein by small molecules.

The basic-helix-loop-helix-leucine-zipper domains of the c-Myc oncoprotein and its obligate partner Max are intrinsically disordered (ID) monomers that undergo coupled folding and binding upon heterodimerization. We have identified the binding sites and determined the structural means by which two unrelated small molecules, 10058-F4 and 10074-G5, bind c-Myc and stabilize the ID monomer over the highly ordered c-Myc-Max heterodimer. In solution, the molecules bind to distinct regions of c-Myc and thus limit its ability to interact with Max and assume a more rigid and defined conformation.

Improved low molecular weight Myc-Max inhibitors.

Compounds that selectively prevent or disrupt the association between the c-Myc oncoprotein and its obligate heterodimeric partner Max (Myc-Max compounds) have been identified previously by high-throughput screening of chemical libraries. Although these agents specifically inhibit the growth of c-Myc-expressing cells, their clinical applicability is limited by their low potency. We describe here several chemical modifications of one of these original compounds, 10058-F4, which result in significant improvements in efficacy.