Targeted stabilization of Munc18-1 function via pharmacological chaperones.

Heterozygous de novo mutations in the neuronal protein Munc18-1 cause syndromic neurological symptoms, including severe epilepsy, intellectual disability, developmental delay, ataxia, and tremor. No disease-modifying therapy exists to treat these disorders, and while chemical chaperones have been shown to alleviate neuronal dysfunction caused by missense mutations in Munc18-1, their required high concentrations and potential toxicity necessitate a Munc18-1-targeted therapy. Munc18-1 is essential for neurotransmitter release, and mutations in Munc18-1 have been shown to cause neuronal dysfunction via aggregation and co-aggregation of the wild-type protein, reducing functional Munc18-1 levels well below hemizygous levels. Here, we identify two pharmacological chaperones via structure-based drug design, that bind to wild-type and mutant Munc18-1, and revert Munc18-1 aggregation and neuronal dysfunction in vitro and in vivo, providing the first targeted treatment strategy for these severe pediatric encephalopathies.

Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c).

The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity.

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. In the presence of p22 Max the effective inhibitor concentration is lower than in the presence of p21 Max, as the p22 Max homodimer formation affects the thermodynamics by competing against the c-Myc-Max heterodimerization event.