Small molecules play critical roles in human health. These molecular architectures, inspired by natural or unnatural scaffolds, can intervene in specific pathological events while remaining orthogonal to normal physiology. While the potential for small molecule therapy is widely recognized, the role of small molecules as endogenous players in human health is underexplored. The same low molecular weight entities that can intercept pathogens also serve immense and unrecognized roles in native human biology. In the Ondrus lab, we merge the utility of chemical design with discoveries in human chemistry. Our aim is to reveal the dimension of biological information that is, and can be, templated in small molecule structures. To pursue this aim, we use an integrated approach of complex small molecule synthesis, biochemical analysis, and chemical genetics in mammalian cells.

As a launching point, we study the Hedgehog (Hh) signaling pathway, a circuit that links metabolism to development thorough the activities of specific cholesterol metabolites.  Defects within this pathway are embryonic lethal or result in severe congenital syndromes such as holoprosencephaly, polydactyly, and cyclopia. Aberrant re-activation of Gli transcription factors within the Hh pathway is oncogenic and the principal driver of basal cell carcinoma, the most common human cancer. We use this important facet of human physiology to ask questions about how nature operates through chemistry.

What unexplored chemistry exists in nature? One of our favorite proteins is the namesake of the Hh pathway, the Hh morphogen, which undergoes an autocatalytic, covalent ligation to cholesterol.  We have built the first comprehensive model for the domain of the Hh protein known as the Sterol Recognition Region (SRR), which is responsible for chaperoning cholesterol to the active site.  Considering the poor nucleophilicity of cholesterol’s C3-hydroxyl group and its extreme hydrophobicity, we hypothesized that the SRR must serve at least two functions: (1) to recruit the Hh protein to cholesterol-containing cellular membranes, and (2) to facilitate nucleophilic attack of cholesterol during ligation.  Armed with a reactivity portrait of the SRR, we are embarking on the design of tools to study and manipulate Hh cholesterolysis. Our goal is to arrive at a blueprint for immolative ligation of natural and unnatural molecules to control protein activity in living cells.

How does nature leverage small molecule structure to control biological processes? To pursue this question in the context of mammalian biology, we are using the unique activity of specific oxysterols (OHCs) to activate the 7TM receptor Smoothened (Smo).  Smo is susceptible to binding of various cholesterol metabolites in vivo, in a manner that is stereospecific, dynamic, and responsive to cellular metabolism. We seek to investigate this exquisite functional selectivity as a paradigm for stereospecific small molecule-protein interactions in cells.  By identifying the residue-level structural motifs that engage in OHC recognition, these studies will reveal the molecular architecture that defines the roles of OHCs in diverse processes such as viral infection, cancer, and immune response.

How can we use small molecules to reimagine cancer therapeutics? Today we benefit from high-resolution methods in genetic and cellular analysis. Given the complex transcriptional landscapes of the Gli oncogenes and the enormous need for small molecules to intercept their activity, we seek to target specific Gli activities in human cancer. Accordingly, we have capitalized on a recently discovered connection between Gli activity and PKC isozymes that target the Gli oncogenes.  What has emerged from our studies is potent Gli inhibition by a class of cyclic dipeptide molecules that block Gli activity with a potency equal to clinical inhibitors but evade known resistance mechanisms.  We are currently examining the mechanism of Gli inhibition, with the aim of establishing therapeutic inroads to a broad spectrum of Gli driven cancers. Moreover, we aim to pursue this chemical genetics approach to define the activities of molecules in specific transcriptional disease states.

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