There is a strong link between lipoprotein(a) levels in plasma and cardiovascular disease. Several of the pathological effects associated with lipoprotein(a) are expected to be mediated via kringle domains on apolipoprotein(a). Therefore, we set out to identify inhibitors targeting the small polar lysine binding sites on the kringles. The hit identification campaign followed by structure-based drug design resulted in a compound that specifically bound to kringle IV-10.

We describe here methods and equations to fit ligand affinity from irreversible protein denaturation. Irreversible denaturation occurs for most proteins, particularly in the space of human therapeutics, but equations to fit these data have eluded investigators for many years. These results suggest the kinetic energy barrier for unfolding is similar across proteins; application of these findings should allow investigators to calculate ligand affinity from a single thermal denaturation data point.

Sensitive protein stability assays are crucial to structural and biophysical studies. Here, we describe novel high-throughput 384-well FRET and BRET-based thermostability assay allowing for the ultrasensitive determination of GPCR stability. These assays are functional in crude lysates, without any requirement for protein purification enabling the profiling of molecules at orphan GPCRs for which tracers do not currently exist. 

At JBL, SPR based assay is used for screening fragments to identify binders and classified as HITS. Subsequently these fragment HITS are subjected to X-ray crystallography. Footprints are further harvested by molecular modelling/Medchem team in designing a molecule by linking or and growing approaches from very fragment HITS.

I will discuss how and when we use different biophysical tools (NMR, SPR, ITC, TSA and MST) to validate HTS hits, understand their binding modes and enable lead optimizations. I will present 2 to 3 different case studies from the papers that we have published recently.

Retinoic inducible gene (RIG)-I senses differences between endogenous and viral RNA for triggering immune response through induction of type I interferons. We present structural biophysical characterization of conformational signatures of 5’-ppp versus 5’-OH dsRNA bound forms of full length RIG-I. Our results were analyzed in the context of the recently published SAXS data, and laid foundation for the hypothesis that motif IVa can be involved in RIG-I activation.

We leverage nuclear magnetic resonance in solution to provide fresh insights into the structural mechanisms of partial agonism in human GPCRs. We also describe NMR studies of endogenous GPCR allosteric modulators (e.g. lipids) and their impact on function-related dynamics.