Understanding the Nature of Selective Inhibition of G-Protein βγ Subunit Signaling Pathways by Small Molecules

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Pathology and Laboratory Medicine, 2010.Guanine nucleotide exchange proteins (G proteins) are heterotrimeric proteins composed of three subunits α, β, γ that initiate intracellular signaling cascades. Previously our laboratory identified small molecules that bind to Gβγ and interfere with or enhance binding of select effectors with Gβγ. To understand the molecular mechanisms of selectivity we adopted a hypothesis of different compounds binding directly to different sub-surfaces on the Gβγ ‘hot spot’. To assess binding of compounds directly to Gβγ we developed a Surface Plasmon resonance (SPR) method utilizing immobilized Gβγ subunits. The results indicated that multiple compounds bound directly and non-covalently to Gβγ with affinities in the range of 400 nM to 10 mM. The binding was reversible, but with surprisingly slow on and off rate kinetics. For example; the Kd for binding of a small molecule gallein to Gβ1γ2 was 420 nM with kon=1 x 103 M-1s-1, koff= 4 x 10-4 s-1. To define the location and binding modes for these small molecules we used a combination of X-ray crystallography and Alanine-scanning mutagenesis. The mutagenesis strategy involved separately purifying Gβγ subunits with single amino acids within the Gβγ hotspot were mutated to alanine, and then assessing small molecule binding by SPR. The results showed that at least two groups of small molecules were affected by mutations on two critical hydrophobic residues, Leu117 and Tyr145, while another group remained unaffected confirming our initial hypothesis. We also obtained co-crystals of several small molecules bound to Gβγ that diffracted X-rays between 2-3Å resolution. One of these was a co-crystal of the protein in complex with NSC201400 (M201) and Gβ1γ2C68S that we solved and refined to a resolution of 2.12 Å. The asymmetric unit of this crystal form comprises a dimer with a nearly perfect dyad with the hot spots oriented in direct opposition. M201 bridges the dimer of Gβγ molecules, binding to the symmetry related amino acids in the hot spots of each subunit. Although the electron density suggested multiple conformational states, the tri-heterocylic structure of M201 was seen to bind within a distinct hydrophobic pocket on Gβγ that lines the central cavity of the β propeller domain. One conformation showed hydrogen bonding of a specific amine on M201 to a network of hydrogen bonded water molecules within the central tunnel of Gβγ. Structure activity relationship analysis using SPR and an in vitro reconstituted phospholipase Cβ2 (PLCβ2) assay confirmed that the compound bound to Leu117 and Tyr145 on Gβγ (as predicted from the X-ray structure) and the importance of the amine, removal of which resulted in attenuation of binding and functional effects. We were further able to determine that this binding mode is shared by at least one other class of small molecules all of which were able to potentiate Gβγ’s ability to increase PLCβ2 enzyme activity, a result achieved without increasing Gβγ’s affinity towards PLCβ2. The insights gained from this study on the molecular mechanisms of inhibition of Gβγ signaling by specific small molecules should lead to the development of more potent and selective drugs that target human diseases resulting from altered G-protein signaling