A novel series of beta-amino amides incorporating fused heterocycles, i.e., triazolopiperazines, were synthesized and evaluated as inhibitors of dipeptidyl peptidase IV (DPP-IV) for the treatment of type 2 diabetes. (2R)-4-Oxo-4-[3-(trifluoromethyl)-5,6-dihydro[1,2,4]triazolo[4,3-a]pyrazin-7(8H)-yl]-1-(2,4,5-trifluorophenyl)butan-2-amine (1) is a potent, orally active DPP-IV inhibitor (IC(50) = 18 nM) with excellent selectivity over other proline-selective peptidases, oral bioavailability in preclinical species, and in vivo efficacy in animal models. MK-0431, the phosphate salt of compound 1, was selected for development as a potential new treatment for type 2 diabetes.

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Summary The insulin receptor (IR) is a dimeric protein that plays a crucial role in controlling glucose homeostasis, regulating lipid, protein and carbohydrate metabolism, and modulating brain neurotransmitter levels1,2. IR dysfunctions have been associated with many diseases, including diabetes, cancer, and Alzheimer’s1,3,4. The primary sequence has been known since the 1980s5, and is composed of an extracellular portion (ectodomain, ECD), a single transmembrane helix and an intracellular tyrosine kinase domain. Insulin binding to the dimeric ECD triggers kinase domain auto-phosphorylation and subsequent activation of downstream signaling molecules. Biochemical and mutagenesis data have identified two putative insulin binding sites (S1 and S2)6. While insulin bound to an ECD fragment containing S1 and the apo ectodomain have been characterized structurally7,8, details of insulin binding to the full receptor and the signal propagation mechanism are still not understood. Here we report single particle cryoEM reconstructions for the 1:2 (4.3 Å) and 1:1 (7.4 Å) IR ECD dimer:Insulin complexes. The symmetric 4.3 Å structure shows two insulin molecules per dimer, each bound between the Leucine-rich sub domain L1 of one monomer and the first fibronectin-like domain (FnIII-1) of the other monomer, and making extensive interactions with the α subunit C-terminal helix (α-CT helix). The 7.4 Å structure has only one similarly bound insulin per receptor dimer. The structures confirm the S1 binding interactions and define the full S2 binding site. These insulin receptor states suggest that recruitment of the α-CT helix upon binding of the first insulin changes the relative sub domain orientations and triggers downstream signal propagation.

The quinazolinone and pyridol-pyrimidine classes of p38 MAP kinase inhibitors have a previously unseen degree of specificity for p38 over other MAP kinases. Comparison of the crystal structures of p38 bound to four different compounds shows that binding of the more specific molecules is characterized by a peptide flip between Met109 and Gly110. Gly110 is a residue specific to the alpha, beta and gamma isoforms of p38. The delta isoform and the other MAP kinases have bulkier residues in this position. These residues would likely make the peptide flip energetically unfavorable, thus explaining the selectivity of binding. To test this hypothesis, we constructed G110A and G110D mutants of p38 and measured the potency of several compounds against them. The results confirm that the selectivity of quinazolinones and pyridol-pyrimidines results from the presence of a glycine in position 110. This unique mode of binding may be exploited in the design of new p38 inhibitors.

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.