Giovanna Scapin

The crystal structure of human hypoxanthine-guanine phosphoribosyltransferase with bound GMP.

The crystal structure of HGPRTase with bound GMP has been determined and refined to 2.5 A resolution. The enzyme has a core alpha/beta structure resembling the nucleotide-binding fold of dehydrogenases, and a second lobe composed of residues from the amino and carboxy termini. The GMP molecule binds in an anti conformation in a solvent-exposed cleft of the enzyme. Lys-165, which forms a hydrogen bond to O6 of GMP, appears to be critical for determining the specificity for guanine and hypoxanthine over adenine. The location of active site residues also provides evidence for a possible mechanism for general base-assisted HGPRTase catalysis. A rationalization of the effects on stability and activity of naturally occurring single amino acid mutations of HGPRTase is presented, including a discussion of several mutations at the active site that lead to Lesch-Nyhan syndrome.

Structural studies on human muscle fatty acid binding protein at 1.4 å resolution: binding interactions with three C18 fatty acids

BACKGROUND Muscle fatty acid binding protein (M-FABP) is one of a family of cytosolic lipid-binding proteins involved in fatty acid processing. In order to investigate the precise interactions between M-FABP and its ligands and to understand the structural basis of differential binding affinity, we have compared the structures of M-FABP in complex with three C18 fatty acids. RESULTS We describe the crystal structures of M-FABP in complex with n-octadecanoate (stearate), trans-delta 9-octadecenoate (elaidate) and cis-delta 9-octadecenoate (oleate). These structures were refined using least-squares positional and anisotropic temperature factor refinement to final R-factors of 11.4%, 12.1% and 13.2% respectively for all the data between 8.0 A and 1.4 A resolution. CONCLUSIONS Stearate, elaidate and oleate each adopt highly similar U-shaped conformations when they bind to M-FABP within a large interior binding cavity, which also contains 13 ordered water molecules. The atomic structure of the protein is virtually identical, regardless of the nature of the bound ligand. The fatty acid is thought to enter the interior cavity of the protein via a portal in its surface while interior solvent is released through a secondary opening. The ligand affinity can be correlated with the conformational energy and the solubility of the bound ligand.

Omarigliptin (MK-3102): A Novel Long-Acting DPP-4 Inhibitor for Once-Weekly Treatment of Type 2 Diabetes.

In our effort to discover DPP-4 inhibitors with added benefits over currently commercially available DPP-4 inhibitors, MK-3102 (omarigliptin), was identified as a potent and selective dipeptidyl peptidase 4 (DPP-4) inhibitor with an excellent pharmacokinetic profile amenable for once-weekly human dosing and selected as a clinical development candidate. This manuscript summarizes the mechanism of action, scientific rationale, medicinal chemistry, pharmacokinetic properties, and human efficacy data for omarigliptin, which is currently in phase 3 clinical development.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix α7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region α7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Discovery of Potent and Selective Dipeptidyl Peptidase IV Inhibitors Derived from [beta]-Aminoamides Bearing Subsituted Triazolopiperazines

A series of beta-aminoamides bearing triazolopiperazines have been discovered as potent, selective, and orally active dipeptidyl peptidase IV (DPP-4) inhibitors by extensive structure-activity relationship (SAR) studies around the triazolopiperazine moiety. Among these, compound 34b with excellent in vitro potency (IC50 = 4.3 nM) against DPP-4, high selectivity over other enzymes, and good pharmacokinetic profiles exhibited pronounced in vivo efficacy in an oral glucose tolerance test (OGTT) in lean mice. On the basis of these properties, compound 34b has been profiled in detail. Further refinement of the triazolopiperazines resulted in the discovery of a series of extremely potent compounds with subnanomolar activity against DPP-4 (42b- 49b), that is, 4-fluorobenzyl-substituted compound 46b, which is notable for its superior potency (IC50 = 0.18 nM). X-ray crystal structure determination of compounds 34b and 46b in complex with DPP-4 enzyme revealed that (R)-stereochemistry at the 8-position of triazolopiperazines is strongly preferred over (S) with respect to DPP-4 inhibition.

Conformation-assisted Inhibition of Protein-tyrosine Phosphatase-1B Elicits Inhibitor Selectivity over T-cell Protein-tyrosine Phosphatase

PTP-1B represents an attractive target for the treatment of type 2 diabetes and obesity. Given the role that protein phosphatases play in the regulation of many biologically relevant processes, inhibitors against PTP-1B must be not only potent, but also selective. It has been extremely difficult to synthesize inhibitors that are selective over the highly homologous TCPTP. We have successfully exploited the conservative Leu119 to Val substitution between the two enzymes to synthesize a PTP-1B inhibitor that is an order of magnitude more selective over TCPTP. Structural analyses of PTP-1B/inhibitor complexes show a conformation-assisted inhibition mechanism as the basis for selectivity. Such an inhibitory mechanism may be applicable to other homologous enzymes.

EXPRESSION PURIFICATION, AND CRYSTALLIZATION OF MESO-DIAMINOPIMELATE DEHYDROGENASE FROM CORYNEBACTERIUM GLUTAMICUM

The gene encoding the meso‐diaminopimelate dehydrogenase (DAPDH) from Corynebacterium glutamicum was overexpressed and purified to homogeneity. Crystals of the binary DAPDH‐NADP+ complex were obtained from solutions of polyethylene glycol 8000, 100 mM sodium cacodylate, pH 6.5, and 150–300 mM Mg(OAc)2. The crystals diffract to 2.2 Å, belong to the orthorhombic space group P21, and contain two molecules per asymmetric unit.

Crystallization of κ-bungarotoxin: Preliminary X-ray data obtained from the venom-derived protein

Abstract κ-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to α-bungarotoxin and other α-neurotoxins, but differs sharply in physiologic action. The α-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The κ-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The κ-neurotoxins display a low level of interaction with receptors that are strongly affected by α-neurotoxins, but α-neurotoxins are completely without effect on receptors that are affected by κ-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of κ-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A , c = 39.6 A , and angles of α = β = 90° and γ = 120°. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of κ-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the α-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.

A comparative study of the binding properties, dipeptidyl peptidase-4 (DPP-4) inhibitory activity and glucose-lowering efficacy of the DPP-4 inhibitors alogliptin, linagliptin, saxagliptin, sitagliptin and vildagliptin in mice

Since 2006, DPP‐4 inhibitors have become established therapy for the treatment of type 2 diabetes. Despite sharing a common mechanism of action, considerable chemical diversity exists amongst members of the DPP‐4 inhibitor class, raising the question as to whether structural differences may result in differentiated enzyme inhibition and antihyperglycaemic activity.