Identification of an Orally Efficacious GPR40/FFAR1 Receptor Agonist.

GPR40/FFAR1 is a G protein-coupled receptor predominantly expressed in pancreatic ß-cells and activated by long-chain free fatty acids, mediating enhancement of glucose-stimulated insulin secretion. A novel series of substituted 3-(4-aryloxyaryl)propanoic acid derivatives were prepared and evaluated for their activities as GPR40 agonists, leading to the identification of compound 5, which is highly potent in in vitro assays and exhibits robust glucose lowering effects during an oral glucose tolerance test in nSTZ Wistar rat model of diabetes (ED50 = 0.8 mg/kg; ED90 = 3.1 mg/kg) with excellent pharmacokinetic profile, and devoid of cytochromes P450 isoform inhibitory activity.

A flexible domain is essential for the large step size and processivity of myosin VI.

Myosin VI moves processively along actin with a larger step size than expected from the size of the motor. Here, we show that the proximal tail (the approximately 80-residue segment following the IQ domain) is not a rigid structure but, rather, a flexible domain that permits the heads to separate. With a GCN4 coiled coil inserted in the proximal tail, the heads are closer together in electron microscopy (EM) images, and the motor takes shorter processive steps. Single-headed myosin VI S1 constructs take nonprocessive 12 nm steps, suggesting that most of the processive step is covered by a diffusive search for an actin binding site. Based on these results, we present a mechanical model that describes stepping under an applied load.

Dynamics of the core tryptophan during the formation of a productive molten globule intermediate of barstar.

The evolution of the nanosecond dynamics of the core tryptophan, Trp53, of barstar has been monitored during the induction of collapse and structure formation in the denatured D form at pH 12, by addition of increasing concentrations of the stabilizing salt Na(2)SO(4). Time-resolved fluorescence methods have been used to monitor the dynamics of Trp53 in the intermediates that are populated during the salt-induced transition of the D form to the molten globule B form. The D form approximates a random coil and displays two rotational correlation times. A long rotational correlation time of 2.54 ns originates from segmental mobility, and a short correlation time of 0.26 ns originates from independent motion of the tryptophan side chain. Upon addition of approximately 0.1 M Na(2)SO(4), the long rotational correlation time increases to approximately 6.4 ns, as the chain collapses and the segmental motions merge to reflect the global tumbling motion of a pre-molten globule P form. The P form exists as an expanded form with approximately 30% greater volume than the native (N) state. The persistence of an approximately 50% contribution to anisotropy decay by the short rotational correlation time suggests that the core of the P form is highly molten and permits free rotation of the Trp side chain. With increasing salt concentrations, tight core packing is achieved before secondary and tertiary structure formation is complete, an observation which agrees well with earlier kinetic folding studies. Thus, the equilibrium model developed here for describing the formation of structure during folding faithfully captures snapshots of transient kinetic intermediates observed on the folding pathway of barstar. A comparison of the refolding kinetics at pH 7, when refolding is initiated from the D, P, and B forms, suggests that formation of a collapsed state with a rigid core and approximately 30% secondary and tertiary structure, which presumably defines a coarse native-like topology, constitutes the intrinsic barrier in the folding of barstar.

Mechanism of formation of a productive molten globule form of barstar.

Structural analysis of the initial steps in protein folding is difficult because of the swiftness with which these steps occur. Hence, the link between initial polypeptide chain collapse and formation of secondary and other specific structures remains poorly understood. Here, an equilibrium model has been developed for characterizing the initial steps of folding of the small protein barstar, which lead to the formation of a productive molten globule in the folding pathway. In this model, the high-pH-unfolded form (D form) of barstar, which is shown to be as unstructured as the urea-denatured form, is transformed progressively into a molten globule B form by incremental addition of the salt Na(2)SO(4) at pH 12. At very low concentrations of Na(2)SO(4), the D form collapses into a pre-molten globule (P) form, whose volume exceeds that of the native (N) state by only 20%, and which lacks any specific structure as determined by far- and near-UV circular dichroism. At higher concentrations of Na(2)SO(4), the P form transforms into the molten globule (B) form in a highly noncooperative transition populated by an ensemble of at least two intermediates. The B form is a dry molten globule in which water is excluded from the core, and in which secondary structure develops to 65% and tertiary contacts develop to 40%, relative to that of the native protein. Kinetic refolding experiments carried out at pH 7 and at high Na(2)SO(4) concentrations, in which the rate of folding of the D form to the N state is compared to that of the B form to the N state, indicate conclusively that the B form is a productive intermediate that forms on the direct pathway of folding from the D form to the N state.