Alan M. Mathiowetz

Short Hydrophobic Peptides with Cyclic Constraints Are Potent Glucagon-like Peptide-1 Receptor (GLP-1R) Agonists.

Cyclic constraints are incorporated into an 11-residue analogue of the N-terminus of glucagon-like peptide-1 (GLP-1) to investigate effects of structure on agonist activity. Cyclization through linking side chains of residues 2 and 5 or 5 and 9 produced agonists at nM concentrations in a cAMP assay. 2D NMR and CD spectra revealed an N-terminal β-turn and a C-terminal helix that differentially influenced affinity and agonist potency. These structures can inform development of small molecule agonists of the GLP-1 receptor to treat type 2 diabetes.

On-resin N-methylation of cyclic peptides for discovery of orally bioavailable scaffolds

Backbone N-methylation is common among peptide natural products and has a significant impact on both the physical properties and the conformational states of cyclic peptides. However, the specific impact of N-methylation on passive membrane diffusion in cyclic peptides has not been investigated systematically. Here we report a method for the selective, on-resin N-methylation of cyclic peptides to generate compounds with drug-like membrane permeability and oral bioavailability. The selectivity and degree of N-methylation of the cyclic peptide was determined by backbone stereochemistry, suggesting that conformation dictates the regiochemistry of the N-methylation reaction. The permeabilities of the N-methyl variants were corroborated by computational studies on a 1024-member virtual library of N-methyl cyclic peptides. One of the most permeable compounds, a cyclic hexapeptide (MW = 755) with three N-methyl groups, showed an oral bioavailability of 28% in rat.

Human liver glycogen phosphorylase inhibitors bind at a new allosteric site

BACKGROUND Glycogen phosphorylases catalyze the breakdown of glycogen to glucose-1-phosphate for glycolysis. Maintaining control of blood glucose levels is critical in minimizing the debilitating effects of diabetes, making liver glycogen phosphorylase a potential therapeutic target. RESULTS The binding site in human liver glycogen phosphorylase (HLGP) for a class of promising antidiabetic agents was identified crystallographically. The site is novel and functions allosterically by stabilizing the inactive conformation of HLGP. The initial view of the complex revealed key structural information and inspired the design of a new class of inhibitors which bind with nanomolar affinity and whose crystal structure is also described. CONCLUSIONS We have identified the binding site of a new class of allosteric HLGP inhibitors. The crystal structure revealed the details of inhibitor binding, led to the design of a new class of compounds, and should accelerate efforts to develop therapeutically relevant molecules for the treatment of diabetes.

Optimizing PK properties of cyclic peptides: the effect of side chain substitutions on permeability and clearance

A series of cyclic peptides were designed and prepared to investigate the physicochemical properties that affect oral bioavailabilty of this chemotype in rats. In particular, the ionization state of the peptide was examined by the incorporation of naturally occurring amino acid residues that are charged in differing regions of the gut. In addition, data was generated in a variety of in vitro assays and the usefulness of this data in predicting the subsequent oral bioavailability observed in the rat is discussed.

Addressing Limitations with the MM-GB/SA Scoring Procedure using the WaterMap Method and Free Energy Perturbation Calculations

The MM-GB/SA scoring technique has become an important computational approach in drug design. We, and others, have demonstrated that for congeneric molecules the correlation with experimental data obtained with the physics-based scoring is usually superior to scoring functions from typical docking algorithms. Despite showing good accuracy when applied within a series, much work is necessary to improve the MM-GB/SA method in order to gain greater efficiency in drug design. Here, we investigate the poor estimation of protein desolvation provided by the GB/SA solvation model and the large dynamic range observed in the MM-GB/SA scoring compared to that of the experimental data. In the former, replacing the GB/SA protein desolvation in the MM-GB/SA method by the free energy associated with displacing binding site waters upon ligand binding estimated by WaterMap provides the best results when ranking congeneric series of factor Xa and cyclin-dependent kinase 2 (CDK2) inhibitors. However, the improvement is modest over results obtained with the MM-GB/SA and WaterMap methods individually, apparently due to the high correlation between the free energy liberation of the displaced solvent and the protein-ligand van der Waals interactions, which in turn may be interpretable as estimates of the hydrophobic effect and hydrophobic-like interactions, respectively. As for the large dynamic range, comparisons between MM-GB/SA and FEP calculations indicate that for the factor Xa test set this problem has its origin in the lack of shielding effects of protein--ligand electrostatic interactions; that overly favors ligands that engage in hydrogen bonds with the protein.

Probing the Physicochemical Boundaries of Cell Permeability and Oral Bioavailability in Lipophilic Macrocycles Inspired by Natural Products

Cyclic peptide natural products contain a variety of conserved, nonproteinogenic structural elements such as d-amino acids and amide N-methylation. In addition, many cyclic peptides incorporate γ-amino acids and other elements derived from polyketide synthases. We hypothesized that the position and orientation of these extended backbone elements impact the ADME properties of these hybrid molecules, especially their ability to cross cell membranes and avoid metabolic degradation. Here we report the synthesis of cyclic hexapeptide diastereomers containing γ-amino acids (e.g., statines) and systematically investigate their structure-permeability relationships. These compounds were much more water-soluble and, in many cases, were both more membrane permeable and more stable to liver microsomes than a similar non-statine-containing derivative. Permeability correlated well with the extent of intramolecular hydrogen bonding observed in the solution structures determined in the low-dielectric solvent CDCl3, and one compound showed an oral bioavailability of 21% in rat. Thus, the incorporation of γ-amino acids offers a route to increase backbone diversity and improve ADME properties in cyclic peptide scaffolds.

A potentiator of orthosteric ligand activity at GLP-1R acts via covalent modification

We report that 4-(3-(benzyloxy)phenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), which behaves as a positive allosteric modulator at the glucagon-like peptide-1 receptor (GLP-1R), covalently modifies cysteines 347 and 438 in GLP-1R. C347, located in intracellular loop 3 of GLP-1R, is critical to the activity of BETP and a structurally distinct GLP-1R ago-allosteric modulator, N-(tert-butyl)-6,7-dichloro-3-(methylsulfonyl)quinoxalin-2-amine. We further show that substitution of cysteine for phenylalanine 345 in the glucagon receptor is sufficient to confer sensitivity to BETP.

Use of 3D Properties to Characterize Beyond Rule-of-5 Property Space for Passive Permeation

The application of conformationally dependent measures of size and polarity to characterize beyond rule-of-5 (Ro5) space for passive permeation was investigated. Specifically, radius of gyration, an alternative to molecular weight, and three-dimensional polar surface area and the generalized Born/surface area dehydration free energy, alternatives to hydrogen-bond donor and acceptor counts, were computed on models of the permeating conformations of over 35 000 molecules. The resulting guidelines for size and polarity, described by the 3D properties, should aid the design of Ro5 violators with passive permeability.

Allosteric inhibition of fructose-1,6-bisphosphatase by anilinoquinazolines

Anilinoquinazolines currently of interest as inhibitors of tyrosine kinases have been found to be allosteric inhibitors of the enzyme fructose 1,6-bisphosphatase. These represent a new approach to inhibition of F16BPase and serve as leads for further drug design. Enzyme inhibition is achieved by binding at an unidentified allosteric site.

Biaryl-bridged macrocyclic peptides: conformational constraint via carbogenic fusion of natural amino acid side chains.

A general method for constraining peptide conformations via linkage of aromatic sidechains has been developed. Macrocyclization of suitably functionalized tri-, tetra- and pentapeptides via Suzuki-Miyaura cross-coupling has been used to generate side chain to side chain, biaryl-bridged 14- to 21-membered macrocyclic peptides. Biaryl bridges possessing three different configurations, meta-meta, meta-ortho, and ortho-meta, were systematically explored through regiochemical variation of the aryl halide and aryl boronate coupling partners, allowing fine-tuning of the resultant macrocycle conformation. Suzuki-Miyaura macrocyclizations were successfully achieved both in solution and on solid phase for all three sizes of peptide. This approach constitutes a means of constraining peptide conformation via direct carbogenic fusion of side chains of naturally occurring amino acids such as phenylalanine and tyrosine, and so is complementary to strategies involving non-natural, for example, hydrocarbon, bridges.