Multivalent scaffolds for affinity maturation of small molecule cell surface binders and their application to prostate tumor targeting.

Adamantane scaffolds for affinity maturation of prostate cancer specific ligands of low molecular mass are described. These scaffolds are modular and can be used for conjugation of up to three ligands and an additional effector molecule by standard peptide coupling techniques. The potential of the scaffolds is demonstrated with the multimerization of GPI 1, a prostate cancer specific small molecule. A detailed study of multimerized GPI conjugates with near-infrared fluorophores and their binding properties to different prostate cancer cell lines shows the specific binding of these conjugates to cell types positive for prostate specific membrane antigen (PSMA). We demonstrate that these conjugates allow the sensitive imaging of prostate cancer cells with NIR methodology and suggest that our adamantane scaffolds might be generally useful for affinity maturation of small molecules targeting cell surface epitopes.

Synthesis and biological evaluation of D-amino acid oxidase inhibitors.

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including d-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[ d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chloro-benzo[ d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with d-serine enhanced the plasma and brain levels of d-serine in rats compared to the oral administration of d-serine alone.

Structural basis of interactions between human glutamate carboxypeptidase II and its substrate analogs.

Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal transduction and intestinal folate absorption by means of the hydrolysis of its two natural substrates, N-acetyl-aspartyl-glutamate and folyl-poly-gamma-glutamates, respectively. During the past years, tremendous efforts have been made toward the structural analysis of GCPII. Crystal structures of GCPII in complex with various ligands have provided insight into the binding of these ligands, particularly to the S1' site of the enzyme. In this article, we have extended structural characterization of GCPII to its S1 site by using dipeptide-based inhibitors that interact with both S1 and S1' sites of the enzyme. To this end, we have determined crystal structures of human GCPII in complex with phosphapeptide analogs of folyl-gamma-glutamate, aspartyl-glutamate, and gamma-glutamyl-glutamate, refined at 1.50, 1.60, and 1.67 A resolution, respectively. The S1 pocket of GCPII could be accurately defined and analyzed for the first time, and the data indicate the importance of Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1) substrate residues. Direct interactions between the positively charged guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor provide mechanistic explanation of GCPII preference for acidic dipeptides. Additionally, observed conformational flexibility of the Arg463 and Arg536 side chains likely regulates GCPII affinity toward different inhibitors and modulates GCPII substrate specificity. The biochemical experiments assessing the hydrolysis of several GCPII substrate derivatives modified at the P1 position, also included in this report, further complement and extend conclusions derived from the structural analysis. The data described here form an a solid foundation for the structurally aided design of novel low-molecular-weight GCPII inhibitors and imaging agents.

Azetidine-based inhibitors of dipeptidyl peptidase IV (DPP IV).

The structure-activity relationships of azetidine-based DPP IV inhibitors will be discussed in detail in the following review. The azetidine-based DPP IV inhibitors can be divided into three main subtypes, the 2-cyanoazetidines, 3-fluoroazetidines and 2-ketoazetidines. These subtypes have been explored and structure-activity relationships have been established by several groups. Several compounds within each of these subtypes display sub micromolar potency against DPP IV. The most potent cyanoazetidines and ketoazetidines have large, hydrophobic amino acid groups bound to the azetidine nitrogen and display activities below 100 nM. DPP IV inhibition is not sensitive to stereochemistry at the 2-position as both 2-(R)- and 2-(S)-cyano and -keto azetidines display similar inhibitory potencies. While these "warhead"-based cyano- and ketoazetidines have the potential for covalent, bond-forming inhibition, they can also react to internally cyclize into inactive ketopiperazines and dihydroketopyrazine. Thus, chemical instability was also explored for compounds in these two subtypes and certain members of the cyanoazetidine series display aqueous stability comparable to the closely related cyanopyrrolidines. Select 3-fluoroazetidines also display inhibitory potencies below 1 microM without the propensity for cyclization and chemical instability associated with the other subseries.

Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors.

In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1' binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the alpha-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6h.

Design and synthesis of poly ADP-ribose polymerase-1 inhibitors. 2. Biological evaluation of aza-5[H]-phenanthridin-6-ones as potent, aqueous-soluble compounds for the treatment of ischemic injuries.

A series of aza-5[H]-phenanthridin-6-ones were synthesized and evaluated as inhibitors of poly ADP-ribose polymerase-1 (PARP-1). Inhibitory potency of the unsubstituted aza-5[H]-phenanthridin-6-ones (i.e., benzonaphthyridones) was dependent on the position of the nitrogen atom within the core structure. The A ring nitrogen analogues (7-, 8-, and 10-aza-5[H]-phenanthridin-6-ones) were an order of magnitude less potent than C ring nitrogen analogues (1-, 2-, 3-, and 4-aza-5[H]-phenanthridin-6-ones). Preliminary stroke results from 1- and 2-aza-5[H]-phenanthridin-6-one prompted structure-activity relationships to be established for several 2- and 3-substituted 1-aza-5[H]-phenanthridin-6-ones. The 2-substituted 1-aza-5[H]-phenanthridin-6-ones were designed to improve the solubility and pharmacokinetic profiles for this series of PARP-1 inhibitors. Most importantly, three compounds from this series demonstrated statistically significant protective effects in rat models of stroke and heart ischemia.

Synthesis and biological evaluation of thiol-based inhibitors of glutamate carboxypeptidase II: discovery of an orally active GCP II inhibitor.

A series of 2-(thioalkyl)pentanedioic acids were synthesized and evaluated as inhibitors of glutamate carboxypeptidase II (GCP II, EC 3.4.17.21). The inhibitory potency of these thiol-based compounds against GCP II was found to be dependent on the number of methylene units between the thiol group and pentanedioic acid. A comparison of the SAR of the thiol-based inhibitors to that of the phosphonate-based inhibitors provides insight into the role of each of the two zinc-binding groups in GCP II inhibition. The most potent thiol-based inhibitor, 2-(3-mercaptopropyl)pentanedioic acid (IC(50) = 90 nM), was found to be orally bioavailable in rats and exhibited efficacy in an animal model of neuropathic pain following oral administration.