Michael Robert Wiley

Small-molecule direct thrombin inhibitors

Because of its central role in the control of thrombus formation, the trypsin-like serine protease thrombin is an important target for therapeutic intervention in thrombotic disease. The patent literature from the past several years illustrates that the design of low molecular weight, active-site thrombin inhibitors has followed several major themes. These include tripeptides and peptide mimimics of the cleavage site of fibrinogen, analogues of tertiary arginine amides, and non-peptide inhibitors derived from screening and rational design. This report reviews the concurrent development of these approaches.

D-Phe-Pro-p-Amidinobenzylamine: A potent and highly selective thrombin inhibitor

Abstract The design, synthesis, and enzyme inhibitory profile of D-Phe-Pro-p-Amidinobenzylamine are presented. This compound has inhibitory activity equivalent to D-Phe-Pro-Arg-H, two orders of magnitude more potent than D-Phe-Pro-Agmatine. The results indicate that binding energy provided by the covalent bond of a transition-state analog can be replaced with noncovalent interactions.

Serine protease selectivity of the thrombin inhibitor D-Phe-Pro-Agmatine and its homologs

Abstract Analogs of D-Phe-Pro-Agmatine were assayed for inhibititory activity versus thrombin, trypsin, plasmin, n-tPA and urokinase. The X-ray structure of the thrombin/D-Phe-Pro-Agmatine co-crystal revealed that the agmatine and analogous arginals have very similar bound conformations.

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine

OBJECTIVE Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

A four component coupling strategy for the synthesis of d-phenylglycinamide-derived non-covalent factor Xa inhibitors

A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.

The design of potent, selective, non-covalent, peptide thrombin inhibitors utilizing imidazole as a S1 binding element

Modeling of neutral or mildly basic functional groups in the S1 site of thrombin led to the targeting of imidazole as a S1 binding element and correctly predicted the optimal chain length for connecting this group with the S2 and S3 binding elements. Derivatives of 4-(3-aminopropyl)-imidazole can be selective inhibitors of thrombin demonstrating potent anticoagulant activity.

Identification of potent and selective hydantoin inhibitors of aggrecanase-1 and aggrecanase-2 that are efficacious in both chemical and surgical models of osteoarthritis.

A disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS-4) and ADAMTS-5 are zinc metalloproteases commonly referred to as aggrecanase-1 and aggrecanase-2, respectively. These enzymes are involved in the degradation of aggrecan, a key component of cartilage. Inhibitors of these enzymes could be potential osteoarthritis (OA) therapies. A series of hydantoin inhibitors of ADAMTS-4 and ADAMTS-5 were identified from a screening campaign and optimized through structure-based drug design to give hydantoin 13. Hydantoin 13 had excellent selectivity over other zinc metalloproteases such as TACE, MMP2, MMP3, MMP13, and MMP14. The compound also produced efficacy in both a chemically induced and surgical model of OA in rats.

Preparation of 2-hydroxybenzamidines from 3-aminobenzisoxazoles

Abstract 2-Hydroxybenzamidines have been prepared from 3-aminobenzisoxazoles by reductive cleavage of the nitrogen–oxygen bond using catalytic hydrogenation, Zn/AcOH or NiCl 2 /NaBH 4 . This ring-opening reaction can be accomplished chemoselectively in the presence of a variety of hydrogenation-sensitive functional groups including an aryl bromide, benzyl carbamate, and olefin.

N-substituted glycines as replacements for proline in tripeptide aldehyde thrombin inhibitors

Abstract We have prepared a series of tripeptide arginine aldehydes in which the P2 proline has been replaced with a variety of N -substituted glycines. The effects of these modifications on thrombin inhibitory potency and serine protease selectivity were evaluated.