Trypsin inhibitors for the treatment of pancreatitis.

Proline-based trypsin inhibitors occupying the S1-S2-S1' region were identified by an HTS screening campaign. It was discovered that truncation of the P1' moiety and appropriate extension into the S4 region led to highly potent trypsin inhibitors with excellent selectivity against related serine proteases and a favorable hERG profile.

Discovery of Orally Active Hydroxyethylamine Based SPPL2a Inhibitors.

SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely described in the literature. Recently, we have disclosed a selective, small molecular weight agent SPL-707 which confirmed that pharmacological inhibition of SPPL2a leads to the accumulation of its substrate CD74/p8 and as a consequence to a reduction in the number of B-cells as well as myeloid dendritic cells in mice. In this paper we describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit, several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410) which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.

Discovery of the First Potent, Selective, and Orally Bioavailable Signal Peptide Peptidase-Like 2a (SPPL2a) Inhibitor Displaying Pronounced Immunomodulatory Effects In Vivo.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1'-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.

Fluorescence Lifetime-Based Competitive Binding Assays for Measuring the Binding Potency of Protease Inhibitors In Vitro.

Fluorescence lifetime (FLT)-based assays have developed to become highly attractive tools in drug discovery. All recently published examples of FLT-based assays essentially describe their use for monitoring enzyme-mediated peptide modifications, such as proteolytic cleavage or phosphorylation/dephosphorylation. Here we report the development of competitive binding assays as novel, inhibitor-centric assays, principally employing the FLT of the acridone dye Puretime 14 (PT14) as the readout parameter. Exemplified with two case studies on human serine proteases, the details of the rationale for both the design and synthesis of probes (i.e., active site-directed low-molecular-weight inhibitors conjugated to PT14) are provided. Data obtained from testing inhibitors with the novel assay format match those obtained with alternative formats such as FLT-based protease activity and time-resolved fluorescence resonance energy transfer-based competitive binding assays.

In vivo imaging with fluorescent smart probes to assess treatment strategies for acute pancreatitis.

BACKGROUND AND AIMS: Endoprotease activation is a key step in acute pancreatitis and early inhibition of these enzymes may protect from organ damage. In vivo models commonly used to evaluate protease inhibitors require animal sacrifice and therefore limit the assessment of dynamic processes. Here, we established a non-invasive fluorescence imaging-based biomarker assay to assess real-time protease inhibition and disease progression in a preclinical model of experimental pancreatitis. METHODS: Edema development and trypsin activation were imaged in a rat caerulein-injection pancreatitis model. A fluorescent "smart" probe, selectively activated by trypsin, was synthesized by labeling with Cy5.5 of a pegylated poly-L-lysine copolymer. Following injection of the probe, trypsin activation was monitored in the presence or absence of inhibitors by in vivo and ex vivo imaging. RESULTS: We established the trypsin-selectivity of the fluorescent probe in vitro using a panel of endopeptidases and specific inhibitor. In vivo, the probe accumulated in the liver and a region attributed to the pancreas by necropsy. A dose dependent decrease of total pancreatic fluorescence signal occurred upon administration of known trypsin inhibitors. The fluorescence-based method was a better predictor of trypsin inhibition than pancreatic to body weight ratio. CONCLUSIONS: We established a fluorescence imaging assay to access trypsin inhibition in real-time in vivo. This method is more sensitive and dynamic than classic tissue sample readouts and could be applied to preclinically optimize trypsin inhibitors towards intrapancreatic target inhibition.

Design, synthesis, and evaluation of indolinones as inhibitors of the transforming growth factor ß receptor I (TGFßRI).

Inhibition of transforming growth factor ß (TGFß) type I receptor (Alk5) offers a novel approach for the treatment of fibrotic diseases and cancer. Indolinones substituted in position 6 were identified as a new chemotype inhibiting TGFßRI concomitant with a low cross-reactivity among the human kinome. A subset of compounds showed additional inhibition of platelet-derived growth factor receptor alpha (PDGFRα), contributing to an interesting pharmacological profile. In contrast, p38 kinase, which is often inhibited by TGFßRI inhibitors, was not targeted by derivatives based on the indolinone chemotype. Guided by an X-ray structure of lead compound 5 (BIBF0775) soaked into the kinase domain of TGFßRI, optimization furnished potent and selective inhibitors of TGFßRI. Potent inhibition translated well into good inhibition of TGFßRI-mediated phosphorylation of Smad2/3, demonstrating efficacy in a cellular setting. Optimized compounds were extensively profiled on a 232-kinase panel and showed low cross-reactivities within the human kinome.