Discovery of TP0628103: A Highly Potent and Selective MMP-7 Inhibitor with Reduced OATP-Mediated Clearance Designed by Shifting Isoelectric Points.

Matrix metalloproteinase-7 (MMP-7) has been shown to play an important role in pathophysiological processes such as cancer and fibrosis. We previously discovered selective MMP-7 inhibitors by molecular hybridization and structure-based drug design. However, the systemic clearance (CLtot) of the biologically active lead compound was very high. Because our studies revealed that hepatic uptake by organic anion transporting polypeptide (OATP) was responsible for the high CLtot, we found a novel approach to reducing their uptake based on isoelectric point (IP) values as an indicator for substrate recognition by OATP1B1/1B3. Our "IP shift strategy" to adjust the IP values culminated in the discovery of TP0628103 (18), which is characterized by reduced in vitro OATP-mediated hepatic uptake and in vivo CLtot. Our in vitro-in vivo extrapolation of OATP-mediated clearance and the "IP shift strategy" provide crucial insights for a new medicinal chemistry approach to reducing the systemic clearance of OATP1B1/1B3 substrates.

Structure-Based Optimization and Biological Evaluation of Potent and Selective MMP-7 Inhibitors for Kidney Fibrosis.

Matrix metalloproteinase-7 (MMP-7) has been shown to play important roles in pathophysiological processes involved in the development/progression of diseases such as cancer and fibrosis. We discovered selective MMP-7 inhibitors composed of arylsulfonamide, carboxylate, and short peptides by a molecular hybridization approach. These compounds interacted with MMP-7 via multiple hydrogen bonds in the cocrystal structures. To obtain compounds for in vivo evaluation, we attempted structural optimization, particularly targeting Tyr167 at the S3 subsite through structure-based drug design, and identified compound 15 as showing improved MMP-7 potency and MMP subtype selectivity. A novel π-π stacking interaction with Tyr167 was achieved when 4-pyridylalanine was introduced as the P3 residue. Compound 15 suppressed the progression of kidney fibrosis in a dose-dependent manner in a mouse model of unilateral ureteral obstruction. Thus, we demonstrated, for the first time, that potent and selective MMP-7 inhibitors could prevent the progression of kidney fibrosis.

Discovery of Novel Pyrazolylpyridine Derivatives for 20-Hydroxyeicosatetraenoic Acid Synthase Inhibitors with Selective CYP4A11/4F2 Inhibition.

20-Hydroxyeicosatetraenoic acid (20-HETE) is one of the major oxidized arachidonic acid (AA) metabolites produced by cytochrome P450 (CYP) 4A11 and CYP4F2 isozymes in the human liver and kidney. Numerous studies have suggested the involvement of 20-HETE in the pathogenesis of renal diseases, and suppression of 20-HETE production by inhibition of CYP4A11 and CYP4F2 may be an attractive therapeutic strategy for renal diseases. At first, we identified methylthiazole derivative 2 as a potent dual inhibitor of CYP4A11 and CYP4F2. An optimization study of a series of derivatives with a molecular weight of around 300 to improve aqueous solubility and selectivity against drug-metabolizing CYPs while maintaining the CYP4A11- and CYP4F2-inhibitory activities led to the identification of acetylpiperidine compound 11c. Compound 11c inhibited 20-HETE production in both human and rat renal microsomes and exhibited a favorable pharmacokinetic profile. Furthermore, 11c also significantly inhibited renal 20-HETE production in Sprague-Dawley rats after oral dosing at 0.1 mg/kg.

Discovery of Highly Potent and Selective Matrix Metalloproteinase-7 Inhibitors by Hybridizing the S1' Subsite Binder with Short Peptides.

Matrix metalloproteinase-7 (MMP-7) has emerged as a protein playing important roles in both physiological and pathophysiological processes. Despite the growing interest in MMP-7 as a potential therapeutic target for diseases including cancer and fibrosis, potent and selective MMP-7 inhibitors have yet to be identified. Compound 1, previously reported by Edman and co-workers, binds to the S1' subsite of MMP-7, exhibiting moderate inhibitory activity and selectivity. To achieve both higher inhibitory activity and selectivity, we conceived hybridizing 1 with short peptides. The initially designed compound 6, which was a hybrid molecule between 1 and a tripeptide (Ala-Leu-Met) derived from an MMP-2-inhibitory peptide (APP-IP), showed enhanced MMP-7-inhibitory activity. Subsequent optimization of the peptide moiety led to the development of compound 18 with remarkable potency for MMP-7 and selectivity over other MMP subtypes.

SGL5213, a novel and potent intestinal SGLT1 inhibitor, suppresses intestinal glucose absorption and enhances plasma GLP-1 and GLP-2 secretion in rats.

Sodium-glucose cotransporter 1 (SGLT1) is the primary transporter for glucose absorption from digested nutrients in the gastrointestinal tract. Intestinal SGLT1 inhibition reduces post-prandial hyperglycemia and enhances the increase of plasma glucagon-like peptide-1 (GLP-1) levels. SGL5213 is a novel and potent intestinal SGLT1 inhibitor. This study characterizes the pharmacological profiles of SGL5213 in rodents. Orally administered SGL5213 was hardly absorbed and its distribution was restricted to the gastrointestinal lumen. SGL5213 significantly improved post-prandial hyperglycemia in streptozotocin (STZ)-induced diabetic rats at doses of 1 mg/kg or more. After the oral administration of starch, SGL5213 increased the amount of residual glucose in the small intestine at 1-3 h and in the cecum and colon at 3-9 h by inhibiting glucose absorption and allowing the unabsorbed glucose to be delivered into the lower-gastrointestinal tract. In the vehicle group, the plasma total GLP-1 (tGLP-1) and tGLP-2 levels increased at 15 min and the plasma total glucose-dependent insulinotropic polypeptide (tGIP) level increased at 1 h after meal loading. SGL5213 at doses of 0.1 mg/kg or more enabled the plasma levels of tGLP-1 and tGLP-2 to be retained for a period of 1-6 h, compared with the vehicle group. In contrast, SGL5213 at doses of 0.3 mg/kg or more suppressed the plasma tGIP elevation after meal loading. This study demonstrated for the first time that an intestinal SGLT1 inhibitor enhanced post-prandial plasma GLP-2 secretion. These results suggest that SGL5213 might exhibit a useful pharmacological efficacy through the physiological actions of GLP-1 and GLP-2.

In vitro characterization of luseogliflozin, a potent and competitive sodium glucose co-transporter 2 inhibitor: Inhibition kinetics and binding studies.

In this study, we evaluated an inhibition model of luseogliflozin on sodium glucose co-transporter 2 (SGLT2). We also analyzed the binding kinetics of the drug to SGLT2 protein using [(3)H]-luseogliflozin. Luseogliflozin competitively inhibited human SGLT2 (hSGLT2)-mediated glucose uptake with a Ki value of 1.10 nM. In the absence of glucose, [(3)H]-luseogliflozin exhibited a high affinity for hSGLT2 with a Kd value of 1.3 nM. The dissociation half-time was 7 h, suggesting that luseogliflozin dissociates rather slowly from hSGLT2. These profiles of luseogliflozin might contribute to the long duration of action of this drug.

A Strategy for assessing potential drug-drug interactions of a concomitant agent against a drug absorbed via an intestinal transporter in humans.

A strategy for assessing potential drug-drug interactions (DDIs) based on a simulated intestinal concentration is described. The proposed prediction method was applied to the DDI assessment of luseogliflozin, a novel antidiabetic drug, against miglitol absorbed via the intestinal sodium-glucose cotransporter 1 (SGLT1). The method involves four steps: collection of physicochemical and pharmacokinetic parameters of luseogliflozin for use in a computer simulation; evaluation of the validity of these parameters by verifying the goodness of fit between simulated and observed plasma profiles; simulation of the intestinal luseogliflozin concentration-time profile using the Advanced Compartment Absorption and Transit (ACAT) model in a computer program and estimation of the time spent above a value 10-fold higher than the IC50 value (TAIC) for SGLT1; and evaluation of the DDI potential of luseogliflozin by considering the percentage of TAIC against the miglitol Tmax (time for Cmax) value (TAIC/Tmax). An initial attempt to prove the validity of this method was performed in rats. The resulting TAIC/Tmax in rats was 32%, suggesting a low DDI potential of luseogliflozin against miglitol absorption. The validity was then confirmed using an in vivo interaction study in rats. In humans, luseogliflozin was expected to have no DDI potential against miglitol absorption, since the TAIC/Tmax in humans was lower than that in rats. This prediction was proven, as expected, in a clinical interaction study. In conclusion, the present strategy based on a simulation of the intestinal concentration-time profile using dynamic modeling would be useful for assessing the clinical DDI potential of a concomitant agent against drugs absorbed via an intestinal transporter.

Molecular dissection of the mechanisms of substrate recognition and F-actin-mediated activation of cofilin-phosphatase Slingshot-1.

Slingshot-1 (SSH1), a member of a dual-specificity protein phosphatase family, regulates actin dynamics by dephosphorylating and reactivating cofilin, an actin-depolymerizing factor. SSH1 has the SSH family-specific, N-terminal, noncatalytic (SSH-N) domain, consisting of the A and B subdomains. SSH1 is activated by binding to actin filaments. In this study, we examined the mechanisms of SSH1 substrate recognition of phospho-cofilin (P-cofilin) and SSH1 activation by F-actin. We found that P-cofilin binds to a phosphatase-inactive mutant, SSH1(CS), in which the catalytic Cys-393 is replaced by Ser. Using a series of deletion mutants, we provided evidence that both the phosphatase (P) domain and the adjacent B domain are indispensable for P-cofilin binding of SSH1(CS) and cofilin-phosphatase activity of SSH1. In contrast, the A domain is required for the F-actin-mediated activation of SSH1, but not for P-cofilin binding or basal cofilin-phosphatase activity. The P domain alone is sufficient for the phosphatase activity toward p-nitrophenyl phosphate (pNPP), indicating that the SSH-N domain is not essential for the basal phosphatase activity of SSH1. Addition of F-actin increased the cofilin-phosphatase activity of SSH1 more than 1200-fold, but the pNPP-phosphatase activity only 2.2-fold, which suggests that F-actin principally affects the cofilin-specific phosphatase activity of SSH1. When expressed in cultured cells, SSH1, but not its mutant deleted of SSH-N, accumulated in the rear of the lamellipodium. Together, these findings suggest that the conserved SSH-N domain plays critical roles in P-cofilin recognition, F-actin-mediated activation, and subcellular localization of SSH1.

Actin filaments-stabilizing and -bundling activities of cofilin-phosphatase Slingshot-1.

Slingshot-1 (SSH1) is known to regulate actin filament dynamics by dephosphorylating and activating cofilin, an actin-depolymerizing factor. SSH1 binds to filamentous (F-) actin through its multiple F-actin-binding sites and its cofilin-phosphatase activity is enhanced by binding to F-actin. In this study, we demonstrate that SSH1 has F-actin-stabilizing and -bundling activities. In vitro actin depolymerization assays revealed that SSH1 suppressed spontaneous and cofilin-induced actin depolymerization in a dose-dependent manner. SSH1 inhibited F-actin binding and severing activities of cofilin. Low-speed centrifugation assays combined with fluorescence and electron microscopic analysis revealed that SSH1 has F-actin-bundling activity, independently of its cofilin-phosphatase activity. Deletion of N- or C-terminal regions of SSH1 significantly reduced its F-actin-stabilizing and -bundling activities, indicating that both regions are critical for these functions. As SSH1 does not form a homodimer, it probably bundles F-actin through its multiple F-actin-binding sites. Knockdown of SSH1 expression by RNA interference significantly suppressed stress fiber formation in C2C12 myoblast cells, indicating a role for SSH1 in stress fiber formation or stabilization in cells. SSH1 thus has the potential to regulate actin filament dynamics and organization in cells via F-actin-stabilizing and -bundling activities, in addition to its ability to dephosphorylate cofilin.