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 Aryloxyphenyl-Heptapeptide Hybrids as Potent and Selective Matrix Metalloproteinase-2 Inhibitors for the Treatment of Idiopathic Pulmonary Fibrosis.

Matrix metalloproteinase-2 (MMP2) is a zinc-dependent endopeptidase that plays important roles in the degradation of extracellular matrix proteins. MMP2 is considered to be an attractive target for the treatment of various diseases such as cancer, arthritis, and fibrosis. In this study, we have developed a novel class of MMP2-selective inhibitors by hybridizing the peptide that binds to a zinc ion and S2-S5 pockets with small molecules that bind to the S1' pocket. Structural modifications based on X-ray crystallography revealed that the introduction of 2,4-diaminobutanoic acid (Dab) at position 4 dramatically enhanced MMP2 selectivity by forming an electrostatic interaction with Glu130. After improving the metabolic and chemical stability, TP0556351 (9) was identified. It exhibited potent MMP2 inhibitory activity (IC50 = 0.20 nM) and extremely high selectivity. It suppressed the accumulation of collagen in a bleomycin-induced idiopathic pulmonary fibrosis model in mice, demonstrating the efficacy of MMP2-selective inhibitors for fibrosis.

EAAT1 variants associated with glaucoma.

Glaucoma is one of the leading causes of blindness characterized by progressive loss of retinal ganglion cells (RGCs) and their axons. We reported that glutamate/aspartate transporter (GLAST) knockout mice showed progressive RGC loss and optic nerve degeneration that are similar to glaucoma. To explore the possibility that rare variants in the EAAT1 gene (the human homolog of GLAST) cause susceptibility to glaucoma, we performed targeted sequencing of EAAT1 in 440 patients with glaucoma and 450 control subjects. We identified 8 rare variants in 20 out of 440 patients, including 4 synonymous and 4 missense variants located at protein coding regions. One of these rare variants (rs117295512) showed significant association with the risk of glaucoma (OR = 10.44, P = 0.005). Furthermore, the allele frequency for loss-of-function EAAT1 variants, pAla169Gly and pAla329Thr, was 5.5 folds higher in the glaucoma (1.1%) compared with the control cohort (0.2%). These findings suggest that these rare variants may contribute to the pathogenesis of glaucoma and that loss-of-function variants in EAAT1 are present in a small number of patients with glaucoma.

Cyclohepta[ b]indole Synthesis through [5 + 2] Cycloaddition: Bifunctional Indium(III)-Catalyzed Stereoselective Construction of 7-Membered Ring Fused Indoles.

A new approach for the synthesis of highly functionalized tetrahydrocyclohepta[ b]indoles through [5 + 2] cycloaddition was developed. Two carbon-carbon bonds were formed by the simple addition of an indium catalyst, which acted as both a π-Lewis acid and σ-Lewis acid to activate the alkyne and unsaturated ester, respectively. The reaction could be applied to various substrates and proceeded regio- and diastereoselectively in all cases.

Comparison of Radioiodine- or Radiobromine-Labeled RGD Peptides between Direct and Indirect Labeling Methods.

Radiolabeled cyclic peptides containing the (Arg-Gly-Asp) RGD sequence for use in positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, and targeted radionuclide therapy of cancer have been reported. In this study, RGD was used as a model carrier peptide for diagnosis and therapy of cancer. To evaluate the characteristics of radiohalogen-labeled peptides, several kinds of labeled RGD peptides [125I-c(RGDyK), 77Br-c(RGDyK), [125I]SIB-c(RGDfK), [77Br]SBrB-c(RGDfK), [125I]SIB-EG2-c(RGDfK), and [77Br]SBrB-EG2-c(RGDfK)] were designed, prepared, and evaluated. In these initial studies, 77Br (t1/2=57.0 h) and 125I (t1/2=59.4 d) were used because of their longer half-lives. Precursor peptides were synthesized using a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase methodology. Radiolabeled peptides were prepared by chloramine-T method or conjugation of RGD peptides with [125I]N-succinimidyl 3-iodobenzoate ([125I]SIB) or [77Br]N-succinimidyl 3-bromobenzoate ([77Br]SBrB). Measurement of the partition coefficients, integrin binding assay, and biodistribution experiments in tumor-bearing mice were performed. 125I and 77Br labeling were successfully performed using similar methods, and in vitro characteristics and biodistributions were similar between the 125I-labeled and corresponding 77Br-labeled peptides. [125I]SIB- and [77Br]SBrB-conjugated RGD peptides showed higher partition coefficients, lower tumor uptakes, and higher intestinal uptake than 125I-c(RGDyK) and 77Br-c(RGDyK). [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK), which possess an ethylene glycol linker, decreased lipophilicity and uptake in intestine compared with [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK), which possess no linker. However, the improvement in biodistribution of [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK)] was insufficient. In conclusion, directly radiohalogenated c(RGDyK) peptides are potentially more useful for tumor imaging and therapy than indirectly radiohalogenated ones.

Relationships Between Trunk Movement Patterns During Lifting Tasks Compared With Unloaded Extension From a Flexed Posture.

OBJECTIVES: The purpose of this study was to investigate between movement patterns of trunk extension from full unloaded flexion and lifting techniques, which could provide valuable information to physical therapists, doctors of chiropractic, and other manual therapists. METHODS: A within-participant study design was used. Whole-body kinematic and kinetic data during lifting and full trunk flexion were collected from 16 healthy male participants using a 3-dimensional motion analysis system (Vicon Motion Systems). To evaluate the relationships of joint movement between lifting and full trunk flexion, Pearson correlation coefficients were calculated. RESULTS: There was no significant correlation between the amount of change in the lumbar extension angle during the first half of the lifting trials and lumbar movement during unloaded trunk flexion and extension. However, the amount of change in the lumbar extension angle during lifting was significantly negatively correlated with hip movement during unloaded trunk flexion and extension (P < .05). CONCLUSIONS: The findings that the maximum hip flexion angle during full trunk flexion had a greater influence on kinematics of lumbar-hip complex during lifting provides new insight into human movement during lifting. All study participants were healthy men; thus, findings are limited to this group.

Trunk lean gait decreases multi-segmental coordination in the vertical direction.

[Purpose] The strategy of trunk lean gait to reduce external knee adduction moment (KAM) may affect multi-segmental synergy control of center of mass (COM) displacement. Uncontrolled manifold (UCM) analysis is an evaluation index to understand motor variability. The purpose of this study was to investigate how motor variability is affected by using UCM analysis on adjustment of the trunk lean angle. [Subjects and Methods] Fifteen healthy young adults walked at their preferred speed under two conditions: normal and trunk lean gait. UCM analysis was performed with respect to the COM displacement during the stance phase. The KAM data were analyzed at the points of the first KAM peak during the stance phase. [Results] The KAM during trunk lean gait was smaller than during normal gait. Despite a greater segmental configuration variance with respect to mediolateral COM displacement during trunk lean gait, the synergy index was not significantly different between the two conditions. The synergy index with respect to vertical COM displacement during trunk lean gait was smaller than that during normal gait. [Conclusion] These results suggest that trunk lean gait is effective in reducing KAM; however, it may decrease multi-segmental movement coordination of COM control in the vertical direction.

The fluorescently responsive 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine discriminates cytidine via the DNA minor groove.

A new environmentally responsive fluorescent nucleoside, 3-(naphthalen-1-ylethynyl)-3-deaza-2'-deoxyguanosine (3nzG), has been synthesized. The nucleoside, 3nzG, exhibited solvatochromic properties and when introduced into ODN probes it was able to recognize 2'-deoxycytidine in target strands by a distinct change in its emission wavelength through probing microenvironmental changes in the DNA minor groove. Thus, 3nzG has the potential for use as a fluorescent probe molecule for micro-structural studies of nucleic acids including the detection of single-base alterations in target DNA sequences.

Construction of Optically Active Isotwistanes and Aminocyclitols Using Chiral Cyclohexadiene as a Common Intermediate.

We have developed a new method for synthesizing chiral isotwistane and homoisotwistane skeletons as well as aminocyclitols in a highly stereoselective manner. These results were achieved through the use of a common intermediate, which was derived from the ytterbium-catalyzed asymmetric Diels-Alder reaction of Danishefsky diene.

Influences of trunk flexion on mechanical energy flow in the lower extremities during gait.

[Purpose] The time-series waveforms of mechanical energy generation, absorption, and transfer through the joints indicate how movements are produced and controlled. Previous studies have used these waveforms to evaluate and describe the efficiency of human movements. The purpose of this study was to examine the influence of trunk flexion on mechanical energy flow in the lower extremities during gait. [Subjects and Methods] The subjects were 8 healthy young males (mean age, 21.8 ± 1.3 years, mean height, 170.5 ± 6.8 cm, and mean weight, 60.2 ± 6.8 kg). Subjects walked at a self-selected gait speed under 2 conditions: normal gait (condition N), and gait with trunk flexion formed with a brace to simulate spinal curvature (condition TF). The data collected from initial contact to the mid-stance of gait was analyzed. [Results] There were no significant differences between the 2 conditions in the mechanical energy flow in the knee joint and negative mechanical work in the knee joint. However, the positive mechanical work of the knee joint under condition TF was significantly less than that under condition N. [Conclusion] Trunk flexion led to knee flexion in a standing posture. Thus, a strategy of moving of center of mass upward by knee extension using less mechanical energy was selected during gait in the trunk flexed posture.

Design and synthesis of environmentally sensitive fluorescent 2-naphthylethynylated 2'-deoxyadenosines: Detection of target DNA via changes in fluorescence intensity and wavelength.

Various C2-naphthylethynylated 2'-deoxyadenosines were synthesized as environmentally sensitive fluorescent (ESF) nucleosides and their photophysical properties were examined. Among the ESF nucleosides synthesized, four exhibited strong solvatochromicity, two of which were incorporated into oligodeoxynucleotides (ODNs). These ODN probes were able to detect target DNA through distinct changes in fluorescence intensity and wavelength and acted as effective reporter probes.

Catalytic Asymmetric Nazarov Cyclization of Heteroaryl Vinyl Ketones through a Crystallographically Defined Chiral Dinuclear Nickel Complex.

A Ni(NTf2)2 and tetradentate bisimino-bisquinoline ligand complex catalyzed the enantioselective Nazarov cyclization of heteroaryl vinyl ketones. An X-ray-quality crystal was obtained from a mixture of the Ni complex and the substrate, which was the dinuclear chiral Ni complex. From information regarding the structure of the complex, the substrate was distorted to form a helical shape, and the carbon atoms involved in bond formation were close to each other. In addition, mechanistic studies revealed that the configuration of the olefin moiety was isomerized before bond formation.

Single mutations introduced in the essential ribosomal proteins L3 and S10 cause a sporulation defect in Bacillus subtilis.

We introduced single mutations into the rplC and rpsJ genes, which encode the essential ribosomal proteins L3 (RplC) and S10 (RpsJ), respectively, and are located in the S10 gene cluster of the gram-positive, endospore-forming bacterium Bacillus subtilis, and examined whether these mutations affected their growth rate, sporulation, competence development and 70S ribosome formation. Mutant cells harboring the G52D mutation in the L3 ribosomal protein, which is located at the peptidyl transferase center of 50S, accumulated 30S subunit at 45°C, probably due to a defect in 50S formation, and exhibited a reduction in the sporulation frequency at high temperature. On the other hand, mutant cells harboring the H56R mutation in the S10 protein, which is located near the aminoacyl-tRNA site of 30S, showed severe growth defect and deficiency in spore formation, and also exhibited significant delay in competence development.

Boundary-element calculations for amplification of effects of low-frequency electric fields in a doublet-shaped biological cell.

To examine the amplification of the effects of low-frequency electric fields due to the junction between the cells, the amplitude of transmembrane potential P(TMP0), the time-averaged normal stress sigma(n) and the outward force density sigma(out) on the shell-phase, and the squared intensity I(ee) of electric fields in the inner and the outer phases induced by uniform external ac fields were calculated with the boundary-element method for doublet-shaped cell models consisting of two spheres of the same size connected by the junction; the results were compared with those in a spherical model. When the external fields were parallel to the long axis of the doublet-shaped models, P(TMP0), sigma(n) and sigma(out) at the pole were greater than those in the spherical model, and sigma(out) and I(ee) at the junction increased with the decrease in the junction-radius. The external fields perpendicular to the long axis caused I(ee) greater than that at the center of the spherical model and negative sigma(out), at the junction. The amplification of P(TMP0), sigma(n), sigma(out) and I(ee) took place within restricted frequency-regions that could be specified by the characteristic frequencies for the frequency-dependence of the polarization factor of the models.