Discovery of TP0597850: A Selective, Chemically Stable, and Slow Tight-Binding Matrix Metalloproteinase-2 Inhibitor with a Phenylbenzamide-Pentapeptide Hybrid Scaffold.

Matrix metalloproteinase-2 (MMP2) is a zinc-dependent endopeptidase and a promising target for various diseases, including cancer and fibrosis. Herein, we report the discovery of a novel MMP2-selective inhibitor with high chemical stability and slow tight-binding features. Based on the degradation mechanism of our small-molecule-peptide hybrid 1, the tripeptide linker {5-aminopentanoic acid [Ape(5)]-Glu-Asp} of 1 was replaced by a shorter linker (γ-D-Glu). Phenylbenzamide was suitable for the new generation of MMP2 inhibitors as an S1' pocket-binding group. The introduction of (4S)-aminoproline dramatically increased the chemical stability while maintaining high subtype selectivity because of its interaction with Glu130. TP0597850 (18) exhibited high stability over a wide range of pH values as well as potent MMP2 inhibition (Ki = 0.034 nM) and ≥2000-fold selectivity determined using the inhibition constants. A kinetic analysis revealed that it possesses slow tight-binding nature with a long MMP2 dissociative half-life (t1/2 = 265 min).

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.

Discovery of a potent dual inhibitor of wild-type and mutant respiratory syncytial virus fusion proteins through the modulation of atropisomer interconversion properties.

The development of effective respiratory syncytial virus (RSV) fusion glycoprotein (F protein) inhibitors against both wild-type and the D486N-mutant F protein is urgently required. We recently reported a 15-membered macrocyclic pyrazolo[1,5-a]pyrimidine derivative 4 that exhibited potent anti-RSV activities against not only wild-type, but also D486N-mutant F protein. However, NMR studies revealed that the 15-membered derivative 4 existed as a mixture of atropisomers. An optimization study of the linker moiety between the 2-position of the benzoyl moiety and the 7-position of the pyrazolo[1,5-a]pyrimidine scaffold identified a 16-membered derivative 42c with an amide linker that showed a rapid interconversion of atropisomers. Subsequent optimization of the 5-position of the pyrazolo[1,5-a]pyrimidine scaffold and the 5-position of the benzoyl moiety resulted in the discovery of a potent clinical candidate 60b for the treatment of RSV infections.

Lead optimization of 8-(methylamino)-2-oxo-1,2-dihydroquinolines as bacterial type II topoisomerase inhibitors.

The global increase in multidrug-resistant pathogens has caused severe problems in the treatment of infections. To overcome these difficulties, the advent of a new chemical class of antibacterial drug is eagerly desired. We aimed at creating novel antibacterial agents against bacterial type II topoisomerases, which are well-validated targets. TP0480066 (compound 32) has been identified by using structure-based optimization originated from lead compound 1, which was obtained as a result of our previous lead identification studies. The MIC90 values of TP0480066 against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), and genotype penicillin-resistant Streptococcus pneumoniae (gPRSP) were 0.25, 0.015, and 0.06 µg/mL, respectively. Hence, TP0480066 can be regarded as a promising antibacterial drug candidate of this chemical class.

Lead Identification of 8-(Methylamino)-2-oxo-1,2-dihydroquinoline Derivatives as DNA Gyrase Inhibitors: Hit-to-Lead Generation Involving Thermodynamic Evaluation.

DNA gyrase and topoisomerase IV are well-validated pharmacological targets, and quinolone antibacterial drugs are marketed as their representative inhibitors. However, in recent years, resistance to these existing drugs has become a problem, and new chemical classes of antibiotics that can combat resistant strains of bacteria are strongly needed. In this study, we applied our hit-to-lead (H2L) chemistry for the identification of a new chemical class of GyrB/ParE inhibitors by efficient use of thermodynamic parameters. Investigation of the core fragments obtained by fragmentation of high-throughput screening hit compounds and subsequent expansion of the hit fragment was performed using isothermal titration calorimetry (ITC). The 8-(methylamino)-2-oxo-1,2-dihydroquinoline derivative 13e showed potent activity against Escherichia coli DNA gyrase with an IC50 value of 0.0017 µM. In this study, we demonstrated the use of ITC for primary fragment screening, followed by structural optimization to obtain lead compounds, which advanced into further optimization for creating novel antibacterial agents.

Palmitate induces apoptosis and inhibits osteogenic differentiation of human periodontal ligament stem cells.

OBJECTIVE: The aim of the present study was to investigate the effect of palmitate on human periodontal ligament stem cells (PDLSCs). DESIGN: PDLSCs were isolated from the third molars of healthy adult donors, and cultured in normal or osteogenic medium supplemented with palmitate (0, 100, or 250 µM) for 21 days. Cell proliferation was evaluated by measuring the amount of formazan at 6, 24, 48, and 72 h. Apoptosis was detected by ELISA and terminal deoxynucleotidyl transferase dUTP nick end labeling assay at days 3 and 7. Osteogenic differentiation was evaluated by measuring the alkaline phosphatase (ALP) activity, production of procollagen type I C-peptide and osteocalcin, mineralization, and mRNA expression of Runx2 at days 3, 7, 14, and 21. In addition, mRNA expression of IL-6 and IL-8 was measured at day 3. RESULTS: Palmitate inhibited the proliferation, ALP activity, production of procollagen type I C-peptide and osteocalcin, mineralization, and mRNA expression of Runx2 in the cultured PDLSCs. Palmitate also induced apoptosis and mRNA expression of IL-6 and IL-8 in the PDLSCs. CONCLUSIONS: The results of the present study demonstrate that palmitate induces apoptosis and inhibits osteogenic differentiation of PDLSCs. These findings may help clarify the relationship between palmitate and periodontal tissue regeneration.

Functional 1,3a,6a-triazapentalene scaffold: Design of fluorescent probes for kinesin spindle protein (KSP).

1,3a,6a-Triazapentalene is a compact fluorescent chromophore. In this study, triazapentalene was used to modify a series of biphenyl-type inhibitors of kinesin spindle protein (KSP) to develop fluorescent probes for the intracellular visualization of this protein. Microscopic studies demonstrated that these novel triazapentalene-labeled compounds exhibited inhibitory activity towards KSP in cultured cells and provided important information concerning the intracellular distribution.

Potential new chemotherapy strategy for human ovarian carcinoma with a novel KSP inhibitor.

Among synthetic kinesin spindle protein (KSP) inhibitor compounds, KPYB10602, a six-member lactam-fused carbazole derivative was the most potent in vitro against cell growth of human ovarian cancer, A2780. KPYB10602 caused dose-dependent suppression of tumor growth in vivo. Mitotic arrest due to KPYB10602 was confirmed in vitro, and was characterized by inhibition of securin degradation. Apoptosis after mitotic arrest was associated with an increase in the ratio of pro-apoptotic Bax to anti-apoptotic Bcl-2. Increase of reactive oxygen species (ROS) and caspase pathway were also involved. Furthermore, KPYB10602 caused little neurotoxicity in vivo. Therefore, KPYB10602 could be a promising candidate as an anti-tumor agent with reduced adverse events for treating human ovarian cancer.

Kinesin spindle protein inhibitors with diaryl amine scaffolds: crystal packing analysis for improved aqueous solubility.

Diaryl amine derivatives have been designed and synthesized as novel kinesin spindle protein (KSP) inhibitors based on planar carbazole-type KSP inhibitors with poor aqueous solubility. The new generation of inhibitors was found to show comparable inhibitory activity and high selectivity for KSP, and this was accompanied with improved solubility. Kinetic analysis and molecular modeling studies suggested that these inhibitors work in an ATP-competitive manner via binding to the secondary allosteric site formed by α4 and α6 helices of KSP. Comparative structural investigations on a series of compounds revealed that the higher solubility of diaryl amine-type inhibitors was attributed to fewer van der Waals interactions in the crystal packing and the hydrogen-bond acceptor nitrogen of the aniline moiety for favorable solvation.

Optimization of diaryl amine derivatives as kinesin spindle protein inhibitors.

Structure-activity relationship studies of diaryl amine-type KSP inhibitors were carried out. Diaryl amine derivatives with a pyridine ring or urea group were less active when compared with the parent carboline and carbazole derivatives. Optimization studies of a lactam-fused diphenylamine-type KSP inhibitor revealed that the aniline NH group and 3-CF3 phenyl group were indispensable for potent KSP inhibition. Modification with a seven-membered lactam-fused phenyl group and a 4-(trifluoromethyl)pyridin-2-yl group improved aqueous solubility while maintaining potent KSP inhibitory activity. From these studies, we identified novel diaryl amine-type KSP inhibitors with a favorable balance of potency and solubility.

Structure-activity relationship study of phenylpyrazole derivatives as a novel class of anti-HIV agents.

The structure-activity relationship of phenylpyrazole derivative 1 was investigated for the development of novel anti-HIV agents. Initial efforts revealed that the diazenyl group can be replaced by an aminomethylene group. In addition, we synthesized various derivatives by the reductive amination of benzaldehydes with 5-aminopyrazoles and carried out parallel structural optimization on the benzyl group and the pyrazole ring. This optimization led to a six-fold more potent derivative 32j than the lead compound 1, and this derivative has a 3',4'-dichloro-(1,1'-biphenyl)-3-yl group.

Structure-activity relationships of carboline and carbazole derivatives as a novel class of ATP-competitive kinesin spindle protein inhibitors.

The kinesin spindle protein (KSP) is a mitotic kinesin involved in the establishment of a functional bipolar mitotic spindle during cell division. It is considered to be an attractive target for cancer chemotherapy with reduced side effects. Based on natural product scaffold-derived fused indole-based inhibitors and known biphenyl-type KSP inhibitors, various carboline and carbazole derivatives were synthesized and biologically evaluated. ß-Carboline and lactam-fused carbazole derivatives exhibited remarkably potent KSP inhibitory activity and mitotic arrest in prometaphase with formation of an irregular monopolar spindle. The planar tri- and tetracyclic analogs inhibited KSP ATPase in an ATP-competitive manner just like biphenyl-type inhibitors.