Discovery of novel indole derivatives as potent and selective inhibitors of proMMP-9 activation.

Matrix metalloproteinase-9 (MMP-9) is a secreted zinc-dependent endopeptidase that degrades the extracellular matrix and basement membrane of neurons, and then contributes to synaptic plasticity by remodeling the extracellular matrix. Inhibition of MMP-9 activity has therapeutic potential for neurodegenerative diseases such as fragile X syndrome. This paper reports the molecular design, synthesis, and in vitro studies of novel indole derivatives as inhibitors of proMMP-9 activation. High-throughput screening (HTS) of our internal compound library and subsequent merging of hit compounds 1 and 2 provided compound 4 as a bona-fide lead. X-ray structure-based design and subsequent lead optimization led to the discovery of compound 33, a highly potent and selective inhibitor of proMMP-9 activation.

Lead identification of novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid derivative as a potent heparanase-1 inhibitor.

Heparanase-1 (HPSE1) is an endo-ß-d-glucuronidase that cleaves heparan sulfate proteoglycans into short-chain heparan sulfates (HS). The inhibition of HPSE1 has therapeutic potential for proteinuric diseases such as nephrotic syndrome because increased HPSE1 expression is associated with the loss of HS in the glomerular basement membrane, leading to the development of proteinuria. The present study examined the generation of a lead compound focusing on chemical structures with a sugar moiety, such as glycosides and sugar analogs, taking their physical properties into consideration. Compound 10, an exo-ß-d-glucuronidase (GUSß) inhibitor, was found to have a weak inhibitory activity against endo-ß-d-glucuronidase HPSE1. A structure-activity relationship study using the X-ray co-crystal structure of 10 and HPSE1 resulted in 12a, which showed a more than 14-fold increase in HPSE1 inhibitory activity compared with that of 10. Compound 12a could be a novel lead compound for the development of a potent HPSE1 inhibitor.

Structure-based lead optimization to improve potency and selectivity of a novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid series of heparanase-1 inhibitor.

Heparanase-1 (HPSE1) is an endo-ß-d-glucuronidase that is the only mammalian enzyme known to cleave heparan sulfate (HS) of heparan sulfate proteoglycans (HSPG), a key component of the glycocalyx layer of the vascular endothelium matrix. Inhibition of HPSE1 has therapeutic potential for cancer and proteinuric kidney diseases. We previously reported that 2 showed a moderate potency as an HPSE1 inhibitor and an issue of selectivity against exo-ß-d-glucuronidase (GUSß) and glucocerebrosidase (GBA) remained. A structure-based lead optimization of 2 using X-ray co-crystal structure analysis and fragment molecular orbital calculation resulted in 4e, which showed a more than 7-fold increase in HPSE1 inhibitory activity. The subsequent introduction of a methyl group into the 6-hydroxy group of 4e resulted in 18 with reduced inhibitory activities against GUSß and GBA while maintaining the inhibitory activity against HPSE1. The inhibitory activities of 18 against serum HPSE1 in mice were significant and lasted for 4 h at doses of 3, 30, and 100 mg/kg. Compound 18 could be a novel lead compound for HPSE1 inhibitors with improved inhibitory activity against HPSE1 and increased HPSE1 selectivity over GUSß and GBA.

Lead optimization of 2-hydroxymethyl imidazoles as non-hydroxamate LpxC inhibitors: Discovery of TP0586532.

Infectious diseases caused by resistant Gram-negative bacteria have become a serious problem, and the development of therapeutic drugs with a novel mechanism of action and that do not exhibit cross-resistance with existing drugs has been earnestly desired. UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a drug target that has been studied for a long time. However, no LpxC inhibitors are available on the market at present. In this study, we sought to create a new antibacterial agent without a hydroxamate moiety, which is a common component of the major LpxC inhibitors that have been reported to date and that may cause toxicity. As a result, a development candidate, TP0586532, was created that is effective against carbapenem-resistant Klebsiella pneumoniae and does not pose a cardiovascular risk.

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.

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).

Unique structure of ozoralizumab, a trivalent anti-TNFα NANOBODY® compound, offers the potential advantage of mitigating the risk of immune complex-induced inflammation.

Biologics have become an important component of treatment strategies for a variety of diseases, but the immunogenicity of large immune complexes (ICs) and aggregates of biologics may increase risk of adverse events is a concern for biologics and it remains unclear whether large ICs consisting of intrinsic antigen and therapeutic antibodies are actually involved in acute local inflammation such as injection site reaction (ISR). Ozoralizumab is a trivalent, bispecific NANOBODY® compound that differs structurally from IgGs. Treatment with ozoralizumab has been shown to provide beneficial effects in the treatment of rheumatoid arthritis (RA) comparable to those obtained with other TNFα inhibitors. Very few ISRs (2%) have been reported after ozoralizumab administration, and the drug has been shown to have acceptable safety and tolerability. In this study, in order to elucidate the mechanism underlying the reduced incidence of ISRs associated with ozoralizumab administration, we investigated the stoichiometry of two TNFα inhibitors (ozoralizumab and adalimumab, an anti-TNFα IgG) ICs and the induction by these drugs of Fcγ receptor (FcγR)-mediated immune responses on neutrophils. Ozoralizumab-TNFα ICs are smaller than adalimumab-TNFα ICs and lack an Fc portion, thus mitigating FcγR-mediated immune responses on neutrophils. We also developed a model of anti-TNFα antibody-TNFα IC-induced subcutaneous inflammation and found that ozoralizumab-TNFα ICs do not induce any significant inflammation at injection sites. The results of our studies suggest that ozoralizumab is a promising candidate for the treatment of RA that entails a lower risk of the IC-mediated immune cell activation that leads to unwanted immune responses.

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.

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.

Fragment-Based Discovery of Novel Non-Hydroxamate LpxC Inhibitors with Antibacterial Activity.

UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) is a zinc metalloenzyme that catalyzes the first committed step in the biosynthesis of Lipid A, an essential component of the cell envelope of Gram-negative bacteria. The most advanced, disclosed LpxC inhibitors showing antibacterial activity coordinate zinc through a hydroxamate moiety with concerns about binding to other metalloenzymes. Here, we describe the discovery, optimization, and efficacy of two series of compounds derived from fragments with differing modes of zinc chelation. A series was evolved from a fragment where a glycine moiety complexes zinc, which achieved low nanomolar potency in an enzyme functional assay but poor antibacterial activity on cell cultures. A second series was based on a fragment that chelated zinc through an imidazole moiety. Structure-guided design led to a 2-(1S-hydroxyethyl)-imidazole derivative exhibiting low nanomolar inhibition of LpxC and a minimum inhibitory concentration (MIC) of 4 µg/mL against Pseudomonas aeruginosa, which is little affected by the presence of albumin.

Crystallization and preliminary X-ray crystallographic analysis of Ca2+-free primary Ca2+-sensor of Na+/Ca2+ exchanger.

The plasma-membrane Na(+)/Ca(2+) exchanger (NCX) regulates intracellular Ca(2+) levels in cardiac myocytes. Two Ca(2+)-binding domains (CBD1 and CBD2) exist in the large cytosolic loop of NCX. The binding of Ca(2+) to CBD1 results in conformational changes that stimulate exchange to exclude Ca(2+) ions, whereas CBD2 maintains the structure, suggesting that CBD1 is the primary Ca(2+)-sensor. In order to clarify the structural scaffold for the Ca(2+)-induced conformational transition of CBD1 at the atomic level, X-ray structural analysis of its Ca(2+)-free form was attempted; the structure of the Ca(2+)-bound form is already available. Recombinant CBD1 (NCX1 372-508) with a molecular weight of 16 kDa was crystallized by the sitting-drop vapour-diffusion method at 293 K. The crystals belonged to the hexagonal space group P6(2)22 or P6(4)22, with unit-cell parameters a = b = 56.99, c = 153.86 A, beta = 120 degrees , and contained one molecule per asymmetric unit (V(M) = 2.25 A(3) Da(-1)) with a solvent content of about 55% (V(S) = 45.57%). Diffraction data were collected within the resolution range 27.72-3.00 A using an R-AXIS detector and gave a data set with an overall R(merge) of 10.8% and a completeness of 92.8%.