A new brain-penetrant glucosylceramide synthase inhibitor as potential Therapeutics for Gaucher disease.

Gaucher disease (GD), the most common lysosomal storage disorders, is caused by GBA gene mutations resulting in glycosphingolipids accumulations in various tissues, such as the brain. While suppressing glycosphingolipid accumulation is the central strategy for treating peripheral symptoms of GD, there is no effective treatment for the central nervous system symptoms. As glycosphingolipid biosynthesis starts from ceramide glycosylation by glucosylceramide synthase (GCS), inhibiting GCS in the brain is a promising strategy for neurological GD. Herein, we discovered T-036, a potent and brain-penetrant GCS inhibitor with a unique chemical structure and binding property. T-036 does not harbor an aliphatic amine moiety and has a noncompetitive inhibition mode to the substrates, unlike other known inhibitors. T-036 exhibited sufficient exposure and a significant reduction of glucosylsphingolipids in the plasma and brain of the GD mouse model. Therefore, T-036 could be a promising lead molecule for treating central nervous system symptoms of GD.

Ameliorative effect of phosphodiesterase 4 and 5 inhibitors in deoxycorticosterone acetate-salt hypertensive uni-nephrectomized KKAy mice.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD). Hypertension increases kidney stress, which deteriorates function, and leads to peripheral renal vascular resistance. Long-term hypoperfusion promotes interstitial fibrosis and glomerular sclerosis, resulting in nephrosclerosis. Although hypertension and DN are frequent ESRD complications, relevant animal models remain unavailable. We generated a deoxycorticosterone acetate (DOCA)-salt hypertensive uni-nephrectomized (UNx) KKAy mouse model demonstrating hypertension, hyperglycemia, cardiac hypertrophy, kidney failure, increased urinary albumin creatinine ratio (UACR), and increased renal PDE4D and cardiac PDE5A mRNA levels. We hypothesized that the novel PDE4 selective inhibitor, compound A, and PDE5 inhibitor, sildenafil, exhibit nephroprotective, and cardioprotective effects in this new model. Compound A, sildenafil, and the angiotensin II receptor blocker, irbesartan, significantly reduced ventricular hypertrophy and pleural effusion volume. Meanwhile, compound A and sildenafil significantly suppressed the UACR, urinary kidney injury molecule-1, and monocyte chemoattractant protein-1 levels, as well as that of renal pro-fibrotic marker mRNAs, including collagen 1A1, fibronectin, and transforming growth factor-beta (TGF-ß). Moreover, compound A significantly suppressed TGF-ß-induced pro-fibrotic mRNA expression in vitro in all major kidney lesions, including within the glomerular mesangial region, podocytes, and epithelial region. Hence, PDE4 and PDE5 inhibitors may be promising treatments, in combination with irbesartan, for DN with hypertension as they demonstrate complementary mechanisms.

Improvement of pulmonary arterial hypertension, inflammatory response, and epithelium injury by dual activation of cAMP/cGMP pathway in a rat model of monocrotaline-induced pulmonary hypertension.

Pulmonary hypertension (PH) is a life-threatening lung disease. PH with concomitant lung diseases, e.g., idiopathic pulmonary fibrosis, is associated with poor prognosis. Development of novel therapeutic vasodilators for treatment of these patients is a key imperative. We evaluated the efficacy of dual activation of cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) using an active, small-molecule phosphodiesterase (PDE4)/PDE5 dual inhibitor (Compound A). Compound A increased both cAMP and cGMP levels in WI-38 lung fibroblasts and suppressed the expressions of type-1 collagen α1 chain and fibronectin. Additionally, compound A reduced right ventricular weight/left ventricular weight+septal weight ratio, brain natriuretic peptide expression levels in right ventricle, C─C motif chemokine ligand 2 expression levels in lung, and plasma surfactant protein D. Our data indicate that dual activation of cAMP/cGMP pathways may be a novel treatment strategy for PH.

Discovery of novel serine palmitoyltransferase inhibitors as cancer therapeutic agents.

We pursued serine palmitoyltransferase (SPT) inhibitors as novel cancer therapeutic agents based on a correlation between SPT inhibition and growth suppression of cancer cells. High-throughput screening and medicinal chemistry efforts led to the identification of structurally diverse SPT inhibitors 4 and 5. Both compounds potently inhibited SPT enzyme and decreased intracellular ceramide content. In addition, they suppressed cell growth of human lung adenocarcinoma HCC4006 and acute promyelocytic leukemia PL-21, and displayed good pharmacokinetic profiles. Reduction of 3-ketodihydrosphingosine, the direct downstream product of SPT, was confirmed under in vivo settings after oral administration of compounds 4 and 5. Their anti-tumor efficacy was observed in a PL-21 xenograft mouse model. These results suggested that SPT inhibitors might have potential to be effective cancer therapeutics.

Antitumor activity of a novel and orally available inhibitor of serine palmitoyltransferase.

Metabolic reprogramming is an essential hallmark of neoplasia. Therefore, targeting cancer metabolism, including lipid synthesis, has attracted much interest in recent years. Serine palmitoyltransferase (SPT) plays a key role in the initial and rate-limiting step of de novo sphingolipid biosynthesis, and inhibiting SPT activity prevents the proliferation of certain cancer cells. Here, we identified a novel and orally available SPT inhibitor, compound-2. Compound-2 showed an anti-proliferative effect in several cancer cell models, reducing the levels of the sphingolipids ceramide and sphingomyelin. In the presence of compound-2, exogenously added S1P partially compensated the intracellular sphingolipid levels through the salvage pathway by partially rescuing compound-2-induced cytotoxicity. This suggested that the mechanism underlying the anti-proliferative effect of compound-2 involved the reduction of sphingolipid levels. Indeed, compound-2 promoted multinuclear formation with reduced endogenous sphingomyelin levels specifically in a compound-2-sensitive cell line, indicating that the effect was induced by sphingolipid reduction. Furthermore, compound-2 showed potent antitumor activity without causing significant body weight loss in the PL-21 acute myeloid leukemia mouse xenograft model. Therefore, SPT may be an attractive therapeutic anti-cancer drug target for which compound-2 may be a promising new drug.

Synthesis and evaluation of hedgehog signaling inhibitor with novel core system.

As we previously reported, N-methylpyrrolo[3,2-c]pyridine derivatives 1 (TAK-441) was discovered as a clinical candidate of hedgehog (Hh) signaling inhibitor by modification of the upper part. We next focused on modification of the lower part including core skeletons to discover new Hh signaling inhibitors with novel core rings. Efforts to find novel chemotypes by using X-ray single crystal structure analysis led to some potent Hh signaling inhibitors (2c, 2d, 2e, 2f) with novel core ring systems, which had benzamide moiety at the 5-position as a key component for potent activity. The suppression of Gli1 expression with these new Hh signaling inhibitors were weaker than that of compound 1 (TAK-441) because of low pharmacokinetic property. We recognized again TAK-441 is a good compound as clinical candidate with good structural and pharmacokinetic advantages.

Pharmacokinetic and pharmacodynamic modeling of hedgehog inhibitor TAK-441 for the inhibition of Gli1 messenger RNA expression and antitumor efficacy in xenografted tumor model mice.

6-Ethyl-N-[1-(hydroxyacetyl)piperidin-4-yl]-1-methyl-4-oxo-5-(2-oxo-2-phenylethyl)-3-(2,2,2-trifluoroethoxy)-4,5-dihydro-1H-pyrrolo[3,2-c]pyridine-2-carboxamide (TAK-441) is a potent, selective hedgehog signaling pathway inhibitor that binds to Smo and is being developed for the treatment of cancer. The objectives of these studies were to explore the possibility of establishing of a link between the pharmacokinetics of TAK-441 and the responses of Gli1 mRNA in tumor-associated stromal or skin cells and the antitumor effect of hedgehog inhibition. To this end, we built pharmacokinetic and pharmacodynamic models that describe the relationship of the concentrations of TAK-441 plasma to the responses of Gli1 mRNA in the tumor (target) and skin (surrogate) and to tumor growth inhibition in mice bearing xenografts of human pancreatic tumors (PAN-04). The responses of Gli1 mRNA and tumor growth were described by an indirect response model and an exponential tumor growth model, respectively. The IC50 values for Gli1 mRNA inhibition in the tumor and skin by TAK-441 were estimated to be 0.0457 and 0.113 µg/ml, respectively. The IC90 value for tumor growth inhibition was estimated to be 0.68 µg/ml. These results suggest that a >83% inhibition of Gli1 mRNA expression in the skin or a >94% inhibition of Gli1 mRNA expression in the tumor would be required to sufficiently inhibit (>90%) hedgehog-related tumor growth in the xenografted model mice. We conclude that Gli1 mRNA expression in the tumor and skin could be a useful biomarker for predicting the antitumor effect of hedgehog inhibitors.

Discovery of the investigational drug TAK-441, a pyrrolo[3,2-c]pyridine derivative, as a highly potent and orally active hedgehog signaling inhibitor: modification of the core skeleton for improved solubility.

We recently reported the discovery of the novel pyrrolo[3,2-c]quinoline-4-one derivative 1 as a potent inhibitor of Hedgehog (Hh) pathway signaling. However, the PK evaluation of 1 at high dosage (100 mg/kg) revealed the C(max) value 3.63 µg/mL, likely due to poor solubility of this compound. Efforts to improve solubility by reducing the aromatic ring count of the core system led to N-methylpyrrolo[3,2-c]pyridine derivative 11. Further optimization of the 3-alkoxy group led to compound 11d with acceptable solubility and potent Hh inhibitory activity. Compound 11d suppressed transcription factor Gli1 mRNA expression in tumor-associated stromal tissue and inhibited tumor growth (treatment/control ratio, 3%) in a mouse medulloblastoma allograft model owing to the improved PK profile based on increased solubility. Compound 11d (TAK-441) is currently in clinical trials for the treatment of advanced solid tumors.

Discovery of pyrrolo[3,2-c]quinoline-4-one derivatives as novel hedgehog signaling inhibitors.

The Hedgehog (Hh) signaling pathway plays a significant role in the regulation of cell growth and differentiation during embryonic development. Since activation of the Hh signaling pathway is implicated in several types of human cancers, inhibitors of this pathway could be promising anticancer agents. Using high throughput screening, thieno[3,2-c]quinoline-4-one derivative 9a was identified as a compound of interest with potent in vitro activity but poor metabolic stability. Our efforts focused on enhancement of in vitro inhibitory activity and metabolic stability, including core ring conversion and side chain optimization. This led to the discovery of pyrrolo[3,2-c]quinoline-4-one derivative 12b, which has a structure distinct from previously reported Hh signaling inhibitors. Compound 12b suppressed stromal Gli1 mRNA expression in a murine model and demonstrated antitumor activity in a murine medulloblastoma allograft model.

Identification of the first highly selective inhibitor of human lactate dehydrogenase B.

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor.

Replication stress (RS) is a cancer hallmark; chemotherapeutic drugs targeting RS are widely used as treatments for various cancers. To develop next-generation RS-inducing anticancer drugs, cell division cycle 7 (CDC7) has recently attracted attention as a target. We have developed an oral CDC7-selective inhibitor, TAK-931, as a candidate clinical anticancer drug. TAK-931 induced S phase delay and RS. TAK-931-induced RS caused mitotic aberrations through centrosome dysregulation and chromosome missegregation, resulting in irreversible antiproliferative effects in cancer cells. TAK-931 exhibited significant antiproliferative activity in preclinical animal models. Furthermore, in indication-seeking studies using large-scale cell panel data, TAK-931 exhibited higher antiproliferative activities in RAS-mutant versus RAS-wild-type cells; this finding was confirmed in pancreatic patient-derived xenografts. Comparison analysis of cell panel data also demonstrated a unique efficacy spectrum for TAK-931 compared with currently used chemotherapeutic drugs. Our findings help to elucidate the molecular mechanisms for TAK-931 and identify potential target indications.

CETSA quantitatively verifies in vivo target engagement of novel RIPK1 inhibitors in various biospecimens.

The proof of target engagement (TE) is a key element for evaluating potential investment in drug development. The cellular thermal shift assay (CETSA) is expected to facilitate direct measurement of intracellular TE at all stages of drug development. However, there have been no reports of applying this technology to comprehensive animal and clinical studies. This report demonstrates that CETSA can not only quantitatively evaluate the drug-TE in mouse peripheral blood, but also confirm TE in animal tissues exemplified by using the receptor interacting protein 1 kinase (RIPK1) lead compound we have developed. Our established semi-automated system allows evaluation of the structure-activity relationship using native RIPK1 in culture cell lines, and also enables estimation of drug occupancy ratio in mouse peripheral blood mononuclear cells. Moreover, optimized tissue homogenisation enables monitoring of the in vivo drug-TE in spleen and brain. Our results indicate that CETSA methodology will provide an efficient tool for preclinical and clinical drug development.

Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one Derivatives as Orally eIF4A3-Selective Inhibitors.

Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed antitumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of compounds represents promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.

Loss of lysophospholipase 3 increases atherosclerosis in apolipoprotein E-deficient mice.

Human LCAT-like lysophospholipase (LLPL), or lysophospholipase 3, was first identified in vitro, in foam cells derived from THP-1 cells. We demonstrated that LLPL was present in foam cells in the severe atherosclerotic lesions that develop in apolipoprotein E-null (apoE(-/-)) mice. This indicated that LLPL might affect lipid metabolisms in foam cells and, therefore, atherogenesis. Accordingly, we created LLPL-knockout mice by gene targeting and crossed them with apoE(-/-) mice. We showed that the absence of LLPL increased lesion formation markedly in apoE(-/-) mice but had little effect on the plasma-lipid profile. In addition, LLPL-deficient peritoneal macrophages were more sensitive to apoptosis induced by exposure to oxidized low-density lipoprotein. LLPL might provide a link between apoptosis in macrophages and atherogenesis. Our data demonstrate that LLPL activity is anti-atherogenic and indicate that the regulation of this enzyme might be a novel drug target for the treatment of atherosclerosis.