Inhibition of protein arginine deiminase 4 prevents inflammation-mediated heart failure in arthritis.

Rheumatoid arthritis is a prototypic inflammatory condition with affected patients being at greater risk of incident heart failure (HF). Targeting innate immune cell function in the pathogenesis of HF bears the potential to guide the development of future therapies. A collagen-induced arthritis (CIA) model in DBA/1 J mice was used to generate arthritis. Mice with CIA developed concentric hypertrophic myocardial remodeling, left ventricular (LV) diastolic dysfunction, and HF with elevated plasma B-type natriuretic peptide levels but preserved LV ejection fraction. Key features of HF in CIA were increased infiltration of activated neutrophils, deposition of neutrophil extracellular traps in the myocardium, and increased tissue levels of the proinflammatory cytokine IL-1ß. Specific inhibition of protein arginine deiminase 4 (PAD4) by an orally available inhibitor (JBI-589), administered after the onset of clinical arthritis, prevented HF with reduced neutrophil infiltration. We identify PAD4-mediated neutrophil activation and recruitment as the key thromboinflammatory pathway driving HF development in arthritis. Targeting PAD4 may be a viable therapeutic approach for the prevention of HF secondary to chronic inflammation.

Alleviation of arthritis through prevention of neutrophil extracellular traps by an orally available inhibitor of protein arginine deiminase 4.

Protein arginine deiminases (PAD) 4 is an enzyme that catalyzes citrullination of protein and its role in autoimmune diseases has been established through clinical genetics and gene knock out studies in mice. Further, studies with PAD4 - deficient mice have shown that PAD4 deficiency does not lead to increased infection or immune suppression, which makes PAD4 an attractive therapeutic target for auto-immune and inflammatory diseases. PAD4 has critical enzymatic role of promoting chromatin decondensation and neutrophil extracellular traps (NETs) formation that is associated with a number of immune-mediated pathological conditions. Here, we present a non-covalent PAD4 inhibitor JBI-589 with high PAD4 isoform selectivity and delineated its binding mode at 2.88 Å resolution by X-ray crystallography. We confirmed its effectiveness in inhibiting NET formation in vitro. Additionally, by using two mouse arthritis models for human rheumatoid arthritis (RA), the well-known disease associated with PAD4 clinically, we established its efficacy in vivo. These results suggest that JBI-589 would be beneficial for both PAD4 and NET-associated pathological conditions.

Novel dual LSD1/HDAC6 inhibitor for the treatment of cancer.

Dually targeting the epigenetic proteins lysine specific demethylase 1 (LSD1) and histone deacetylases (HDACs) that play a key role in cancer cells by modulating gene repressor complexes including CoREST will have a profound effect in inhibiting tumour growth. Here, we evaluated JBI-097 a dual LSD1/HDAC6 inhibitor, for its in vitro and in vivo activities in various tumor models. In vitro, JBI-097 showed a strong potency in inhibiting LSD1 and HDAC6 enzymatic activities with the isoform selectivity over other HDACs. Cell-based experiments demonstrated a superior anti-proliferative profile against haematological and solid tumor cell lines. JBI-097 also showed strong modulation of HDAC6 and LSD1 specific biomarkers, alpha-tubulin, CD86, CD11b, and GFi1b. In vivo, JBI-097 showed a stronger effect in erythroleukemia, multiple myeloma xenograft models, and in CT-26 syngeneic model. JBI-097 also showed efficacy as monotherapy and additive or synergistic efficacy in combination with the standard of care or with immune checkpoint inhibitors. These and other findings suggest that JBI-097 could be a promising molecule for targeting the LSD1 and HDAC6. Further studies are warranted to elucidate the mechanism of action.

A Novel Selective Inhibitor JBI-589 Targets PAD4-Mediated Neutrophil Migration to Suppress Tumor Progression.

Neutrophils are closely involved in the regulation of tumor progression and formation of premetastatic niches. However, the mechanisms of their involvement and therapeutic regulation of these processes remain elusive. Here, we report a critical role of neutrophil peptidylarginine deiminase 4 (PAD4) in neutrophil migration in cancer. In several transplantable and genetically engineered mouse models, tumor growth was accompanied by significantly elevated enzymatic activity of neutrophil PAD4. Targeted deletion of PAD4 in neutrophils markedly decreased the intratumoral abundance of neutrophils and led to delayed growth of primary tumors and dramatically reduced lung metastases. PAD4-mediated neutrophil accumulation by regulating the expression of the major chemokine receptor CXCR2. PAD4 expression and activity as well as CXCR2 expression were significantly upregulated in neutrophils from patients with lung and colon cancers compared with healthy donors, and PAD4 and CXCR2 expression were positively correlated in neutrophils from patients with cancer. In tumor-bearing mice, pharmacologic inhibition of PAD4 with the novel PAD4 isoform-selective small molecule inhibitor JBI-589 resulted in reduced CXCR2 expression and blocked neutrophil chemotaxis. In mouse tumor models, targeted deletion of PAD4 in neutrophils or pharmacologic inhibition of PAD4 with JBI-589 reduced both primary tumor growth and lung metastases and substantially enhanced the effect of immune checkpoint inhibitors. Taken together, these results suggest a therapeutic potential of targeting PAD4 in cancer. SIGNIFICANCE: PAD4 regulates tumor progression by promoting neutrophil migration and can be targeted with a small molecule inhibitor to suppress tumor growth and metastasis and increase efficacy of immune checkpoint blockade therapy.

Novel tricyclic small molecule inhibitors of Nicotinamide N-methyltransferase for the treatment of metabolic disorders.

Nicotinamide N-methyltransferase (NNMT) is a metabolic regulator that catalyzes the methylation of nicotinamide (Nam) using the co-factor S-adenosyl-L-methionine to form 1-methyl-nicotinamide (MNA). Overexpression of NNMT and the presence of the active metabolite MNA is associated with a number of diseases including metabolic disorders. We conducted a high-throughput screening campaign that led to the identification of a tricyclic core as a potential NNMT small molecule inhibitor series. Elaborate medicinal chemistry efforts were undertaken and hundreds of analogs were synthesized to understand the structure activity relationship and structure property relationship of this tricyclic series. A lead molecule, JBSNF-000028, was identified that inhibits human and mouse NNMT activity, reduces MNA levels in mouse plasma, liver and adipose tissue, and drives insulin sensitization, glucose modulation and body weight reduction in a diet-induced obese mouse model of diabetes. The co-crystal structure showed that JBSNF-000028 binds below a hairpin structural motif at the nicotinamide pocket and stacks between Tyr-204 (from Hairpin) and Leu-164 (from central domain). JBSNF-000028 was inactive against a broad panel of targets related to metabolism and safety. Interestingly, the improvement in glucose tolerance upon treatment with JBSNF-000028 was also observed in NNMT knockout mice with diet-induced obesity, pointing towards the glucose-normalizing effect that may go beyond NNMT inhibition. JBSNF-000028 can be a potential therapeutic option for metabolic disorders and developmental studies are warranted.

Novel Inhibitors of Nicotinamide-N-Methyltransferase for the Treatment of Metabolic Disorders.

Nicotinamide-N-methyltransferase (NNMT) is a cytosolic enzyme catalyzing the transfer of a methyl group from S-adenosyl-methionine (SAM) to nicotinamide (Nam). It is expressed in many tissues including the liver, adipose tissue, and skeletal muscle. Its expression in several cancer cell lines has been widely discussed in the literature, and recent work established a link between NNMT expression and metabolic diseases. Here we describe our approach to identify potent small molecule inhibitors of NNMT featuring different binding modes as elucidated by X-ray crystallographic studies.

Novel dual LSD1/HDAC6 inhibitors for the treatment of multiple myeloma.

Lysine specific demethylase 1 (LSD1) and HDAC6 are epigenetic proteins associated with several diseases, including cancer and combined inhibition of these proteins could be highly beneficial in treating some cancers such as AML, MM and solid tumors. Multiple myeloma (MM) is a challenging cancer with fast relapse rate where novel treatment options are the need of the hour. We have designed and developed novel, LSD1 and HDAC6 selective dual inhibitors to target MM. Our dual inhibitor compound 1 shows superior potency in multiple MM cell lines. In MM.1S xenograft model compound 1 shows superior efficacy compared to single agent LSD1 and HDAC6 inhibitors by oral administration and is well tolerated. Further evaluation of the molecule in other cancers is in progress.

Validated LC-MS/MS Method for Simultaneous Quantitation of SAFit-1 and SAFit-2 in Mice Plasma: Application to a Pharmacokinetic Study.

SAFit-1 and SAFit-2 are selective FKBP51 (FK506-binding protein 51) ligands. In this paper, we present the development and validation data of an LC-MS/MS method for the simultaneous quantitation of SAFit-1 and SAFit-2 in mice plasma as per FDA regulatory guideline. SAFit-1 and SAFit-2 along with internal standard were extracted from mice plasma using liquid-liquid extraction method. Chromatographic resolution of SAFit-1, SAFit-2 and the internal standard (warfarin) was achieved on an X-Terra phenyl column using 0.2% formic acid:acetonitrile (20:80, v/v) as an eluent, which was delivered at a flow-rate of 0.9 mL/min. The MS/MS ion transitions monitored were m/z 748.4→420.4, 803.7→384.3 and 309.2 →163.2 for SAFit-1, SAFit-2 and the internal standard, respectively. The linearity range was 2.45-2446 ng/mL for both SAFit-1 and SAFit-2. The intra- and inter-day accuracy and intra- and inter-day precision were in the range of 0.90-1.07 and 2.38-10.8%, respectively for SAFit-1; 0.97-1.15 and 0.23-12.5%, respectively for SAFit-2. Both SAFit-1 and SAFit-2 were found to be stable in stability studies (up to three freeze-thaw cycles and for long-term at -80°C for 30 days) and processed (bench-top for 3 h and in in-injector for 16 h) samples. The application of the validated method was shown in a pharmacokinetic study in mice.

Novel KV7 ion channel openers for the treatment of epilepsy and implications for detrusor tissue contraction.

Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual KV7 isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a less significant role in bladder contraction and further investigation is needed.

A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

Novel nicotinamide analog as inhibitor of nicotinamide N-methyltransferase.

Nicotinamide N-methyltransferase (NNMT) has been linked to obesity and diabetes. We have identified a novel nicotinamide (NA) analog, compound 12 that inhibited NNMT enzymatic activity and reduced the formation of 1-methyl-nicotinamide (MNA), the primary metabolite of NA by ∼80% at 2 h when dosed in mice orally at 50 mg/kg.

Crystal structures of monkey and mouse nicotinamide N-methyltransferase (NNMT) bound with end product, 1-methyl nicotinamide.

Nicotinamide N-methyltransferase (NNMT) is a S-adenosyl-l-methionine (SAM)-dependent enzyme that catalyzes N-methylation of nicotinamide (NA) and other pyridines to form N-methyl pyridinium ions. Here we report the first ternary complex X-ray crystal structures of monkey NNMT and mouse NNMT in bound form with the primary endogenous product, 1-methyl nicotinamide (MNA) and demethylated cofactor, S-adenosyl-homocysteine (SAH) determined at 2.30 Å and 1.88 Å respectively. The structural fold of these enzymes is identical to human NNMT. It is known that the primary endogenous product catalyzed by NNMT, MNA is a specific inhibitor of NNMT. Our data clearly indicates that the MNA binds to the active site and it would be trapped in the active site due to the formation of the bridge between the pole (long helix, α3) and long C-terminal loop. This might explain the mechanism of MNA acting as a feedback inhibitor of NNMT.

Validated LC-MS-MS Method for Determination of SF0034 in Mice Plasma: Application to a Pharmacokinetic Study in Mice.

A rapid and sensitive assay method has been developed and validated using liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the positive-ion mode for the estimation of SF0034 in mice plasma. The assay procedure involves a simple protein precipitation of SF0034 and tolbutamide (internal standard, IS) from mice plasma. Chromatographic separation was performed on an Atlantis dC18 column using an isocratic mobile phase comprising 0.2% formic acid:acetonitrile (10:90, v/v) at a flow rate of 0.60 mL/min. The total run time was 2.5 min. For mass spectrometric detection, the multiple reaction monitoring was used and ion transitions monitored were m/z 322 → 248 for SF0034 and 271 → 155 for IS. Method validation was performed as per regulatory guidelines and the results met the acceptance criteria. A calibration curve was constructed in the range of 2.08-2,078 ng/mL. The intra- and inter-day precision was in the range of 1.06-14.4% and 7.16-11.7%, respectively. This novel method has been applied to a pharmacokinetic study in mice.

Novel hydroxamates potentiated in vitro activity of fluconazole against Candida albicans.

A set of 12 novel hydroxamate compounds (NHCs), structurally designed as inhibitors of histone deacetylase (HDAC) enzyme, were synthesized at our facility. These were adamantane derivatives with N-hydroxyacetamide as pharmacophore, and each of these compounds was tested for potentiating activity on fluconazole. The concentration of fluconazole which completely inhibited (concentration of complete inhibition [CCI]) the growth of Candida albicans ATCC 90028 and C. albicans ATCC 64550 was determined by micro-dilution method in the absence and presence of NHCs. The CCI of fluconazole without the NHC combination was 64 µg/ml and 1024 µg/ml against C. albicans ATCC 90028 and C. albicans ATCC 64550, respectively. The majority of the NHCs potentiated the fluconazole activity markedly as CCI of fluconazole against C. albicans ATCC 90028 reduced to 0.25 µg/ml. Similarly, CCI of fluconazole against C. albicans ATCC 64550 reduced to 4-8 µg/ml in combination with majority of NHCs while the best activity was displayed by the compound 1 with a reduction of CCI to 0.5 µg/ml. The study results revealed the potential usage of hydroxamate derivatives, structurally designed as HDAC inhibitors to enhance the activity of fluconazole against C. albicans.

Validation of an LC-MS/MS method for simultaneous detection of four HDAC inhibitors - belinostat, panobinostat, rocilinostat and vorinostat in mouse plasma and its application to a mouse pharmacokinetic study.

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of four HDAC inhibitors, namely belinostat (BST), panobinostat (PST), rocilinostat (RST) and vorinostat (VST), in mouse plasma as per regulatory guidelines. The analytes and internal standard were extracted from 50 µL mouse plasma by protein precipitation, followed by chromatographic separation using an Atlantis C18 column with an isocratic mobile phase comprising 0.1% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.5 mL/min within 2.5 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 319 → 93, 350 → 158, 434 → 274 and 265 → 232 for BST, PST, RST and VST, respectively, in the positive ionization mode. The calibration curves were linear from 2.92 to 2921 ng/mL for BST and PST and from 1.01 to 1008 ng/mL for RST and VST with r2 ≥ 0.99 for all of the analytes. The intra- and inter-batch accuracy and precision (CV) across quality controls varied from 85.5 to 112% and from 2.30 to 12.5, respectively, for all of the analytes. Analytes were found to be stable under different stability conditions. The method was applied to an i.v. pharmacokinetic study in mice.

In vivo effective dibenzo[b,d]furan-1-yl-thiazoles as novel PDE-4 inhibitors.

Herein we report the synthesis, PDE-4B and TNF-α inhibitory activities of a few dibenzo[b,d]furan-1-yl-thiazole derivatives. The hydroxycyclohexanol amide derivatives 14, 18, 24, 29, 31 and 33 exhibited promising in vitro PDE-4B and TNF-α inhibitory activities. Compound 24 showed good systemic availability in preclinical animal models and was also found to be non-toxic (exploratory mutagenicity test). Further it exhibited promising results in in vivo asthma/COPD and Uveitis models.

Sensitive method for the determination of rocilinostat in small volume mouse plasma by LC-MS/MS and its application to a pharmacokinetic study in mice.

A highly sensitive, specific and rapid LC-ESI-MS/MS method has been developed and validated for the quantification of rocilinostat in small volume mouse plasma (20 µL) using vorinostat as an internal standard (IS) as per regulatory guidelines. Sample preparation was accomplished through a protein precipitation procedure with acetonitrile. Chromatography was achieved on Prodigy ODS-2 column using a binary gradient using mobile phase A (0.2% formic acid in water) and B (acetonitrile) at a flow rate of 0.38 mL/min. The total chromatographic run time was 4.1 min and the elution of rocilinostat and IS occurred at ~3.2 and 2.9 min, respectively. A linear response function was established in the concentration range of 0.28-1193 ng/mL in mouse plasma. The intra- and inter-day accuracy and precisions were in the ranges of 3.12-8.93 and 6.41-11.6%, respectively. This novel method has been applied to a pharmacokinetic study in mice. Copyright © 2016 John Wiley & Sons, Ltd.

Orally available stilbene derivatives as potent HDAC inhibitors with antiproliferative activities and antitumor effects in human tumor xenografts.

Herein we report the synthesis and activity of a novel class of HDAC inhibitors based on 2, 3-diphenyl acrylic acid derivatives. The compounds in this series have shown to be potent HDAC inhibitors possessing significant antiproliferative activity. Further compounds in this series were subjected to metabolic stability in human liver microsomes (HLM), mouse liver microsomes (MLM), and exhibits promising stability in both. These efforts culminated with the identification of a developmental candidate (5a), which displayed desirable PK/PD relationships, significant efficacy in the xenograft models and attractive ADME profiles.

Quinolone-based HDAC inhibitors.

HDAC inhibitors emerged as promising drug candidates in combating wide variety of cancers. At present, two of the compounds SAHA and Romidepsin were approved by FDA for cutaneous T-cell lymphoma and many are in various clinical phases. A new quinolone cap structure was explored with hydroxamic acid as zinc-binding group (ZBG). The pan HDAC inhibitory and antiproliferative activities against three human cancer cell lines HCT-116 (colon), NCI-H460 (lung) and U251 (glioblastoma) of the compounds (4a-4w) were evaluated. Introduction of heterocyclic amines in CAP region increased the enzyme inhibitory and antiproliferative activities and few of the compounds tested are metabolically stable in both MLM and HLM.

Discovery of adamantane based highly potent HDAC inhibitors.

Herein, we report the development of highly potent HDAC inhibitors for the treatment of cancer. A series of adamantane and nor-adamantane based HDAC inhibitors were designed, synthesized and screened for the inhibitory activity of HDAC. A number of compounds exhibited GI50 of 10-100 nM in human HCT116, NCI-H460 and U251 cancer cells, in vitro. Compound 32 displays efficacy in human tumour animal xenograft model.