Discovery of Novel Class of METTL3 Inhibitors with a Pyridin-2(1H)‑one Moiety.

<b>Abstract ID 19484</b> <b>Poster Board 566</b> Schizophrenia is a devastating neuropsychiatric illness impacting approximately 1% of the global population and is the 15^th leading cause of disability worldwide. Schizophrenia is characterized by positive (hallucinations, delusions, paranoia), negative (flat affect, decreased motivation) and cognitive symptoms. Current therapies treat mostly the positive symptoms and are associated with a plethora of off-target side effects such as sedation, weight gain, and diabetes, among others. All FDA-approved antipsychotic medications target the D2 dopamine receptor (D2R) but also exhibit poly-pharmacology with other receptors. Further, there are few D2R antagonists that can selectively inhibit the D2R without also antagonizing the D3 and D4 dopamine receptors (D3R, D4R, respectively). We recently identified a D2R-selective antagonist scaffold, MLS6916, from a high throughput screen of the D2R. When counter-screened against 168 GPCRs using β-arrestin recruitment as a functional readout, 10 uM MLS6916 only inhibited the D2R, and to a lesser extent, the D4R. Further, using radioligand binding competition assays, MLS6916 was &gt;200-fold D2R&gt;D3R selective and 12-fold D2R&gt;D4R selective. Despite its promising D2R selectivity, MLS6916 was found to exhibit poor metabolic stability when assayed using rat liver microsomes. Interestingly, we found high species variability with respect to metabolic stability using rat, mouse, and human liver microsomes. While many analogs exhibited poor metabolic stability in rat liver microsomes, we observed equal or worse stability in mouse liver microsomes, but dramatically higher stability in human liver microsomes. High metabolic stability in humans is essential for moving a compound into the clinic, but preclinical studies will require at least moderate metabolic stability in rodents to employ animal models that are predictive of antipsychotic efficacy and/or adverse side effects. Thus, to chemically optimize this scaffold and explore its structure-activity relationships, greater than 100 analogs were synthesized to identify modifications that might result in improved metabolic stability. All analogs were also evaluated for D2R, D3R, and D4R activities using radioligand binding competition and β-arrestin recruitment assays. Lead compounds were identified that possessed D2R Ki values of &lt;100 nM, were highly selective versus the D3R and D4R, and exhibited improved metabolic stability in mouse and/or human liver microsomes. We further determined the pharmacokinetic profiles of the most promising compounds in mice since this species has good models for predicting antipsychotic efficacy. After injecting 30 mg/kg i.p., we found that the compounds exhibited t_½ values of 5-6 hr in both plasma and brain. Importantly, the compounds exhibited 1:1 brain-plasma ratios with Cmax values &gt;10 uM indicating excellent brain penetration. In summary, we have identified lead candidate compounds that have exceptional D2R-selectivity, excellent metabolic stabilities in human liver microsomes, and sufficient metabolic stability in mice to conduct behavioral studies. Future studies will investigate preclinical antipsychotic efficacy in mouse models as well as the potential for on-target side effects such as catalepsy. These advanced leads may have the potential to treat neuropsychiatric disorders with greatly reduced off-target side effects.

A convenient transesterification method for synthesis of AT2 receptor ligands with improved stability in human liver microsomes

Objectives: Pharmacokinetic issues were the leading cause of drug attrition, accounting for approximately 40% of all cases before the turn of the century. To this end, several high-throughput in vitro assays like microsomal stability have been developed to evaluate the pharmacokinetic profiles of compounds in the early stages of drug discovery. At NCATS, a single-point rat liver microsomal (RLM) stability assay is used as a Tier I assay, while human liver microsomal (HLM) stability is employed as a Tier II assay. We experimentally screened and collected data on over 30,000 compounds for RLM stability and over 7000 compounds for HLM stability. Although HLM stability screening provides valuable insights, the increasing number of hits generated, along with the time- and resource-intensive nature of the assay, highlights the need for alternative strategies. One promising approach is leveraging in silico models trained on these experimental datasets. Methods: We describe the development of an HLM stability prediction model using our in-house HLM stability dataset. Results: Employing both classical machine learning methods and advanced techniques, such as neural networks, we achieved model accuracies exceeding 80%. Moreover, we validated our model using external test sets and found that our models are comparable to some of the best models in literature. Additionally, the strong correlation observed between our RLM and HLM data was further reinforced by the fact that our HLM model performance improved when using RLM stability predictions as an input descriptor. Conclusions: The best model along with a subset of our dataset (PubChem AID: 1963597) has been made publicly accessible on the ADME@NCATS website for the benefit of the greater drug discovery community. To the best of our knowledge, it is the largest open-source model of its kind and the first to leverage cross-species data.

2-Aminopyrimidine derivatives as selective dual inhibitors of JAK2 and FLT3 for the treatment of acute myeloid leukemia

Structural modifications at the 6-position of thieno[2,3-d]pyrimidines and their effects on potency at FLT3 for treatment of acute myeloid leukemia

Objectives: Acute myeloid leukemia (AML) is a heterogeneous disease with diverse genetic abnormalities present in all ages, but mainly prevalent in an elderly population with an average age of above 60 years. The treatment of elderly AML remains a formidable challenge as the long-term outcomes of elderly AML patients have not improved in the last three decades, with ≤5% overall survival recorded at 5 years, calling for novel treatment options. Epigenetic therapy has a significant impact on the management of hematologic malignancies. Recent findings show that using very low dosages of decitabine depletes DNA methyl transferase 1 (DNMT1) without cytotoxicity. Decitabine-mediated epigenetic therapy does not induce FLT3 (fms-like tyrosine kinase 3) ligand, which hinders the effectiveness of FLT3 inhibitors. The inhibition of FLT3 and DNMT1 is associated with terminal myeloid differentiation of AML cells. Therefore, combining the inhibitors of FLT3 and DNMT1 may be an effective therapeutic approach for the treatment of poor-risk AML. Ponatinib is a third-generation receptor tyrosine kinase inhibitor. Various in vitro and in vivo preclinical studies demonstrated antileukemic activities of ponatinib against AML cells bearing FLT3-ITD mutations. Methods: Apoptosis and CD11b were measured by flow cytometry. FLT3 signaling and DNMT1 levels were analyzed by immunoblotting. Results: FLT3-ITD expressing AML cell lines MV4-11, MOLM-13, and MOLM-14 were used in the study. Co-treatment with decitabine (10-100 nM) and ponatinib (2 nM) in AML cells induced apoptosis in association with PARP cleavage. Increased levels of pro-apoptotic protein BAD and decreased levels of anti-apoptotic protein MCL1 were observed in these treated cells. The combination of decitabine and ponatinib also showed similar effects in primary AML cells expressing FLT3-ITD. The Western blot analysis demonstrated that treatment of decitabine decreased levels of DNMT1 and ponatinib inhibited FLT3 signaling and activation of downstream effectors STAT5, AKT, and ERK1/2 with induction of PU.1 a key regulator of myeloid differentiation. Co-treatment with ponatinib and decitabine using low concentrations in MV4-11 and MOLM14 cells induced myeloid differentiation. The percentage of differentiated cells was measured by increased surface expression of CD11b analyzed by flow cytometry and granulocytic/monocytic morphology examined by Wright-Giemsa staining. Conclusions: Mechanistically, the hypomethylating agent initiates the differentiation process and FLT3 inhibition augments differentiation leading to apoptosis of AML cells. Altogether, these preclinical findings of downregulation of DNMT1 and induction of PU.1 are a novel differentiation approach to induce apoptosis in AML cells and warrant future therapeutic potential for the treatment of AML patients expressing FLT3 mutations. Citation Format: Sudhakiranmayi Kuravi, Myles Taylor, Tara L. Lin, Jensen Roy, Joseph McGuirk, Ramesh Balusu. A targeted differentiation therapy for the treatment of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4821.

A novel class of phosphodiesterase 10A (PDE10A) inhibitors with improved metabolic stability in mouse liver microsomes were designed and synthesized starting from 2-({4-[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]phenoxy}methyl)quinoline (MP-10). Replacement of the phenoxymethyl part of MP-10 with an oxymethyl phenyl unit led to the identification of 2-[4-({[1-methyl-4-(pyridin-4-yl)-1H-pyrazol-3-yl]oxy}methyl)phenyl]quinoline (14), which showed moderate PDE10A inhibitory activity with improved metabolic stability in mouse and human liver microsomes over MP-10. Compound 14 showed high concentrations in plasma and brain after intraperitoneal administration and dose-dependently attenuated the hyperlocomotion induced by phencyclidine in mice, and oral administration of 14 (0.1, 0.3 mg/kg) also improved visual-recognition memory impairment in mice.

Effects of Decitabine and Homoharringtonine in Combination in Leukemia Cell Lines

In this study, the antiproliferative activity of 3 phenyl 4-(2-oxo-3-alkylimidazolidin-1-yl)benzenesulfonates (PAIB-SOs) was assessed in a time-dependent manner together with their hepatic stability and metabolism using human, mouse and rat liver microsomes. CEU-818, -820 and -913 were selected as promising hit compounds. Their antiproliferative activity on human breast carcinoma MCF-7 cells was evaluated using escalating concentrations of drugs at 24, 36 and 48h and the sulforhodamine B assay. Their hepatic stability was evaluated by HPLC-UV of extracts obtained from human, mouse and rat liver microsomes. The antiproliferative activity of PAIB-SOs is concentration and time-dependent and requires between 24 and 36h of contact with MCF-7 cells to detect a significant antiproliferative activity. PAIB-SOs stability in microsomes usually decreases following this order: human ≈ (rat>mouse). The CEU-913 exhibits the longest half-life in rat and human liver microsomes while the CEU-820 exhibits the longest half-life in mouse liver microsomes. Our in vitro results suggest that PAIB-SOs should have a minimum contact time of 24h with the tumour to trigger significant antitumoural activity. The activity of mouse liver microsomes towards PAIB-SOs is higher than rat microsomes and tends to be higher than human liver microsomes.

Microsomal prostaglandin E synthase-1 (mPGES-1) is responsible for the massive prostaglandin E2 (PGE2) formation during inflammation. Increasing evidence reveals mPGES-1 inhibitors as a safe alternative to nonsteroidal anti-inflammatory drugs. Recently, we reported that a novel series of phenylsulfonyl hydrazide derivatives could reduce LPS-induced PGE2 levels in RAW 264.7 macrophage cells via an inhibition of the mPGES-1 enzyme. However, a few of the phenylsulfonyl hydrazide derivatives showed poor metabolic stability in liver microsomes. In order to identify new mPGES-1 inhibitors with improved metabolic stability, therefore, a series of arylsulfonamide derivatives has been synthesized and biologically evaluated against PGE2 production and the mPGES-1 enzyme. Among them, MPO-0186 inhibits the production of PGE2 (IC50 = 0.20 μM) in A549 cells via inhibition of mPGES-1 (IC50 = 0.49 μM in a cell-free assay) together with high selectivity over both COX-1 and COX-2. A molecular docking study theoretically suggests that MPO-0186 could inhibit PGE2 production by blocking the PGH2 binding site of the mPGES-1 enzyme. Furthermore, MPO-0186 demonstrated good metabolic stability in human liver microsomes and no significant inhibition observed in clinically relevant CYP isoforms.

Plant derived natural products have been the major source for treatment of diseases traditionally but with the advent of modern systems of medicine, there is need to explore the active constituents present in it followed by modification for better therapeutic activity, low toxicity and favorable pharmacokinetics to become a drug molecule. A simple, rapid and sensitive high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated according to Food and Drug Administration guidelines for determination of IS01957, a derivative of naturally occurring para coumaric acid in mice plasma. The extraction of the analyte and the internal standard (Carbamazepine) from the plasma samples involved protein precipitation using acetonitrile. Results of validation parameters were met with the acceptance criteria of the FDA guidelines. Method was highly sensitive (5 ng/mL) that could determine very low concentration of compound in plasma The developed and validated method was successfully applied to determine compound’s metabolic stability in mouse liver microsomes (MLM) and human liver microsomes (HLM). Test compound was found to be stable in MLM and HLM in the experimental conditions. Metabolic stability data was extrapolated which was further correlated to pharmacokinetics study in mice through oral, intraperitoneal and intravenous administration. In-vitro half life was found to be greater than 2 h in both MLM and HLM. Hepatic extraction ratio of the compound was found to be in the intermediate range. Pharmacokinetic evaluations revealed that it is a suitable candidate for intraperitoneal as well as oral administration.

We here report on our continued studies of ligands binding to the promising drug target angiotensin II type 2 receptor (AT2R). Two series of compounds were synthesized and investigated. The first series explored the effects of adding small substituents to the phenyl ring of the known selective nonpeptide AT2R antagonist C38, generating small but significant shifts in AT2R affinity. One compound in the first series was equipotent to C38 and showed similar kinetic solubility, and stability in both human and mouse liver microsomes. The second series was comprised of new bicyclic derivatives, amongst which one ligand exhibited a five‐fold improved affinity to AT2R as compared to C38. The majority of the compounds in the second series, including the most potent ligand, were inferior to C38 with regard to stability in both human and mouse microsomes. In contrast to our previously reported findings, ligands with shorter carbamate alkyl chains only demonstrated slightly improved stability in microsomes. Based on data presented herein, a more adequate, tentative model of the binding modes of ligand analogues to the prototype AT2R antagonist C38 is proposed, as deduced from docking redefined by molecular dynamic simulations.

N-Alkylidenearylcarboxamides as new potent and selective CB 2 cannabinoid receptor agonists with good oral bioavailability

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

Targeting the molecular chaperone HSP90 and the anti-apoptotic proteins MCL1 and BCL2 may be a promising novel approach in the treatment of acute myeloid leukemia (AML). The HSP90 inhibitor PU-H71, MCL1 inhibitor S63845, and BCL2 inhibitor venetoclax were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and TP53 mutant AML cell lines and a variety of patient-derived AML cells. Results: PU-H71 and combination treatments with MCL1 inhibitor S63845 or BCL2 inhibitor venetoclax induced cell cycle arrest and apoptosis in susceptible AML cell lines and primary AML. The majority of the primary AML samples were responsive to PU-H71 in combination with BH3 mimetics. Elevated susceptibility to PU-H71 and S63845 was associated with FLT3 mutated AML with CD34 < 20%. Elevated susceptibility to PU-H71 and venetoclax was associated with primary AML with CD117 > 80% and CD11b < 45%. The combination of HSP90 inhibitor PU-H71 and MCL1 inhibitor S63845 may be a candidate treatment for FLT3-mutated AML with moderate CD34 positivity while the combination of HSP90 inhibitor PU-H71 and BCL2 inhibitor venetoclax may be more effective in the treatment of primitive AML with high CD117 and low CD11b positivity.