Potent Reversible Inhibitor Research Articles

Stress degradation study of bortezomib: effect of co-solvent, isolation and characterization of degradation products by UHPLC-Q-TOF-MS/MS and NMR and evaluation of the toxicity of the degradation products

Bortezomib (BTZ) is a first-in-class, potent reversible inhibitor of proteasome used in the treatment of multiple myeloma, the second most common hematological cancer.

Discovery of 8-prenylnaringenin from hop (Humulus lupulus L.) as a potent monoacylglycerol lipase inhibitor for treatments of neuroinflammation and Alzheimer's disease.

Monoacylglycerol lipase (MAGL), a serine hydrolase, converts endocannabinoid 2-arachidonoylglycerol (2-AG) to arachidonic acid (AA) and glycerol in the brain and plays a bidirectional role in controlling nueroinflammation. MAGL, involved in Alzheimer's and Parkinson's diseases, is a promising target for treatment of neurodegenerative disorders. However, the irreversible inhibitors of MAGL lead to the desensitization of CB1 receptors further impairing the benefits associated with the indirect CB1 stimulation. Therefore, development of potent reversible inhibitors from natural products (NPs) and traditional chinese medicines (TCMs) are safer and free from adverse side effects and feasible to avoid drawbacks which irreversible inhibitors cause. Here, we employed pharmacophore-based screening of drug candidates coupled with molecular docking, biochemical assay and Ligplot analyses to identify and characterize inhibitors targeting human MAGL (hMAGL). The built pharmacophore model, Phar-MAGL successfully identified inhibitors NP-2 (IC50 = 9.5 ± 1.2 μM), NP-5 (IC50 = 14.5 ± 1.3 μM), and NP-3 (IC50 = 15.2 ± 1.4 μM), which apparently attenuated the activities of hMAGL in vitro. The evident activities of the identified inhibitors against hMAGL showed that the pharmacophore model, Phar-MAGL is reliable and efficient in screening inhibitors against hMAGL. Our study successfully identified a natrual product inhibitor, NP-2 (8-PN), from the plant Humulus lupulus L. (hops) and its positive effects in neurogenesis and neurodifferentiation along with the evident inhibitory potency against hMAGL revealed the potential for further optimizing and developing into drugs to treat neuroinflammation, Alzheimer's and Parkinson's diseases.

Targeting Enteropeptidase with Reversible Covalent Inhibitors To Achieve Metabolic Benefits.

Inhibition of the serine protease enteropeptidase (EP) opens a new avenue to the discovery of chemotherapeutics for the treatment of metabolic diseases. Camostat has been used clinically for treating chronic pancreatitis in Japan; however, the mechanistic basis of the observed clinical efficacy has not been fully elucidated. We demonstrate that camostat is a potent reversible covalent inhibitor of EP, with an inhibition potency (k inact/KI) of 1.5 × 104 M-1s-1 High-resolution liquid chromatography-mass spectrometry (LC-MS) showed addition of 161.6 Da to EP after the reaction with camostat, consistent with insertion of the carboxyphenylguanidine moiety of camostat. Covalent inhibition of EP by camostat is reversible, with an enzyme reactivation half-life of 14.3 hours. Formation of a covalent adduct was further supported by a crystal structure resolved to 2.19 Å, showing modification of the catalytic serine of EP by a close analog of camostat, leading to formation of the carboxyphenylguanidine acyl enzyme identical to that expected for the reaction with camostat. Of particular note, minor structural modifications of camostat led to changes in the mechanism of inhibition. We observed from other studies that sustained inhibition of EP is required to effect a reduction in cumulative food intake and body weight, with concomitant improved blood glucose levels in obese and diabetic leptin-deficient mice. Thus, the structure-activity relationship needs to be driven by not only the inhibition potency but also the mechanistic and kinetic characterization. Our findings support EP as a target for the treatment of metabolic diseases and demonstrate that reversible covalent EP inhibitors show clinically relevant efficacy. SIGNIFICANCE STATEMENT: Interest in targeted covalent drugs has expanded in recent years, particularly so for kinase targets, but also more broadly. This study demonstrates that reversible covalent inhibition of the serine protease enteropeptidase is a therapeutically viable approach to the treatment of metabolic diseases and that mechanistic details of inhibition are relevant to clinical efficacy. Our mechanistic and kinetic studies outline a framework for detailed inhibitor characterization that is proving essential in guiding discovery efforts in this area.

NADPH-Independent Inactivation of CYP2B6 and NADPH-Dependent Inactivation of CYP3A4/5 by PBD: Potential Implication for Assessing Covalent Modulators for Time-Dependent Inhibition

Pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) has shown broad antitumor properties and potential as a therapeutic agent for cancers. During a routine drug-drug interaction assessment, it was found that PBD is a reversible inhibitor of CYP2C8 (IC50 = 1.1 µM) but not CYP1A2, 2B6, 2C9, 2C19, 2D6, or 3A4/5. Additionally, PBD is a classic time-dependent inhibition (TDI) of CYP3A4/5, with >30-fold shift in IC50 after a preincubation with NADPH. All other CYPs tested did not show evidence for TDI, but potent inhibition of CYP2B6 (IC50 = 1.5 µM) was observed after a preincubation with or without (w/wo) NADPH, which was an unexpected observation given the fact that no inhibition was observed in the direct inhibition assay. No other CYP isoforms were susceptible to this apparent non-NADPH-dependent inhibition, suggesting that PBD may selectively inactivate CYP2B6 without metabolic activation. The washing of the human liver microsome pellet after incubation with PBD did not fully recover CYP2B6 activity, indicating that PBD is covalently bound to CYP2B6, leading to inactivation of the enzyme. To further investigate the mechanism of NADPH-independent inhibition, the IC50 shift was determined for several PBD analogs, and it was found that the compounds without both reactive imines did not show NADPH-independent inhibition of CYP2B6, implying that NADPH-independent inactivation was likely caused by direct covalent binding of PBD to the enzyme in a highly structure-specific manner. These data clearly highlight the need to assess direct and time-dependent inhibition w/wo NADPH to adequately characterize the in vitro CYP inhibitory properties of drug candidates with reactive moieties. SIGNIFICANCE STATEMENT: We described a very unique in vitro CYP inhibition profile of pyrrolo[2,1-c][1,4]benzodiazepine dimer as a potent reversible CYP2C8 inhibitor, an NADPH-dependent CYP3A4/5 time-dependent inhibition (TDI) inhibitor, and an NADPH-independent CYP2B6 TDI inhibitor, and inhibition of CYPs occurs through three distinct mechanisms: reversible drug-enzyme binding, enzyme inactivation via bioactivation, and enzyme inactivation by covalent binding via chemical reactions. Our results suggest that, for compounds with reactive functional moieties, false positives can be reported when the conventional TDI assay is utilized.

The discovery of diazetidinyl diamides as potent and reversible inhibitors of monoacylglycerol lipase (MAGL)

Monoacylglycerol lipase (MAGL) has emerged as an attractive drug target because of its important role in regulating the endocannabinoid 2-arachidonoylglycerol (2-AG) and its hydrolysis product arachidonic acid (AA) in the brain. Herein, we report the discovery of a novel series of diazetidinyl diamide compounds 6 and 10 as potent reversible MAGL inhibitors. In addition to demonstrating potent MAGL inhibitory activity in the enzyme assay, the thiazole substituted diazetidinyl diamides 6d–l and compounds 10 were also effective at increasing 2-AG levels in a brain 2-AG accumulation assay in homogenized rat brain. Furthermore, selected compounds have been shown to achieve good brain penetration after oral administration in an animal study.

Development of potent reversible selective inhibitors of butyrylcholinesterase as fluorescent probes

Brain butyrylcholinesterase (BChE) is an attractive target for drugs designed for the treatment of Alzheimer’s disease (AD) in its advanced stages. It also potentially represents a biomarker for progression of this disease. Based on the crystal structure of previously described highly potent, reversible, and selective BChE inhibitors, we have developed the fluorescent probes that are selective towards human BChE. The most promising probes also maintain their inhibition of BChE in the low nanomolar range with high selectivity over acetylcholinesterase. Kinetic studies of probes reveal a reversible mixed inhibition mechanism, with binding of these fluorescent probes to both the free and acylated enzyme. Probes show environment-sensitive emission, and additionally, one of them also shows significant enhancement of fluorescence intensity upon binding to the active site of BChE. Finally, the crystal structures of probes in complex with human BChE are reported, which offer an excellent base for further development of this library of compounds.

A phase I/II study of epertinib plus trastuzumab with or without chemotherapy in patients with HER2-positive metastatic breast cancer

BackgroundEpertinib (S-222611) is a potent reversible inhibitor of HER2, EGFR and HER4. This trial evaluated the safety, tolerability, pharmacokinetics and antitumour activity of daily oral epertinib combined with trastuzumab (arm A), with trastuzumab plus vinorelbine (arm B) or with trastuzumab plus capecitabine (arm C), in patients with HER2-positive metastatic breast cancer (MBC).MethodsEligible patients, with or without brain metastases, had received prior HER2-directed therapy. A dose-escalation phase determined the tolerability of each combination and established a dose for further study. Further, patients were recruited to expansion cohorts in each of the 3 arms to further explore efficacy and safety.ResultsThe recommended doses of epertinib were 600 mg, 200 mg and 400 mg in arms A, B and C, respectively. The most frequent grade 3/4 adverse event (AE) was diarrhoea in all arms, which was manageable with medical intervention and dose modification. The objective response rate (complete response [CR] plus partial response [PR]) in heavily pre-treated HER2-positive MBC patients at the recommended doses of epertinib combined with trastuzumab was 67% (N = 9), with trastuzumab plus vinorelbine was 0% (N = 5) and with trastuzumab plus capecitabine was 56% (N = 9). Notably, 4 of 6 patients previously treated with T-DM1 responded in the arm A expansion cohort (epertinib plus trastuzumab). In the arm C expansion cohort (epertinib plus trastuzumab plus capecitabine), 4 of 7 patients responded despite previous exposure to capecitabine. Measurable regression of brain metastases was observed in patients with CNS target lesions treated in both arms A and C.ConclusionWe observed safety, tolerability and encouraging antitumour activity of epertinib combined with trastuzumab, or with trastuzumab plus capecitabine. This supports further evaluation of these combinations in patients with pre-treated HER2-positive MBC, with or without brain metastases.Trial registrationEudraCT Number: 2013-003894-87; registered 09-September-2013.

Abstract 317: Vecabrutinib inhibits C481 mutated Bruton's tyrosine kinase and its downstream signaling in vitro

Abstract Inhibition of Bruton’s Tyrosine Kinase (BTK) by ibrutinib, an irreversible inhibitor, has dramatically improved the outcomes in both previously treated and naïve chronic lymphocytic leukemia (CLL) patients. Ibrutinib inactivates BTK through covalently binding to the cysteine 481 residue (C481) which then leads to the inhibition of autophosphorylation of BTK and the inactivation of downstream survival nexus. Although ibrutinib demonstrated >90% overall and event-free survival, about 25% of patients discontinue ibrutinib due to leukemia progression and intolerance. Patients that initially respond to treatment may develop resistance and the most prevalent resistance mechanism of ibrutinib is described as the point mutation affecting the C481 residue of BTK that results in disruption of ibrutinib binding and acquired ibrutinib resistance. Most common mutations are C481S and C481R. Vecabrutinib is a potent reversible BTK inhibitor that binds to BTK through noncovalent interactions. As vecabrutinib does not require binding to C481 residue, it retains its efficacy with mutant BTK in vitro. To better understand differential biology of Wild-Type (WT) and serine and arginine substituted BTK, we labelled MEC-1 cell line with green fluorescence protein (GFP) and overexpressed either BTKWT, BTKC481Sor BTKC481R that contributes to ibrutinib resistance. We selected MEC-1 cell line as it represents CLL disease and the phenotype of the cells share several characteristics of ex vivo CLL cells. We performed Reverse Phase Protein Array (RPPA) and mRNA-sequencing to determine and compare the proteomic and transcriptomic profiles of the MEC-1 cells harboring WT and mutant BTK. Ingenuity Pathway Analysis (IPA) of RPPA data revealed that overexpression of BTK WT leads to the enrichment of protein alterations involved in cell cycle regulation, B cell receptor signaling, PI3K/AKT signaling, PTEN signaling and ERK/MAPK signaling. IPA of RNAseq data upon BTK WT overexpression unraveled the top canonical pathways that include signaling through axonal guidance, ephrin receptor, c-AMP mediated, CXCR4 signaling and PTEN signaling. Comparative analysis of MEC-1 cells with mutant BTK (C481S vs C481R) using IPA distinguished the activated pathways in BTKC481Sharboring cells from cells that express BTKC481R. These results are being validated by western blot and qRT-PCR assays. Immunoblotting results showed that following 24, 48, and 72 hour of vecabrutinib (at 1 µM) treatment reduced p-BTK (Tyr223), p-PLCG2 (Tyr759) and p-ERK (Thr 202/Tyr 204) levels in MEC-1 cells with mutant BTK. These data indicate that vecabrutinib effectively inhibits BTK and its downstream signaling in the presence of mutant BTK, suggesting that vecabrutinib treatment may be a rational approach to overcome ibrutinib resistance. Citation Format: Burcu Aslan, Mikhila Mahendra, Michael D Peoples, Joe R. Marszalek, Christopher P Vellano, Xiaofeng Zheng, Jing Wang, Pietro Taverna, Varsha Gandhi. Vecabrutinib inhibits C481 mutated Bruton's tyrosine kinase and its downstream signaling in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 317.

M-TAP Dance: Targeting PRMT1 and PRMT5 Family Members to Push Cancer Cells Over the Edge

In this issue of Cancer Cell, Fedoriw and colleagues characterize a potent reversible inhibitor of type I PRMTs, GSK3368715, with anti-proliferative effects on numerous cancer types. Using a combination of GSK3368715 with PRMT5 inhibitors, the authors show that a threshold of overall arginine methylation reduction needs to be achieved for synergistic anti-tumor activity.

Oxanosine Monophosphate Is a Covalent Inhibitor of Inosine 5'-Monophosphate Dehydrogenase.

Reactive nitrogen species (RNS) are produced during infection and inflammation, and the effects of these agents on proteins, DNA, and lipids are well recognized. In contrast, the effects of RNS damaged metabolites are less appreciated. 5-Amino-3-β-(d-ribofuranosyl)-3 H-imidazo-[4,5- d][1,3]oxazine-7-one (oxanosine) and its nucleotides are products of guanosine nitrosation. Here we demonstrate that oxanosine monophosphate (OxMP) is a potent reversible competitive inhibitor of IMPDH. The value of Ki varies from 50 to 340 nM among IMPDHs from five different organisms. UV spectroscopy and X-ray crystallography indicate that OxMP forms a ring-opened covalent adduct with the active site Cys (E-OxMP*). Unlike the covalent intermediate of the normal catalytic reaction, E-OxMP* does not hydrolyze, but instead recyclizes to OxMP. IMPDH inhibitors block proliferation and can induce apoptosis, so the inhibition of IMPDH by OxMP presents another potential mechanism for RNS toxicity.

Optimization of a Benzoylpiperidine Class Identifies a Highly Potent and Selective Reversible Monoacylglycerol Lipase (MAGL) Inhibitor.

Monoacylglycerol lipase (MAGL) is the enzyme degrading the endocannabinoid 2-arachidonoylglycerol, and it is involved in several physiological and pathological processes. The therapeutic potential of MAGL is linked to several diseases, including cancer. The development of MAGL inhibitors has been greatly limited by the side effects associated with the prolonged MAGL inactivation. Importantly, it could be preferable to use reversible MAGL inhibitors in vivo, but nowadays only few reversible compounds have been developed. In the present study, structural optimization of a previously developed class of MAGL inhibitors led to the identification of compound 23, which proved to be a very potent reversible MAGL inhibitor (IC50 = 80 nM), selective for MAGL over the other main components of the endocannabinoid system, endowed of a promising antiproliferative activity in a series of cancer cell lines and able to block MAGL both in cell-based as well as in vivo assays.

Osthenol, a prenylated coumarin, as a monoamine oxidase A inhibitor with high selectivity

Osthenol (6), a prenylated coumarin isolated from the dried roots of Angelica pubescens, potently and selectively inhibited recombinant human monoamine oxidase-A (hMAO-A) with an IC50 value of 0.74 µM and showed a high selectivity index (SI > 81.1) for hMAO-A versus hMAO-B. Compound 6 was a reversible competitive hMAO-A inhibitor (Ki = 0.26 µM) with a potency greater than toloxatone (IC50 = 0.93 µM), a marketed drug. Isopsoralen (3) and bakuchicin (1), furanocoumarin derivatives isolated from Psoralea corylifolia L., showed slightly higher IC50 values (0.88 and 1.78 µM, respectively) for hMAO-A than 6, but had low SI values (3.1 for both). Other coumarins tested did not effectively inhibit hMAO-A or hMAO-B. A structural comparison suggested that the 8-(3,3-dimethylallyl) group of 6 increased its inhibitory activity against hMAO-A compared with the 6-methoxy group of scopoletin (4). Molecular docking simulations revealed that the binding affinity of 6 for hMAO-A (−8.5 kcal/mol) was greater than that for hMAO-B (−5.6 kcal/mol) and that of 4 for hMAO-A (−7.3 kcal/mol). Docking simulations also implied that 6 interacted with hMAO-A at Phe208 and with hMAO-B at Ile199 by carbon hydrogen bondings. Our findings suggest that osthenol, derived from natural products, is a selective and potent reversible inhibitor of MAO-A, and can be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.

Computationally driven discovery of phenyl(piperazin-1-yl)methanone derivatives as reversible monoacylglycerol lipase (MAGL) inhibitors

Monoacylglycerol lipase (MAGL) is an attractive therapeutic target for many pathologies, including neurodegenerative diseases, cancer as well as chronic pain and inflammatory pathologies. The identification of reversible MAGL inhibitors, devoid of the side effects associated to prolonged MAGL inactivation, is a hot topic in medicinal chemistry. In this study, a novel phenyl(piperazin-1-yl)methanone inhibitor of MAGL was identified through a virtual screening protocol based on a fingerprint-driven consensus docking (CD) approach. Molecular modeling and preliminary structure-based hit optimization studies allowed the discovery of derivative 4, which showed an efficient reversible MAGL inhibition (IC50 = 6.1 µM) and a promising antiproliferative activity on breast and ovarian cancer cell lines (IC50 of 31–72 µM), thus representing a lead for the development of new and more potent reversible MAGL inhibitors. Moreover, the obtained results confirmed the reliability of the fingerprint-driven CD approach herein developed.

Assay development for determination of DZ2002, a new reversible SAHH inhibitor, and its acid metabolite DZA in blood and application to rat pharmacokinetic study

Methyl (S)-4-(6-amino-9H-purin-9-yl)-2-hydroxybutanoate (DZ2002) is a potent reversible inhibitor of S-adenosyl-L-homocysteine hydrolase (SAHH). Due to its ester structure, DZ2002 is rapidly hydrolyzed in rat blood to 4-(6-amino-9H-purin-9-yl)-2-hydroxybutyric acid (DZA) during and after blood sampling from rats; this hampers accurate determination of the circulating DZ2002 and its acid metabolite DZA in rats. To this end, a method for determining the blood concentrations of DZ2002 and DZA in rats was developed by using methanol to immediately deactivate blood carboxylesterases during sampling. The newly developed bioanalytical assay possessed favorable accuracy and precision with lower limit of quantification of 31 nM for DZ2002 and DZA. This validated assay was applied to a rat pharmacokinetic study of DZ2002. After oral administration, DZ2002 was found to be extensively converted into DZA. The level of systemic exposure to DZ2002 was significantly lower than that of DZA. The apparent oral bioavailability of DZ2002 was 90%–159%. The mean terminal half-lives of DZ2002 and DZA were 0.3–0.9 and 1.3–5.1 h, respectively. The sample preparation method illustrated here may be adopted for determination of other circulating ester drugs and their acid metabolites in rodents.

Development of Novel Monoamine Oxidase-B (MAO-B) Inhibitors with Reduced Blood-Brain Barrier Permeability for the Potential Management of Noncentral Nervous System (CNS) Diseases.

Studies indicate that MAO-B is induced in peripheral inflammatory diseases. To target peripheral tissues using MAO-B inhibitors that do not permeate the blood-brain barrier (BBB) the MAO-B-selective inhibitor deprenyl was remodeled by replacing the terminal acetylene with a CO2H function, and incorporating a para-OCH2Ar motif (compounds 10a-s). Further, in compound 32 the C-2 side chain corresponded to CH2CN. In vitro, 10c, 10j, 10k, and 32 were identified as potent reversible MAO-B inhibitors, and all four compounds were more stable than deprenyl in plasma, liver microsomal, and hepatocyte stability assays. In vivo, they demonstrated greater plasma bioavailability. Assessment of in vitro BBB permeability showed that compound 10k is a P-glycoprotein (P-gp) substrate and 10j displayed mild interaction. Importantly, compounds 10c, 10j, 10k, and 32 displayed significantly reduced BBB permeability after intravenous, subcutaneous, and oral administration. These polar MAO-B inhibitors are pertinent leads for evaluation of efficacy in noncentral nervous system (CNS) inflammatory disease models.

Abstract 1963: ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma

Abstract Background: B-cell receptor (BCR)-mediated signaling plays an important role in the pathogenesis of a subset of diffuse large B-cell lymphoma (DLBCL). Despite major advances in the treatment, ~40% of the relapsed/refractory DLBCL patients still experience early treatment failure after initial response to chemotherapy. ARQ 531, a reversible inhibitor of BTK and BTK-C481S mutant, also potently suppresses BCR signaling. Here we demonstrate that ARQ 531 targets additional kinases and suppresses multiple oncogenic pathways, this inhibitory potency is coupled to broad anti-tumor activity in DLBCL subtypes including tumors resistant to BCR targeted therapy. Methods: Biochemical inhibition and broad kinase profiling were assessed using recombinant proteins. Binding kinetics and the residence time with BTK and BTK-C481S were measured by Surface Plasmon Resonance (SPR) assay. Binding mode of ARQ 531 with BTK was determined by protein crystallography. Pathway inhibition assessments, in vivo efficacy and in vivo target inhibition were performed in DLBCL tumor models derived from TMD8, SUDHL-4 and DOHH-2 cell lines. Results: ARQ 531 potently inhibited BTK (IC50 = 0.85 nM), the binding potency was accompanied by long residence time (51 min). Crystal structure of BTK/ARQ 531 complex showed that ARQ 531 occupies the ATP-binding pocket. Kinase selectivity profile suggested that ARQ 531 inhibits sub-families of Tec, Src, Trk kinases. Significant anti-proliferative activity (GI50 = < 1µM) was observed in hematological malignant cell lines characterized by addiction to BTK signaling and primarily resistant to ibrutinib. Pathway inhibition analysis suggested that ARQ 531 targets multiple oncogenic signaling pathways in both ABC- and GCB-DLBCL cell lines. Unlike ibrutinib, ARQ 531 suppressed both the upstream activating signals (via inhibition of a select member of Src kinase family) and the downstream signaling pathways (via pAKT and pERK kinases). In GCB-DLBCL cell lines (SUDHL-4 and DOHH-2), ARQ 531 potently suppressed expression of anti-apoptotic c-Myc and BCL6 oncoproteins in a dose dependent fashion, and concomitantly induced apoptotic cleavage of PARP protein. In the ibrutinib-resistant SUDHL-4 mouse xenograft model, ARQ 531 potently suppressed tumor growth (>80% inhibition) compared to the control group when dosed orally at 75 mg/kg. Additionally, preliminary data suggest that ARQ 531 crosses the blood, brain-barrier. Conclusion: ARQ 531 is a potent reversible inhibitor of BTK, its distinct kinase selectivity profile offers significant advantage for simultaneous inhibition of multiple therapeutically relevant targets. ARQ 531 attenuated the expression of key oncogenic drivers via inhibition of downstream BCR activating kinases. These results highlight the therapeutic potential of inhibition of BCR signaling inhibition by ARQ 531 in the treatment of DLBCL. Citation Format: Sudharshan Eathiraj, Yi Yu, Ron Savage, Jennifer A. Woyach, Sean D. Reiff, Amy J. Johnson, Brian Schwartz. ARQ 531, a potent reversible BTK inhibitor, exhibits potent antitumor activity in ibrutinib-resistant diffuse large B-cell lymphoma [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 1963.

Complexity of Blocking Bivalent Protein-Protein Interactions: Development of a Highly Potent Inhibitor of the Menin-Mixed-Lineage Leukemia Interaction.

The protein-protein interaction between menin and mixed-lineage leukemia 1 (MLL1) plays an important role in development of acute leukemia with translocations of the MLL1 gene and in solid tumors. Here, we report the development of a new generation of menin-MLL1 inhibitors identified by structure-based optimization of the thienopyrimidine class of compounds. This work resulted in compound 28 (MI-1481), which showed very potent inhibition of the menin-MLL1 interaction (IC50 = 3.6 nM), representing the most potent reversible menin-MLL1 inhibitor reported to date. The crystal structure of the menin-28 complex revealed a hydrogen bond with Glu366 and hydrophobic interactions, which contributed to strong inhibitory activity of 28. Compound 28 also demonstrates pronounced activity in MLL leukemia cells and in vivo in MLL leukemia models. Thus, 28 is a valuable menin-MLL1 inhibitor that can be used for potential therapeutic applications and in further studies regarding the role of menin in cancer.

Challenging medicinal chemistry: ups and downs in a drug discovery project

The ups and downs of a multi-year medicinal chemistry optimisation effort in the quest of identifying inhibitors of human steroid sulfatase as potential topical therapeutics are summarised. The focus lies on dead ends, redirection and mastering of the hurdles rather than detailed structure–activity relationship results of a series. In addition to the usual multi-parameter optimisation towards drug-like properties, unexpected barriers prohibitive for the planned clinical application had to be overcome. While a substrate- and mechanism-based design approach rapidly yielded aryl sulfamates as potent, irreversible enzyme inhibitors, this inhibitor class had to be abandoned due to inherent chemical instability in solution. The rescue of the project was a hit class from a random screening approach, but from there it still took quite some efforts to invent potent reversible inhibitors appropriate for development towards a clinical candidate.

The selective reversible FAAH inhibitor, SSR411298, restores the development of maladaptive behaviors to acute and chronic stress in rodents

Enhancing endogenous cannabinoid (eCB) signaling has been considered as a potential strategy for the treatment of stress-related conditions. Fatty acid amide hydrolase (FAAH) represents the primary degradation enzyme of the eCB anandamide (AEA), oleoylethanolamide (OEA) and palmitoylethanolamide (PEA). This study describes a potent reversible FAAH inhibitor, SSR411298. The drug acts as a selective inhibitor of FAAH, which potently increases hippocampal levels of AEA, OEA and PEA in mice. Despite elevating eCB levels, SSR411298 did not mimic the interoceptive state or produce the behavioral side-effects (memory deficit and motor impairment) evoked by direct-acting cannabinoids. When SSR411298 was tested in models of anxiety, it only exerted clear anxiolytic-like effects under highly aversive conditions following exposure to a traumatic event, such as in the mouse defense test battery and social defeat procedure. Results from experiments in models of depression showed that SSR411298 produced robust antidepressant-like activity in the rat forced-swimming test and in the mouse chronic mild stress model, restoring notably the development of inadequate coping responses to chronic stress. This preclinical profile positions SSR411298 as a promising drug candidate to treat diseases such as post-traumatic stress disorder, which involves the development of maladaptive behaviors.

Inhibition of CYP3A by Antimalarial Piperaquine and Its Metabolites in Human Liver Microsomes With IVIV Extrapolation

The potential of the antimalarial piperaquine and its metabolites to inhibit CYP3A was investigated in pooled human liver microsomes. CYP3A activity was measured by liquid chromatography-tandem mass spectrometry as the rate of 1′-hydroxymidazolam formation. Piperaquine was found to be a reversible, potent inhibitor of CYP3A with the following parameter estimates (%CV): IC50 = 0.76 μM (29), Ki = 0.68 μM (29). In addition, piperaquine acted as a time-dependent inhibitor with IC50 declining to 0.32 μM (28) during 30-min pre-incubation. Time-dependent inhibitor estimates were kinact = 0.024 min−1 (30) and KI = 1.63 μM (17). Metabolite M2 was a highly potent reversible inhibitor with estimated IC50 and Ki values of 0.057 μM (17) and 0.043 μM (3), respectively. M1 and M5 metabolites did not show any inhibitory properties within the limits of assay used. Average (95th percentile) simulated in vivo areas under the curve of midazolam increased 2.2-fold (3.7-fold) on the third which is the last day of piperaquine dosing, whereas for its metabolite M2, areas under the curve of midazolam increased 7.7-fold (13-fold).