Kinase Selectivity Profile Research Articles

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity. Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z9-LYTE) with IC50 determination at a fixed time point. On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB). Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells. Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells. Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates. Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested. Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively. No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. Disclosures Kaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity9s Board of Directors or advisory committees.

Discovery of potent p38α MAPK inhibitors through a funnel like workflow combining in silico screening and in vitro validation

This work describes the rational discovery of novel chemotypes of p38α MAPK inhibitors using a funnel approach consisting of several computer-aided drug discovery methods and biological experiments. Among the identified hits, four compounds belonging to different chemical families showed IC50 values lower than 10 μM. In particular, the 1,4-benzodioxane derivative 5 turned out to be a potent and efficient p38α MAPK inhibitor having IC50 = 0.07 μM, and LEexp and LipE values of 0.38 and 4.8, respectively; noteworthy, the compound had also a promising kinase selectivity profile and the capability to suppress p38α MAPK effects in human immune cells.Overall, the collected findings highlight that the applied strategy has been successful in generating chemical novelty in the inhibitor kinase field, providing suitable chemical candidates for further inhibitor optimization.

Potent bicyclic inhibitors of malarial cGMP-dependent protein kinase: approaches to combining improvements in cell potency, selectivity and structural novelty.

Focussed studies on imidazopyridine inhibitors of Plasmodium falciparum cyclic GMP-dependent protein kinase (PfPKG) have significantly advanced the series towards desirable in vitro property space. LLE-based approaches towards combining improvements in cell potency, key physicochemical parameters and structural novelty are described, and a structure-based design hypothesis relating to substituent regiochemistry has directed efforts towards key examples with well-balanced potency, ADME and kinase selectivity profiles.

Abstract 2194: Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity

Abstract Acalabrutinib is a potent and highly selective, oral covalent inhibitor of Bruton tyrosine kinase (BTK) that received accelerated approval for relapsed/refractory mantle cell lymphoma by the United States Food and Drug Administration in October 2017. ADME studies in humans, rat and dog, revealed extensive metabolism of acalabrutinib. The major circulating metabolite (M27, ACP-5862) was produced by CYP3A oxidation, resulting in a pyrrolidine ring-opened metabolite, with the butynamide electrophile still present. We investigated the on-target BTK inhibition and kinase selectivity profile of ACP-5862 using the same biochemical and cellular assays previously employed to profile acalabrutinib.1 The apparent BTK IC50 was determined over time using the LanthaScreen assay. Results indicate that ACP-5862, like acalabrutinib, is a covalent inhibitor of BTK. Binding kinetics derived from IC50 over time data, indicated that acalabrutinib and ACP-5862 have similar affinity (KI), however the BTK inactivation rate (kinact) for ACP-5862 was half that relative to acalabrutinib. This indicated a covalent BTK inhibition potency for ACP-5862 that was 2-fold lower than acalabrutinib. The overall kinome inhibition profile at 1 µM using KINOMEscan (Eurofins DiscoverX), as well as IC50 determinations on closely related kinases with a Cys in the same position as Cys481 in BTK (ITK, TXK, TEC, BMX, EGFR, ERBB2, ERBB4, BLK, JAK3), both revealed that the kinase selectivity profiles of acalabrutinib and ACP-5862 were similar. On-target inhibition in B cells was investigated measuring inhibition of B-cell antigen receptor-mediated activation of CD69 cell surface expression on human peripheral B cells using human peripheral blood mononuclear cells and human whole blood (hWB). The EC50 for anti-IgD-induced CD69 expression in hWB was 64 ± 6 nM for ACP-5862, compared to 9.2 ± 4.4 nM for acalabrutinib. The hWB EC90, representing near complete inhibition of BTK by acalabrutinib and ACP-5862 was observed at 72 ± 20 nM and 544 ± 376 nM, respectively. In conclusion, the results indicate that ACP-5862, the major metabolite of acalabrutinib, has intrinsic BTK inhibitory activity and a similar kinase selectivity profile as acalabrutinib. The contribution of ACP-5862 to on-target covalent inhibition of BTK in humans is unclear at present, but is most likely limited, since the EC90 in hWB for ACP-5862 approximated observed plasma Cmax in humans dosed with 100 mg acalabrutinib. The relative contribution of acalabrutinib and ACP-5862 to BTK pharmacodynamics is under further investigation. Terry Podoll and J. Greg Slatter are former Acerta Pharma employees. Reference 1. Barf T, Covey T, Izumi R, et al. Acalabrutinib (ACP-196): A covalent Bruton tyrosine kinase inhibitor with a differentiated selectivity and in vivo potency profile. J Pharmacol Exp Ther. 2017;363(2):240-252. Citation Format: Allard Kaptein, Terry Podoll, Gerjan de Bruin, Maaike Emmelot-van Hoek, Anouk de Jong, Bart van Lith, Niels Hoogenboom, Tjeerd Barf, Joseph Ware, J. Greg Slatter. Preclinical pharmacological profiling of ACP-5862, the major metabolite of the covalent BTK inhibitor acalabrutinib, displays intrinsic BTK inhibitory activity [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 2194.

Probing the Inhibition of Microtubule Affinity Regulating Kinase 4 by N-Substituted Acridones

Microtubule affinity regulating kinase 4 (MARK4) becomes a unique anti-cancer drug target as its overexpression is responsible for different types of cancers. In quest of novel, effective MARK4 inhibitors, some acridone derivatives were synthesized, characterized and evaluated for inhibitory activity against human MARK4. Among all the synthesized compounds, three (7b, 7d and 7f) were found to have better binding affinity and enzyme inhibition activity in µM range as shown by fluorescence binding, ITC and kinase assays. Here we used functional assays of selected potential lead molecules with commercially available panel of 26 kinases of same family. A distinctive kinase selectivity profile was observed for each compound. The selective compounds were identified with submicromolar cellular activity against MARK4. Furthermore, in vitro antitumor evaluation against cancerous cells (MCF-7 and HepG2) revealed that compounds 7b, 7d and 7f inhibit cell proliferation and predominantly induce apoptosis in MCF-7 cells, with IC50 values of 5.2 ± 1.2 μM, 6.3 ± 1.2 μM, and 5.8 ± 1.4 μM respectively. In addition, these compounds significantly upsurge the oxidative stress in cancerous cells. Our observations support our approach for the synthesis of effective inhibitors against MARK4 that can be taken forward for the development of novel anticancer molecules targeting MARK4.

Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S.

Precision medicine has revolutionized the treatment of patients in EGFR driven non-small cell lung cancer (NSCLC). Targeted drugs show high response rates in genetically defined subsets of cancer patients and markedly increase their progression-free survival as compared to conventional chemotherapy. However, recurrent acquired drug resistance limits the success of targeted drugs in long-term treatment and requires the constant development of novel efficient inhibitors of drug resistant cancer subtypes. Herein, we present covalent inhibitors of the drug resistant gatekeeper mutant EGFR-L858R/T790M based on the pyrrolopyrimidine scaffold. Biochemical and cellular characterization, as well as kinase selectivity profiling and western blot analysis, substantiate our approach. Moreover, the developed compounds possess high activity against multi drug resistant EGFR-L858R/T790M/C797S in biochemical assays due to their highly reversible binding character, that was revealed by characterization of the binding kinetics. In addition, we present the first X-ray crystal structures of covalent inhibitors in complex with C797S-mutated EGFR which provide detailed insight into their binding mode.

Potency and Selectivity of BTK Inhibitors in Clinical Development for B-Cell Malignancies

Background: Bruton tyrosine kinase (BTK) is a validated target for B-cell malignancies. The BTK inhibitor ibrutinib was approved in chronic lymphocytic leukemia, mantle cell lymphoma (MCL), and Waldenstrom macroglobulinemia. Acalabrutinib is a potent, highly selective, covalent BTK inhibitor with minimal off-target activity; it received accelerated FDA approval in October 2017 for the treatment of patients with MCL having ≥1 prior therapy. In addition to the approved covalent BTK inhibitors ibrutinib and acalabrutinib, clinical data in B-cell malignancies are available for spebrutinib (CC-292), tirabrutinib (ONO/GS-4059) and zanubrutinib (BGB-3111). We performed biochemical and cellular profiling of these 5 BTK inhibitors, investigating potency and selectivity.Methods: Two biochemical kinase assays assessed BTK inhibitor potency, with IC50 determination at a fixed time point (IMAP; Molecular Devices) or over time (LanthaScreen; Invitrogen); the latter was used to calculate binding kinetics. Kinome profiling was performed at a single dose (1 µM) using KINOMEscan (Eurofins DiscoverX). IC50 determinations were made using kinases with a Cys in the same position as the Cys481 residue in BTK, using assays developed in house (using IMAP and LanthaScreen) or at Thermo Fisher Scientific (Z'-LYTE) with IC50 determination at a fixed time point.On-target inhibition of BTK in cellular assays was evaluated using B-cell receptor-mediated activation of CD69 expression on peripheral B cells using human peripheral blood mononuclear cells (hPBMCs) or human whole blood (hWB).Off-target inhibition of epidermal growth factor receptor (EGFR) was evaluated in a cellular assay examining the effect of EGF-induced EGFR phosphorylation in A431 cells.Off-target inhibition of ITK and/or TXK was evaluated using T-cell receptor (TCR)-mediated activation of interleukin-2 expression in Jurkat T cells and CD25 cell surface expression using primary human peripheral T cells.Results: Based on biochemical binding kinetics, ibrutinib and zanubrutinib were the most potent BTK inhibitors (Table 1), followed by spebrutinib; acalabrutinib and tirabrutinib had comparable potency. Differences in potency were largely driven by differences in inactivation rates. However, differences in biochemical potency were lost (in part) in cellular assays using hPBMCs or hWB. EC50 values in hWB were <10 nM for ibrutinib, acalabrutinib and zanubrutinib. Spebrutinib had an EC50 of 140 nM in hWB, showing the greatest loss of potency versus biochemical assays and hPBMCs, going to the physiologically most relevant cellular experimental conditions (Table 2).Differences in overall kinase selectivity were observed among the BTK inhibitors (Figure 1; KINOMEscan). Acalabrutinib had the lowest hit rate; 1.5% of human wild-type kinases were inhibited >65% at 1 µM (excluding BTK). Kinase hit rate was also low for tirabrutinb (2.3%), whereas ibrutinib (9.4%), zanubrutinib (4.3%), and spebrutinib (8.3%) had higher hit rates.Acalabrutinib had a high selectivity for BTK over kinases with a Cys in the same position as the Cys481 residue in BTK (Table 2). Similar results were observed for tirabrutinib, whereas ibrutinib, spebrutinib and zanubrutinib were less selective in this panel of kinases with potential for off-target covalent binding by BTK inhibitors (Table 2). Acalabrutinib also had a higher selectivity for BTK over Src-family kinases than the other BTK inhibitors tested.Acalabrutinib and tirabrutinib had EC50 values >10 µM in the cellular assay of off-target inhibition of EGFR; ibrutinib, zanubrutinib, and spebrutinib had EC50 values of 0.07, 0.39, and 4.7 µM, respectively.No off-target inhibition was observed for acalabrutinib or tirabrutinib on TCR-mediated activation of T cells up to 10 µM. Ibrutinib, zanubrutinib, and spebrutinib had EC50 values <1 µM in Jurkat T cells. Similar results were observed in primary human peripheral T cells.Conclusion: BTK inhibitors in clinical development for B-cell malignancies had differing potency in biochemical assays, but these differences were lost (in part) in cellular assays, particularly in hWB. Among the BTK inhibitors tested, the greatest differentiation was observed in kinase selectivity profiles; acalabrutinib and tirabrutinib had the highest kinase selectivity. [Display omitted] DisclosuresKaptein:Covaluation Pharma BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership; Apo-T BV: Consultancy. de Bruin:Acerta Pharma: Employment. Emmelot-van Hoek:Acerta Pharma: Employment. van de Kar:Acerta Pharma: Employment. de Jong:Acerta Pharma: Employment. Gulrajani:Acerta Pharma: Employment, Equity Ownership. Demont:Acerta Pharma: Employment. Covey:AstraZeneca: Equity Ownership; Acerta Pharma: Employment. Mittag:Acerta Pharma: Employment, Equity Ownership. Barf:Covaluation Holding BV: Employment, Equity Ownership; Acerta Pharma BV: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Novel selective thiadiazine DYRK1A inhibitor lead scaffold with human pancreatic β-cell proliferation activity

The Dual-Specificity Tyrosine Phosphorylation-Regulated Kinase 1A (DYRK1A) is an enzyme that has been implicated as an important drug target in various therapeutic areas, including neurological disorders (Down syndrome, Alzheimer's disease), oncology, and diabetes (pancreatic β-cell expansion). Current small molecule DYRK1A inhibitors are ATP-competitive inhibitors that bind to the kinase in an active conformation. As a result, these inhibitors are promiscuous, resulting in pharmacological side effects that limit their therapeutic applications. None are in clinical trials at this time. In order to identify a new DYRK1A inhibitor scaffold, we constructed a homology model of DYRK1A in an inactive, DFG-out conformation. Virtual screening of 2.2 million lead-like compounds from the ZINC database, followed by in vitro testing of selected 68 compounds revealed 8 hits representing 5 different chemical classes. We chose to focus on one of the hits from the computational screen, thiadiazine 1 which was found to inhibit DYRK1A with IC50 of 9.41 μM (Kd = 7.3 μM). Optimization of the hit compound 1, using structure-activity relationship (SAR) analysis and in vitro testing led to the identification of potent thiadiazine analogs with significantly improved binding as compared to the initial hit (Kd = 71–185 nM). Compound 3–5 induced human β-cell proliferation at 5 μM while showing selectivity for DYRK1A over DYRK1B and DYRK2 at 10 μM. This newly developed DYRK1A inhibitor scaffold with unique kinase selectivity profiles has potential to be further optimized as novel therapeutics for diabetes.

Abstract 1661: Discovery of potent Abelson kinase inhibitors from the scaffold of pyrano[2,3-d]pyrimidin-7-one

Abstract Tyrosine kinases regulate cell proliferation, survival, invasion and angiogenesis during tumor initiation and progression. The Abelson (ABL) family of protein kinases comprises ABL1 and ABL2, which link diverse extracellular stimuli to signaling pathways that control cell growth, survival, invasion, adhesion and migration. Inhibition of ABLs has been implied in many type of hematopoietic malignancies and solid tumors. The compound PD173955 is a potent ABL kinase inhibitor. It was developed from the scaffold pyrido[2,3-d]pyrimidin-7-one by Parke-Davis (Pfizer). To discover novel ABL inhibitors, we investigated the scaffold pyrano[2,3-d]pyrimidin-7-one, which is structurally analogous to pyrido[2,3-d]pyrimidin-7-one. This novel scaffold has not been extensively studied in synthetic chemistry and pharmacology. The compound LDK1504 generated from this scaffold is a close analog of PD173955. It was synthesized as a prototypical compound from the pyrano[2,3-d]pyrimidin-7-one scaffold. In the enzymatic assays, LDK1504 was demonstrated with an inhibitory activity comparable to PD173955 for ABL1 kinase. It can inhibit the ABL1 kinase with an IC50 of 18.7 nM. Further optimization leads to the identification of LDK1512 (Kd = 0.38 nM; IC50 = 1.27 nM) against ABL1. The preparation of this class of compounds was achieved through a facile synthesis of 6-bromo-2-(methylthio)-7H-pyrano[2,3-d]pyrimidin-7-one followed by Suzuki coupling reaction and derivatization of the C-2 position of the pyrimidine ring. In this presentation, our synthesis, preliminary SAR of this class of compounds, and kinase selectivity profiling will be discussed. Citation Format: Zhixing Wu, Boqiao Fu, Changjiang Qiao, Ashila Nagaraju, Dai Lu. Discovery of potent Abelson kinase inhibitors from the scaffold of pyrano[2,3-d]pyrimidin-7-one [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 1661.

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 = &amp;lt; 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 (&amp;gt;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.

Design, synthesis, biological evaluation and molecular modeling of novel 1H-pyrazolo[3,4-d]pyrimidine derivatives as BRAFV600E and VEGFR-2 dual inhibitors

Aiming to explore novel BRAFV600E and VEGFR-2 dual inhibitors, a series of 1H-pyrazolo[3,4-d]pyrimidine derivatives were designed, synthesized and biologically evaluated in this study. Most of the synthesized 1H-pyrazolo[3,4-d]pyrimidine compounds displayed moderate to high potent activity in both enzymatic and cellular proliferation assays. Among these compounds, 9e, 9g, 9m and 9u showed remarkably high inhibitory activities against both BRAFV600E and VEGFR-2 kinase comparable to positive control Sorafenib. Particularly, compound 9u also showed potent anti-proliferative activity against BRAFV600E-expressing A375 (IC50 = 1.74 μM) and H-29 (IC50 = 6.92 μM) as well as VEGFR-2-expressing HUVEC (IC50 = 5.89 μM), which was also comparable to Sorafenib. Furthermore, kinase selectivity profile showed that 9u had almost poor or no significant inhibitory activity against wild-type BRAF and 15 other tested protein kinases. Flow cytometric analysis showed that compound 9u mainly arrested the A375 and HUVEC cell lines in the G0/G1 stage with a concentration-dependent effect. In addition, the molecular docking and molecular dynamics simulations suggested that 9u adopted a similar binding pattern with Sorafenib at the ATP-binding sites of BRAFV600E and VEGFR-2. Taken together, these results indicated that compound 9u may serve as novel lead compound in research on more effective BRAFV600E and VEGFR-2 dual inhibitors.

A Dual‐Inhibitor of Soluble Epoxide Hydrolase and p38 Kinase Alleviating Tau Hyperphosphorylation and Amyloid Neurotoxicity for Potential Treatment of Neuroinflammation in Alzheimer's Disease

Alzheimer's disease (AD) is the most common dementia that cannot be prevented, cured or even slowed. Neuroinflammation associated with tau hyperphosphorylation and amyloid toxicity is a major hallmark in AD. Soluble epoxide hydrolase (sEH) and p38 kinase are therapeutic targets involving in inflammatory signaling. Development of selective inhibitors to modulate both sEH and p38 kinase is an attractive strategy for AD. We herein studied a potent and blood‐brain barrier permeable sEH inhibitor, 1‐(1‐propanoylpiperidin‐4‐yl)‐3‐[4‐(trifluoromethoxy)phenyl] urea (TPPU) (half maximum inhibition concentration, IC50, 0.64 nM), on neuroprotection in a human AD neuronal model. TPPU effectively alleviates tau hyperphosphorylation and amyloid neurotoxicity in vitro with a half maximum effective concentration (EC50) value of 49 nM. In vitro kinase selectivity profiling indicated that TPPU is a specific p38β kinase inhibitor with an IC50 value of 0.27 μM. The results suggested that TPPU shows a synergistic effect of neuropharmacology through a dual inhibition of sEH and p38. Additional knowledge of TPPU on the neuroinflammatory protection against tau and amyloid pathologies warrants further investigation.Support or Funding InformationThis work was supported in part by the NIH National Institute on Minority Health and Health Disparities grant 8G12MD007601.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and biological evaluation of 2,6-disubstituted-9H-purine, 2,4-disubstitued-thieno[3,2-d]pyrimidine and -7H-pyrrolo[2,3-d]pyrimidine analogues as novel CHK1 inhibitors

Checkpoint kinase 1 (CHK1) inhibitors can potentiate the effectiveness of deoxyribonucleic acid (DNA) damaging agents in the treatment of cancer. A novel series of 2,6-disubstituted-9H-purine (3a-p, 5a and 5b), 2,4-disubstituted-thieno[3,2-d]pyrimidine (8a-c) and 2,4-disbustituted-7H-pyrrolo[2,3-d]pyrimidine (11a-c) analogues were designed and synthesized as potent CHK1 inhibitors. Compounds (3a, 3d, 3f and 3j-l) with 9H-purine core displayed more potent inhibition against CHK1. The most potent compound (3l) also exhibited low anti-proliferative effects towards HT29 and Hek293 cell lines. In addition, 3l showed strong potentiating effect (7-fold) on the anti-proliferative activity of gemcitabine towards HT29 cells. The results of cell cycle assay indicated that 3l could strikingly affect the cell cycle distribution of the gemcitabine-treated HT29 cells and induce a significant S phase accumulation. The kinase selectivity profile of 3l displayed acceptable selectivity against other kinases. These results rendered 3l a potent lead compound of CHK1 inhibitor for further investigation.

Abstract B152: CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant

Abstract Introduction: Bruton's tyrosine kinase (BTK) is a member of the Tec family of cytoplasmic tyrosine kinase. BTK plays a crucial role in the BCR signaling, essential for B-cell development, and BTK has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved BTK inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of BTK to inhibit BTK enzymatic activity, but recent studies suggested that the C481S mutation in BTK disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent BTK inhibitor is highly demanded to overcome ibrutinib resistance. We identified a highly selective, orally available non-covalent inhibitor of BTK, CB1763. Here, we report the characterization of CB1763, capable of potently inhibiting BTK [C481S] mutant, both in vitro and in vivo. Material and Methods: We produced ibrutinib-resistant BTK [C481S] mutant recombinant enzyme to assess efficacy of compounds against the drug-resistant enzyme. To evaluate cellular potency, phosphorylations of BTK and PLC- γ were analyzed by Western blotting in C481S BTK mutant transfected HEK293 cells. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate antitumor efficacy of BTK inhibitors. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: In addition to optimization of CB988, previously reported by our group, we identified CB1763 as a highly selective non-covalent BTK inhibitor. CB1763 exhibited strong inhibitory activity for both wild type and C481S mutant BTKs with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.85 and 0.99 nM for wild type and C481S, respectively). In cellular assays, CB1763 significantly reduced Tyr223 autophosphorylation of BTK protein at nano-molar concentration in C481S BTK mutant transfected HEK293 cells. Interestingly, persistent inhibition of autophosphorylation was observed after washout of CB1763, while no prolonged inhibition was observed by ibrutinib treatment. CB1763 demonstrated excellent antitumor activity in the BTK driven OCI-Ly10 xenograft mouse model. Conclusions: BTK [C481S] mutation has been reported in ibrutinib-relapsed CLL and MCL patients. Orally available novel non-covalent BTK inhibitor, CB1763, strongly inhibited C481S mutant BTK in vitro and in vivo, suggesting that CB1763 has the potential to treat patients who are relapsed/refractory to ibrutinib. Citation Format: Tokiko Asami, Wataru Kawahata, Shigeki Kashimoto, Masaaki Sawa. CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B152.

Structure-Based Design of 6-Chloro-4-aminoquinazoline-2-carboxamide Derivatives as Potent and Selective p21-Activated Kinase 4 (PAK4) Inhibitors.

Herein, we report the discovery and characterization of a novel class of PAK4 inhibitors with a quinazoline scaffold. Based on the shape and chemical composition of the ATP-binding pocket of PAKs, we chose a 2,4-diaminoquinazoline series of inhibitors as a starting point. Guided by X-ray crystallography and a structure-based drug design (SBDD) approach, a series of novel 4-aminoquinazoline-2-carboxamide PAK4 inhibitors were designed and synthesized. The inhibitors' selectivity, therapeutic potency, and pharmaceutical properties were optimized. One of the best compounds, 31 (CZh226), showed remarkable PAK4 selectivity (346-fold vs PAK1) and favorable kinase selectivity profile. Moreover, this compound potently inhibited the migration and invasion of A549 tumor cells by regulating the PAK4-directed downstream signaling pathways in vitro. Taken together, these data support the further development of 31 as a lead compound for PAK4-targeted anticancer drug discovery and as a valuable research probe for the further biological investigation of group II PAKs.

Discovery and Optimization of Pyrrolopyrimidine Inhibitors of Interleukin-1 Receptor Associated Kinase 4 (IRAK4) for the Treatment of Mutant MYD88L265P Diffuse Large B-Cell Lymphoma.

Herein we report the optimization of a series of pyrrolopyrimidine inhibitors of interleukin-1 receptor associated kinase 4 (IRAK4) using X-ray crystal structures and structure based design to identify and optimize our scaffold. Compound 28 demonstrated a favorable physicochemical and kinase selectivity profile and was identified as a promising in vivo tool with which to explore the role of IRAK4 inhibition in the treatment of mutant MYD88L265P diffuse large B-cell lymphoma (DLBCL). Compound 28 was shown to be capable of demonstrating inhibition of NF-κB activation and growth of the ABC subtype of DLBCL cell lines in vitro at high concentrations but showed greater effects in combination with a BTK inhibitor at lower concentrations. In vivo, the combination of compound 28 and ibrutinib led to tumor regression in an ABC-DLBCL mouse model.

Quantitative Structure-Activity Relationship Modeling of Kinase Selectivity Profiles.

The discovery of selective inhibitors of biological target proteins is the primary goal of many drug discovery campaigns. However, this goal has proven elusive, especially for inhibitors targeting the well-conserved orthosteric adenosine triphosphate (ATP) binding pocket of kinase enzymes. The human kinome is large and it is rather difficult to profile early lead compounds against around 500 targets to gain an upfront knowledge on selectivity. Further, selectivity can change drastically during derivatization of an initial lead compound. Here, we have introduced a computational model to support the profiling of compounds early in the drug discovery pipeline. On the basis of the extensive profiled activity of 70 kinase inhibitors against 379 kinases, including 81 tyrosine kinases, we developed a quantitative structure–activity relation (QSAR) model using artificial neural networks, to predict the activity of these kinase inhibitors against the panel of 379 kinases. The model’s performance in predicting activity ranges from 0.6 to 0.8 depending on the kinase, from the area under the curve (AUC) of the receiver operating characteristics (ROC). The profiler is available online at http://www.meilerlab.org/index.php/servers/show?s_id=23.

Dynamical models of mutated chronic myelogenous leukemia cells for a post-imatinib treatment scenario: Response to dasatinib or nilotinib therapy.

Targeted inhibition of the oncogenic BCR-ABL1 fusion protein using the ABL1 tyrosine kinase inhibitor imatinib has become standard therapy for chronic myelogenous leukemia (CML), with most patients reaching total and durable remission. However, a significant fraction of patients develop resistance, commonly due to mutated ABL1 kinase domains. This motivated development of second-generation drugs with broadened or altered protein kinase selectivity profiles, including dasatinib and nilotinib. Imatinib-resistant patients undergoing treatment with second-line drugs typically develop resistance to them, but dynamic and clonal properties of this response differ. Shared, however, is the observation of clonal competition, reflected in patterns of successive dominance of individual clones. We present three deterministic mathematical models to study the origins of clinically observed dynamics. Each model is a system of coupled first-order differential equations, considering populations of three mutated active stem cell strains and three associated pools of differentiated cells; two models allow for activation of quiescent stem cells. Each approach is distinguished by the way proliferation rates of the primary stem cell reservoir are modulated. Previous studies have concentrated on simulating the response of wild-type leukemic cells to imatinib administration; our focus is on modelling the time dependence of imatinib-resistant clones upon subsequent exposure to dasatinib or nilotinib. Performance of the three computational schemes to reproduce selected CML patient profiles is assessed. While some simple cases can be approximated by a basic design that does not invoke quiescence, others are more complex and require involvement of non-cycling stem cells for reproduction. We implement a new feedback mechanism for regulation of coupling between cycling and non-cycling stem cell reservoirs that depends on total cell populations. A bifurcation landscape analysis is also performed for solutions to the basic ansatz. Computational models reproducing patient data illustrate potential dynamic mechanisms that may guide optimization of therapy of drug resistant CML.

Abstract 3232: Development of novel Btk inhibitor, CB988 targering ibrutinib-resistant Btk C481S mutant

Abstract Introduction: Bruton's tyrosine kinase (Btk) is a member of the Tec family of cytoplasmic tyrosine kinase. Btk plays a crucial role in the BCR signaling, essential for B-cell development, and Btk has been recognized as a validated therapeutic target for B-cell malignancies. Ibrutinib, the first FDA-approved Btk inhibitor, has been appreciated as a promising targeted therapy for patients with B-cell malignancies. Ibrutinib covalently binds to the Cys481 residue of Btk to inhibit Btk enzymatic activity, but recent studies suggested that the C481S mutation in Btk disrupts the irreversible binding of ibrutinib, and resulted in ibrutinib-resistance in patients. Therefore a non-covalent Btk inhibitor is highly demanded to overcome ibrutinib resistance. Material and methods: We produced two conformationally different Btks, an activated form of Btk (Btk[A]) and an unactivated form of Btk (Btk[U]) by treating Btk protein with ATP (autophosphorylation) or with Lambda Protein Phosphatase (dephosphorylation), respectively. To evaluate cellular potency of Btk inhibitors, phosphorylations of Btk and PLC- γ were analyzed by Western blotting in Ramos cells, a human Burkitt’s lymphoma cell line. ABC-type DLBCL cell line, OCI-Ly10 was used to evaluate anti-tumor efficacy of Btk inhibitors. The ibrutinib-resistant Btk[C481S] mutant was produced to assess efficacy of compounds against the drug resistant enzyme. Kinase selectivity profiling was performed to confirm inhibitor selectivity. Results: Based on structure activity relationship studies, we identified a novel Btk inhibitor, CB988 as a highly selective non-covalent Btk inhibitor. CB988 exhibited remarkable activity for the both conformations of Btks with sub-nanomolar enzyme inhibitory potency in a reversible manner (IC50 = 0.78 and 0.09 nM for Btk[A] and Btk[U], respectively). These results suggested that CB988 preferentially binds to an inactive conformation of Btk. In cellular assays, CB988 strongly reduced Tyr223 phosphorylation of Btk at nano-molar concentration, and decreased phosphorylation of PLC-γ in Ramos cells. Furthermore, CB988 significantly reduced the proliferation of OCI-Ly10 cells. More importantly, CB988 potently inhibited the ibrutinib-resistant Btk[C481S] mutant in vitro (IC50 = 0.6 nM), which differs from other covalent Btk inhibitors. Conclusions: Btk[C481S] mutation have been reported in ibrutinib relapsed CLL and MCL patients. Novel non-covalent Btk inhibitor, CB988 strongly inhibited Btk[C481S] mutant enzyme, suggesting that CB988 has the potential to treat patients who are relapsed/refractory to ibrutinib. Citation Format: Wataru Kawahata, Tokiko Asami, Takayuki Irie, Yasuhiro Iwata, Takao Kiyoi, Masaaki Sawa. Development of novel Btk inhibitor, CB988 targering ibrutinib-resistant Btk C481S mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3232. doi:10.1158/1538-7445.AM2017-3232

Modeling comparative selectivity profiles of kinase inhibitors using FEP/MD protocol

Free energy perturbation (FEP)-based molecular modeling simulation of 5-fluoropyrimidine and 1,3,5-triazine derivatives followed by their synthesis and experimental evaluation have been carried out to estimate kinase selectivity profile. 5-Fluoropyrimidine derivatives show similar binding affinity for c-Src, Btk and Jak1 kinases, while 1,3,5-triazine derivatives demonstrate c-Src kinase selectivity.