Kinase Selectivity Profile Research Articles

Abstract 1816: In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma

Abstract Background: Previously we described multitarget CDK4/6-CDK9 inhibitor, LCI133 for the treatment of endometrial carcinoma (EC). We have optimized LCI133’s chemical properties to increase PI3K p110α potency to better target epithelial-derived cancers, resulting in nanomolar potent LCI139. We characterized LCI139’s in vitro potency, target specificity, activity in in vitro EC models, safety against normal cell lines. Methods: LCI139’s chemical structure was determined using nuclear magnetic resonance and high-resolution mass spectrometry. Specific target inhibition was assessed by cell-free kinase hotspot/lipid kinase assay and selectivity profile. LCI139 (0.001-10μM) was screened against EC cell lines AN3CA, RL95-2, Ishikawa, KLE, HEC-1-A and -B for 48 hours; IC50 values determined by CellTiter-Glo Assay. EC cells were treated with LCI139 or single agent control for 24 hours and stained with Annexin V/7-AAD to assess apoptosis by cytometer. Representative PTEN wildtype (HEC-1) and mutant (AN3CA) EC cell lines were chosen for mechanistic studies and treated 6 to 24 hours with multitarget or single agent inhibitor. RNA was isolated to determine gene expression by qRT-PCR, cells were stained with Vybrant DyeCycle Violet to determine cell cycle arrest by cytometer, or cellular lysates were prepared for Western blot (WB). Results: LCI139’s has a molecular weight of 502.57Da and is a highly potent PI3Kα-CDK4/6-CDK9 inhibitor by cell-free kinase assays: 0.070µM (PI3Kα), 0.461µM (PI3Kγ), 0.214µM (PI3Kδ), 0.0015µM (CDK4), 0.0036µM (CDK6), 0.000039µM (CDK9). KINOMEscan revealed a favorable S(35) selectivity score of 0.221 screened against 403 non-mutant kinases. PTEN mutant AN3CA (IC50 243.6nM), RL95-2 (IC50 22.8nM), Ishikawa (IC50 9.8nM) had nanomolar LCI139 IC50 values, while PTEN-wild type cell lines were insensitive up to 10μM; confirmed by dose-dependent apoptosis and colony-forming assays. WB revealed LCI139 treatment ablated MCL-1 expression, decreased phosphorylation of AKT, Rb, Rpb1 (Ser2), induced significant amounts of cPARP. MCL-1 ablation correlated to decreased Mcl1 expression (p<0.0010) assessed 6 hours post treatment. LCI139 generated heterogenous cell line specific effects on cell cycle progression and arrest. LCI139 displayed a favorable toxicity profile against normal human kidney cells (HEK-293) compared to single agent controls. Conclusions: Triple PI3K-CDK4/6-CDK9 inhibitor, LCI139, is nanomolar potent against PTEN mutant EC cell lines and displays decreased toxicity compared with CDK9 inhibitor reference compounds, AZD4573 and flavopiridol. Our results merit further mechanistic dissection of PTEN interaction with PI3K pathway signaling, cell cycle machinery, CDK9-mediated transcriptional control. These data support extensive in vivo potency, toxicity, PK/PD studies to assess LCI139’s clinical utility in treating EC. Citation Format: Vaidehi Mujumdar, Ritchie Delara, Hailey Dryden, Dhananjaya Pal, Krishnaiah Maddeboina, Page Mangum Arditti, Bharath Yada, Robert Wendel Naumann, Yovanni Casablanca, Erin Crane, Jubilee Brown, Donald Durden, Cody McHale. In silico design of novel multitarget small molecule inhibitor LCI139 for the treatment of PTEN-mutant endometrial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1816.

Structure-activity relationship study of 1,6-naphthyridinone derivatives as selective type II AXL inhibitors with potent antitumor efficacy

The role of AXL in various oncogenic processes has made it an attractive target for cancer therapy. Currently, kinase selectivity profiles, especially circumventing MET inhibition, remain a scientific issue of great interest in the discovery of selective type II AXL inhibitors. Starting from a dual MET/AXL-targeted lead structure from our previous work, we optimized a 1,6-naphthyridinone series using molecular modeling-assisted compound design to improve AXL potency and selectivity over MET, resulting in the potent and selective type II AXL-targeted compound 25c. This showed excellent AXL inhibitory activity (IC50 = 1.1 nM) and 343-fold selectivity over the highly homologous kinase MET in biochemical assays. Moreover, compound 25c significantly inhibited AXL-driven cell proliferation, dose-dependently suppressed 4T1 cell migration and invasion, and induced apoptosis. Compound 25c also showed noticeable antitumor efficacy in a BaF3/TEL-AXL xenograft model at well-tolerated doses. Overall, this study presented a potent and selective type II AXL-targeted lead compound for further drug discovery.

Design, synthesis, and in silico insights into dual-inhibition of CDK-6/Aurora A kinase by 2-phenylbenzimidazole-based small molecules

A novel series of 2-phenylbenzimidazole scaffold was designed as dual tyrosine kinase inhibitors based on targeted chemotherapy. All compounds were assessed for their antiproliferative activity against 60 NCI-cancer cell lines, where four compounds 14, 15, 17, and 18 showed superb growth inhibition values, and meanwhile proved safe towards WI-38 normal cells (IC50 range of 39.14–86.66 µM) compared to Doxorubicin (IC50= 6.72 µM). Hence, they were further in vitro evaluated for dual inhibition of Aurora A and CDK-6 enzymes after kinase selectivity profiling, using an extensive docking study to filter off-target enzymes. The four compounds displayed dual enzymatic inhibition, with compounds 14 and 18 being even more potent regarding CDK-6 with IC50 values of 0.197 µM and 0.172 µM, respectively, than Staurosporine (IC50=0.319 µM), and highly potent towards Aurora A, with IC50 values of 74 nM and 62 nM, respectively, compared to 47 nM for Danusertib. Compounds 14, 15, 17, and 18 triggered cell cycle arrest at the G1 phase and induced total apoptosis of HCT-15 cells by 30.96 % - 45.63 %. In Silico studies including molecular docking, physicochemical properties, compliance with Lipinski's rule, and radar plot technique were applied and were found to be in good agreement with the biological results.

Synthesis and biological evaluation of thieno[3,2-c]pyrazol-3-amine derivatives as potent glycogen synthase kinase 3β inhibitors for Alzheimer’s disease

Glycogen synthase kinase 3β (GSK-3β) is a potential target for anti-Alzheimer’s disease (AD) drug development. In this study, a series of novel thieno[3,2-c]pyrazol-3-amine derivatives was synthesized and evaluated as potential GSK-3β inhibitors by structure-based drug design. The thieno[3,2-c]pyrazol-3-amine derivative 54 with a 4-methylpyrazole moiety which interacted with Arg141 by π-cation interaction was identified as a potent GSK-3β inhibitor with an IC50 of 3.4 nM and an acceptable kinase selectivity profile. In the rat primary cortical neurons, compound 54 showed neuroprotective effects on Aβ-induced neurotoxicity. Western blot analysis indicated that 54 inhibited GSK-3β by up-regulating the expression of phosphorylated GSK-3β at Ser9 and down-regulating the expression of phosphorylated GSK-3β at Tyr216. Meanwhile, 54 decreased tau phosphorylation at Ser396 in a dose-dependent way. In astrocytes and microglia cells, 54 inhibited the expression of inducible nitric oxide synthase (iNOS), indicating that 54 showed an anti-neuroinflammatory effect. In the AlCl3-induced zebrafish AD model, 54 significantly ameliorated the AlCl3-induced dyskinesia, demonstrating its anti-AD activity in vivo.

Discovery of imidazo[1,2-b]pyridazine macrocyclic derivatives as novel ALK inhibitors capable of combating multiple resistant mutants

Anaplastic lymphoma kinase (ALK)-tyrosine kinase inhibitor (TKI) often loses effectiveness against non-small cell lung malignancies (NSCLCs) with ALK gene rearrangements (ALK+). 19 novel imidazo[1,2-b]pyridazine macrocyclic derivatives were designed, synthesized, and tested for their biological activities in an effort to develop ALK inhibitors that would overcome second-generation ALK-TKIs, particularly the G1202R mutation and the lorlatinib-resistant L1196M/G1202R double mutations. Of all the target substances, O-10 had the most effective enzymatic inhibitory activity, with IC50 values for ALKWT, ALKG1202R, and ALKL1196M/G1202R of 2.6, 6.4, and 23 nM, respectively. O-10, on the other hand, reduced the growth of ALK-positive Karpas299, BaF3-EML4-ALKG1202R, and BaF3-EML4-ALKL1196M/G1202R cells with IC50 values of 38, 52, and 64 nM, respectively. This was equally effective to the reference drug Repotrectinib (IC50 = 40, 164, and 208 nM). The kinase selectivity profile, liver microsome stability test and in vivo pharmacokinetic properties in SD rats of compound O-10 were further evaluated. O-10 was regarded as an effective ALK inhibitor for the treatment of mutations overall.

Abstract 1821: Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity

Abstract Hematopoietic progenitor kinase 1 (HPK1), also known as MAP4K1, is a member of the STE20 family of serine/threonine kinases, and restrictedly expressed in hematopoietic cells. In T cells, HPK1 negatively regulated TCR signaling by phosphorylating the adaptor protein SLP76 on Ser376, which leads to the dissociation of the SLP-76/LAT signalosomes and downregulation of T cell priming and activation. HPK1 knockout (KO) or kinase-inactive (KI) mice spontaneously reject tumors with an enhanced immune response compared to wild-type ones. Thus, HPK1 is a novel immuno-oncology (I/O) target. In this work, we described the discovery of a potent HPK1 inhibitor starting from comprehensive SAR-based design with guidance from X-ray crystallography and computational modeling. These efforts led to identification of the potent azaindole inhibitor XNW21015, with excellent kinase selectivity and promising PK profile of low clearance and sufficient exposure for in vivo animal studies. XNW21015 dose-dependently inhibited phosphorylation of SLP76(Ser376) in vitro and in vivo in response to anti-CD3 agonist antibody, with concomitant enhancement in T cell activation. Oral administration of XNW21015 in mouse syngeneic tumor models showed favorable PK profile and robust anti-tumor activity as both single agent and in combination with immune checkpoint inhibitors. In a humanized PDX (patient-derived xenograft) model, XNW21015 enhanced tumor-infiltrated lymphocytes (TIL). In summary, we have discovered a novel, highly potent and selective HPK1 inhibitor XNW21015 as promising small molecule I/O agent, as monotherapy or in combination with immune checkpoint inhibitors. Citation Format: Yuchuan Wu, Rui Hao, Xi Chen, Xiao Liu, Yonghua Xie, Shihua Wang, Bingquan Gao, Jichun Wen, Zhenwei Wu, Wengui Wang, Haiyang Wei, Yonghan Hu, Xiaojun Liu, Meijie Le, Jing Qiang. Discovery of XNW21015, a novel, potent HPK1 inhibitor with excellent immune modulatory activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1821.

Anticancer effect of zanubrutinib in HER2-positive breast cancer cell lines

Small molecule Bruton’s tyrosine kinase (BTK) inhibitors have been developed for the treatment of various haemato-oncological diseases, and ibrutinib was approved as the first BTK inhibitor for anticancer therapy in 2013. Previous reports proved the receptor kinase human epidermal growth factor receptor 2 (HER2) to be a valid off-target kinase of ibrutinib and potentially other irreversible BTK inhibitors, as it possesses a druggable cysteine residue in the active site of the enzyme. These findings suggest ibrutinib as a candidate drug for repositioning in HER2-positive breast cancer (BCa). This subtype of breast cancer belongs to one of the most common classes of breast tumours, and its prognosis is characterized by a high rate of recurrence and tumour invasiveness. Based on their similar kinase selectivity profiles, we investigated the anticancer effect of zanubrutinib, evobrutinib, tirabrutinib and acalabrutinib in different BCa cell lines and sought to determine whether it is linked with targeting the epidermal growth factor receptor family (ERBB) pathway. We found that zanubrutinib is a potential inhibitor of the HER2 signalling pathway, displaying an antiproliferative effect in HER2-positive BCa cell lines. Zanubrutinib effectively inhibits the phosphorylation of proteins in the ERBB signalling cascade, including the downstream kinases Akt and ERK, which mediate key signals ensuring the survival and proliferation of cancer cells. We thus propose zanubrutinib as another suitable candidate for repurposing in HER2-amplified solid tumours.

Abstract 2606: ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance

Abstract Inhibition of the DNA damage response (DDR) pathway is an attractive approach to target cancers cells. Unlike normal cells which relay on two cell cycle checkpoints to repair DNA damage, most tumor cells only depend on the G2 checkpoint because their G1 checkpoint is defective. Wee1 regulates the G2/M checkpoint and abrogating the G2 checkpoint by inhibiting Wee-1 in cancer cells with DNA damage can preferentially sensitize cancer cells to undergo mitotic catastrophe. We have developed and characterized ZN-c3, a potent and selective Wee-1 inhibitor with a differentiated kinase selectivity and pharmacokinetics profile. ZN-c3 was evaluated in ER-positive, triple negative, triple negative PARP inhibitor resistant, and Her-2 positive trastuzumab resistant breast cancers. In the ER-positive MCF-7 efficacy model, ZN-c3 demonstrated anti-tumor activity as a single agent as well as in combinations with a CDK4/6 inhibitor (Palbociclib) or ZN-c5, a selective estrogen receptor degrader (SERD). In the MDA-MB-436, a triple negative breast cancer tumor xenograft model, ZN-c3 demonstrated efficacy as a single agent and in combination with the PARP inhibitors Olaparib or Niraparib. We extended these combination studies by evaluating the efficacy of Niraparib in combination with ZN-c3 in patient-derived xenograft (PDX) triple negative breast tumors. ZN-c3 was efficacious as a single agent and the efficacy was enhanced in the combination with Niraparib. PARP inhibitors are effective and have been approved for treatment of some cancer types. However, most patients develop resistance to these agents. We sought to investigate whether ZN-c3 can overcome the acquired resistance to PARP inhibitors (Olaparib and Niraparib). We generated PARP inhibitor resistant breast tumor xenograft models. A significant tumor growth inhibition was observed in these resistant tumor xenograft models when treated with ZN-c3. These data suggest that ZN-c3 as a single agent can overcome the acquired PARP inhibitor resistance. Finally, we also evaluated ZN-c3 activity in a HER-2 positive trastuzumab resistant breast tumor xenograft model (JIMT-1) and ZN-c3 showed anti-tumor activity alone and in combination with trastuzumab. In summary, these results demonstrate that ZN-c3 is effective as a single agent and in combination with other targeted therapies in breast cancer models. Moreover, ZN-c3 can overcome the acquired resistance to PARP inhibitors and trastuzumab. ZN-c3 is currently in clinical trials for several indications and has demonstrated promising clinical activity and good tolerability. Citation Format: Jiali Li, Jianhui Ma, Petrus R. de Jong, Hooman Izadi, Tiffany Hoang, Noah Ibrahim, Brant Boren, Sayee Hegde Hegde, Fernando Doñate, Ahmed A. Samatar. ZN-c3, a potent and selective Wee-1 inhibitor demonstrates anti-tumor activities in combination with other targeted therapies and overcomes PARP inhibitor resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2606.

Abstract 1180: Discovery of CC-91516, a potent and selective ERK/NLK inhibitor, with anti-tumor activity in preclinical cancer models harboring BRAF or CTNNB1 mutation

Abstract CC-91516 (also called CC0776314), a selective and potent inhibitor of ERK1/2 and NLK, was discovered via a phenotypic screen of a kinase-focused library for compounds that synergize with mTOR kinase inhibitor CC-223 to induce apoptosis in combination with CC-223. Broad kinase selectivity profiling identified ERK1/2 and NLK as targets of CC-91516. Crystal structure of CC-91516 in complex with ERK2 reveals that its 2, 4, 6-trichlorophenyl moiety binds to a unique back pocket of the adenosine-5′-triphosphate (ATP) binding site of ERK2, which is not accessible to other ERK inhibitors such as BVD-523 and GDC-0994. This unique binding mode of CC-91516 leads to a slow off-rate for its ERK binding with long residence time disrupting both the active and inactive ERK forms. Consequentially, CC-91516 causes sustained inhibition of the MAPK pathway in BRAF mutant colorectal cancer cells. In addition, CC-91516 also regulates Wnt/β-catenin and YAP pathways in multiple cancer cell lines. CC-91516 shows potent yet selective anti-proliferative activity against a large panel of cancer cell lines. Activating mutations in BRAF or CTNNB1 gene associate with sensitivity to CC-91516-mediated anti-proliferative activity while mutations in RB and PI3K/PTEN pathway associate with resistance. CC-91516 inhibits ex vivo colony formation of PDX models with BRAF and CTNNB1 mutations. In addition, CC-91516 potently induces apoptosis, inhibits survival, and overcomes resistance to MEK inhibitor trametinib of BRAF or CTNNB1 mutant cancer cells in long-term culture assay in vitro. CC-91516 has good oral bioavailability and shows excellent anti-tumor activity in vivo against both BRAF and CTNNB1 mutant xenograft models. DMPK and toxicology studies showed robust oral exposure across preclinical species. In summary, CC-91516 has demonstrated preclinical anti-tumor activities and DMPK and safety profiles in support of its clinical development. Citation Format: Shuichan Xu, Tam Tran, Dan Zhu, Tao Shi, David Mikolon, Jim Leisten, Philip Chamberlain, Laurie LeBrun, Sogole Bahmanyar, Ning Jiang, Jingjing Zhao, Mehnaz Malek, Ellen Filvaroff, Heather Raymon, Robert Hubbard, John Boylan. Discovery of CC-91516, a potent and selective ERK/NLK inhibitor, with anti-tumor activity in preclinical cancer models harboring BRAF or CTNNB1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1180.

Discovery of pyrrolo[1,2-a]quinoxalin-4(5H)-one derivatives as novel non-covalent Bruton’s tyrosine kinase (BTK) inhibitors

Bruton’s tyrosine kinase (BTK) is a promising target in the treatment of B cell malignancies and autoimmune disorders. Developing selective non-covalent BTK inhibitors is an important strategy to overcome the side effects and drug resistance induced by covalent BTK inhibitors. In this article, we designed and synthesized pyrrolo[1,2-a]quinoxalin-4(5H)-one and imidazo[1,2-a]quinoxalin-4(5H)-one based selective noncovalent BTK inhibitors via scaffold hopping from BMS-986142 and investigated their biological activities. Among the synthesized compounds, pyrrolo[1,2-a]quinoxalin-4(5H)-one derivatives 2 and 4 showed great BTK inhibition potency with IC50 value at 7.41 nM and 11.4 nM, respectively. Besides, they showed equivalent or even better potency in U937 and Ramos cells than BMS-986142. The kinase selectivity profiling study illustrated the excellent selectivity of compound 2 against a panel of 468 kinases. In U937 xenograft models, compound 2 could significantly inhibit tumor growth with TGI = 65.61%. In all, we provided a new scaffold as non-covalent selective BTK inhibitors and the representative compounds exhibited potency both in vitro and in vivo.

Pyrazolo[3,4‐d]pyrimidine derivatives as irreversible Bruton's tyrosine kinase inhibitors

Abstract4,6‐Disubstituted pyrazolo[3,4‐d]pyrimidine derivatives were explored as irreversible Bruton's tyrosine kinase (BTK) inhibitors. The structure–activity relationship was established with over 20 derivatives synthesized to determine initial hit compounds, based on activities against BTK enzyme and TMD8 cells. It turned out that introducing 1‐acrylamido‐4‐aminopiperdine (1b) at the C4 position of pyrazolopyrimidine as in 5e, and 3‐acrylamido‐aniline (1j) as 4‐position substituent, such as in 9d, 10d, and 10e, delivered potent in vitro enzyme activities as well as TMD8 cell‐based cytotoxicities. Considering kinase selectivity profiles, 5e was selected for in vivo efficacy studies with a murine xenograft model using TMD8 cells, where 5e exhibited moderate tumor growth inhibition activities. Further optimization of 5e and 9d may lead to clinically useful compounds to overcome B‐cell‐mediated hematologic cancers.

Transforming Type II to Type I c-Met kinase inhibitors via combined scaffold hopping and structure-guided synthesis of new series of 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-one derivatives

Novel 1,3,4-thiadiazolo[2,3-c]-1,2,4-triazin-4-one derivatives 3a-e, 4a-f and 5a-f were designed as Type I c-Met kinase inhibitors based on scaffold hopping of our previous Type II c-Met kinase lead. Target compounds were then synthesized under the guidance of molecular docking analysis to identify the potential inhibitors that fit the binding pocket of c-Met kinase in the characteristic manner as the reported Type I c-Met kinase inhibitors. All synthesized derivatives were evaluated for their c-Met kinase inhibitory activity at 10 µM concentration, where 3d, 5d and 5f displayed >80% inhibition. Further IC50 investigation of these compounds identified 5d as the most potent c-Met kinase inhibitor with IC50 value of 1.95 µM. Moreover, 5d showed selective antitumor activity against c-Met over-expressing colon HCT-116 and lung A549 adenocarcinoma cells with IC50 values of 6.18 and 10.6 µg/ml, respectively. More significantly, 5d effectively inhibited c-Met phosphorylation in the Western blot experiment. Also, 5d induced cellular apoptosis in HCT-116 cancer cells as well as cell cycle arrest with accumulation of cells in G2/M phase. Finally, kinase selectivity profiling of 5d against nine oncogenic kinases revealed its selectivity to only Tyro3 kinase (% inhibition = 80%, IC50 = 3 µM). All these experimental findings clearly demonstrate that 5d is a potential dual acting inhibitor against c-Met and Tyro3 kinases, standing out as a viable lead that deserves further investigation and development to new generation of antitumor agents.

Abstract 1965: Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile

Abstract Genome instability initiated by DNA damage or DNA replication stress is a driver of tumor development. To ensure the accuracy of DNA replication, DNA damage response (DDR) mediated by various cell cycle checkpoints activate the DNA repair system. One such checkpoint system is the G2-M checkpoint which is involved in the repair and prevention of excessive DNA damage. A key regulator of the G2-M checkpoint is the Wee1 kinase which causes cell cycle arrest to prevent damaged cells from entering mitosis. Thus, inhibition of Wee1 should prevent G2-M checkpoint activation which would lead to unscheduled mitotic entry and induction of apoptosis. Small-molecule inhibitors of Wee1 are currently being tested in several tumor types. Preliminary results show promising clinical activity of AZD-1775, a potent inhibitor of Wee1, however, achieving an optimal dose and a schedule that is well-tolerated has been a challenge. Here, we describe the characterization of ZN-c3, a potent, selective and orally bioavailable small-molecule inhibitor of Wee1. ZN-c3 exhibits potent anti-proliferative activity across multiple tumor cell lines. Compared to AZD-1775, ZN-c3 has superior selectivity profile against a panel of kinases. In vivo, ZN-c3 induced tumor regression as a single agent in several tumor models including non-small cell lung cancer, ovarian cancer, and uterine sarcoma. In combination studies, ZN-c3 potentiates the antitumor efficacies of carboplatin and gemcitabine. The pharmacokinetic and efficacy profiles of ZN-c3 and AZD-1775 were compared in tumor xenograft models. ZN-c3 showed higher plasma and tumor exposure than AZD-1775, which correlated with more potent tumor growth inhibition than AZD-1775. In summary, ZN-c3, with its key differentiated pharmacologic and kinase selectivity profile, is a promising new generation of DDR inhibitor. ZN-c3 is currently in clinical development for the treatment of advanced solid cancers. Citation Format: Jiali Li, Brant Boren, Peter Q. Huang, Kevin D. Bunker, Fernando Doñate, Ahmed A. Samatar. Discovery of ZN-c3, a potent Wee-1 inhibitor with a differentiated pharmacologic and kinase selectivity profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1965.

Abstract 1709: Novel small molecule HPK1 inhibitor induces immunogenic anti-tumor effects

Abstract Background Hematopoietic progenitor kinase 1 (HPK1, MAP4K1), a member of the MAP4K family of protein serine/threonine kinases1,2 is a negative regulator of TCR and BCR3. Immunosuppressive mediators PGE2 and adenosine regulate activation of HPK13. In vivo anti-tumor activity by HPK1 gene deletion, kinase dead HPK1 and small molecule HPK1 inhibitors have been demonstrated in multiple immunogenic syngeneic tumor models3,4. Enhanced anti-tumor efficacy in these models was achieved by combining HPK1 inhibitors with immune check-point blockers (ICBs) like anti-PD-1, anti-PD-L1or anti-CTLA4 antibodies3,4. HPK1 inhibition is a target for immuno-oncology treatment in cancers responsive or non-responsive to current ICBs. Methods Creative medicinal chemistry design complemented by structured-based support was used to identify & develop inhibitors of HPK1 with pico/nano-molar potency, optimal kinase selectivity, PK and efficacy profile. Our SAR efforts were guided by biochemical assays, functional read-outs and primary human in vitro T-cell activation assays. In vivo target engagement and pharmacodynamic data was generated using mouse models of the lung (LLC) and colon (MC38 and CT26) cancer. Results We describe novel and potent HPK1 inhibitors that can inhibit pSLP76 and enhance IL-2 production in Jurkat cells, IL-2 and IFN-γ production in human PBMCs, whole blood and primary T cells and TNF-α production by dendritic cells following stimulation. A reversal of PGE2 or adenosine mediated immunosuppression was also achieved by the inhibitors. These compounds on oral dosing showed strong tumor growth inhibition (TGI) in syngeneic models refractory to ICBs (subcutaneous LLC) as well as responsive to ICBs (subcutaneous MC38 and CT26; orthotopic LLC). TGI was accompanied by the immune response of increased cytokine (IL-2/IFNγ) levels and tumor infiltrating lymphocytes (TILs). Combination with ICBs induced enhanced TGI correlating with enhanced cytokine induction and TILs. We confirmed the immune-mediated mechanism of inhibitors by their lack of efficacy in immune-compromised mice. The lead compound demonstrated cardiac safety (hERG assay), lack of genotoxicity and very good safety margins in the mouse exploratory toxicology studies up to 28-days. Conclusion We have discovered a novel, orally active HPK1 inhibitor that demonstrates excellent stand-alone efficacy in multiple tumor models and also offers the potential to enhance current immunotherapy regimens in both responsive and refractory cancers. Further evaluation of our lead molecule towards the clinic is underway. Acknowledgements Sanjib D, Megha M, Jiju M, Sheetal K, Arti J, Swayam M, Srinivas K, Pradeep V, Vikram B, Abhay K, Jagmohan S, Ravi T, Mohammad Y, Rahul B, Ajit J, Sanjay G, Pramod S.

Biochemical, cellular and structural characterization of novel and selective ERK3 inhibitors

Triazolo[4,5-d]pyrimidin-5-amines were identified from kinase selectivity screening as novel ERK3 inhibitors with sub-100 nanomolar potencies in a biochemical assay using MK5 as substrate and with an attractive kinase selectivity profile. ERK3 crystal structures clarified the inhibitor binding mode in the ATP pocket with impact on A-loop, GC-loop and αC-helix conformations suggesting a potential structural link towards MK5 interaction via the FHIEDE motif. The inhibitors also showed sub-100 nM potencies in a cellular ERK3 NanoBRET assay and with excellent correlation to the biochemical IC50s. This novel series provides valuable tool compounds to further investigate the biological function and activation mechanism of ERK3.

α-Tomatin`nin Antiproliferatif Aktivitesi ve Moleküler Hedef Tanımlaması

α-tomatine is a glycoalkaloid derived from tomato varieties that has been reported to possess various anticancer properties. However, its inhibitory effects on epidermal growth factor receptor is still poorly understood. The aim of this study is to investigate the anticancer effect of α-tomatine and its related mechanisms in lung cancer cells. Cytotoxicity and apoptosis induction of α-tomatine were determined by MTT assay and annexin V-FITC staining methods, respectively. For tyrosine kinase activity, TK-1 kinase selectivity profiling assay and molecular modelling study were performed. The DNA cleavage activity of α-tomatine was investigated using agarose gel electrophoretic method. α-tomatine proved to possess an outstanding antiproliferative activity against A549 and Jurkat cells without noticeable toxicity on PBMC. The results indicated that α-tomatine has a significant inhibition effect on both EGFR and HER2. α-tomatine formed some key interaction into ATP binding sites of EGFR and HER2. Furthermore, α-tomatine strongly disintegrated DNA at low concentrations in the presence of iron(II) complexes. The current findings suggest that α-tomatine has a distinguished receptor tyrosine kinase inhibition profile from erlotinib and might be a potential drug candidate for treatment of NSCLC.

Nicotinamide-Ponatinib Analogues as Potent Anti-CML and Anti-AML Compounds.

Ponatinib is a multikinase inhibitor that is used to treat chronic myeloid leukemia patients harboring mutated ABL1(T315I) kinase. Due to the potent inhibition of FLT3, RET, and fibroblast growth factor receptors (FGFRs), it is also being evaluated against acute myeloid leukemia (AML), biliary, and lung cancers. The multikinase inhibition profile of ponatinib may also account for its toxicity, thus analogs with improved kinase selectivity or different kinase inhibition profiles could be better tolerated. The introduction of nitrogen into drug compounds can enhance efficacy and drug properties (a concept called “necessary nitrogen”). Here, we introduce additional nitrogen into the benzamide moiety of ponatinib to arrive at nicotinamide analogs. A nicotinamide analogue of ponatinib, HSN748, retains activity against FLT3, ABL1, RET, and PDGFRα/β but loses activity against c-Src and P38α. MNK1 and 2 are key kinases that phosphorylate eIF4E to regulate the protein translation complex. MNK also modulates mTORC1 signaling and contributes to rapamycin resistance. Inhibitors of MNK1 and 2 are being evaluated for anticancer therapy. Ponatinib is not a potent inhibitor of MNK1 or 2, but the nicotinamide analogs are potent inhibitors of MNKs. This illustrates a powerful demonstration of the necessary nitrogen concept to alter both the potency and selectivity of drugs.

Design and synthesis of Imidazo[1,2-b]pyridazine IRAK4 inhibitors for the treatment of mutant MYD88 L265P diffuse large B-cell lymphoma

Harboring MYD88 L265P mutation triggers tumors growth through the activation of NF-κB by interleukin-1 receptor associated kinase 4 (IRAK4) in diffuse large B-cell lymphoma (DLBCL), highlighting IRAK4 as a therapeutic target for tumors driven by aberrant MYD88 signaling. Herein, we report the design, synthesis, and structure−activity relationships of imidazo[1,2-b]pyridazines as potent IRAK4 inhibitors. The representative compound 5 exhibited excellent IRAK4 potency (IRAK4 IC50 = 1.3 nM) and favorable kinase selectivity profile. It demonstrated cellular selectivity for activated B cell–like (ABC) subtype DLBCL with MYD88 L265P mutation in cytotoxicity assay. The kinase inhibitory efficiency of compound 5 was further validated by Western blot analysis of phosphorylation of IRAK4 and downstream signaling in OCI-LY10 and TMD8 cells. Besides, combination of compound 5 and BTK inhibitor ibrutinib synergistically reduced the viability of TMD8 cells. These results indicated that compound 5 could be a promising IRAK4 inhibitor for the treatment of mutant MYD88 DLBCL.

Hit-to-Lead Optimization of Benzoxazepinoindazoles As Human African Trypanosomiasis Therapeutics.

Human African trypanosomiasis (HAT) is a neglected tropical disease caused by infection with either of two subspecies of the parasite Trypanosoma brucei. Due to a lack of economic incentive to develop new drugs, current treatments have severe limitations in terms of safety, efficacy, and ease of administration. In an effort to develop new HAT therapeutics, we report the structure-activity relationships around T. brucei for a series of benzoxazepinoindazoles previously identified through a high-throughput screen of human kinase inhibitors, and the subsequent in vivo experiments for HAT. We identified compound 18, which showed an improved kinase selectivity profile and acceptable pharmacokinetic parameters, as a promising lead. Although treatment with 18 cured 60% of mice in a systemic model of HAT, the compound was unable to clear parasitemia in a CNS model of the disease. We also report the results of cross-screening these compounds against T. cruzi, L. donovani, and S. mansoni.

Development of Macrocycle Kinase Inhibitors for ALK2 Using Fibrodysplasia Ossificans Progressiva-Derived Endothelial Cells.

ABSTRACTFibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to activins, thereby turning them into bone‐inducing agents. To date, there is no cure for FOP. The further development of FOP patient‐derived models may contribute to the discovery of novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO. We have characterized peripheral blood‐derived endothelial colony‐forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo endothelial‐to‐mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.