Jeffrey Keats

AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance.

Inhibition of BCR-ABL by imatinib induces durable responses in many patients with chronic myeloid leukemia (CML), but resistance attributable to kinase domain mutations can lead to relapse and a switch to second-line therapy with nilotinib or dasatinib. Despite three approved therapeutic options, the cross-resistant BCR-ABL(T315I) mutation and compound mutants selected on sequential inhibitor therapy remain major clinical challenges. We report design and preclinical evaluation of AP24534, a potent, orally available multitargeted kinase inhibitor active against T315I and other BCR-ABL mutants. AP24534 inhibited all tested BCR-ABL mutants in cellular and biochemical assays, suppressed BCR-ABL(T315I)-driven tumor growth in mice, and completely abrogated resistance in cell-based mutagenesis screens. Our work supports clinical evaluation of AP24534 as a pan-BCR-ABL inhibitor for treatment of CML.

Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non‐small‐cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non‐small‐cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib‐resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK‐positive non‐small‐cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib’s narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance.

AP23846, a novel and highly potent Src family kinase inhibitor, reduces vascular endothelial growth factor and interleukin-8 expression in human solid tumor cell lines and abrogates downstream angiogenic processes

c-Src is frequently activated in human malignancies, including colon, breast, and pancreatic carcinomas. Several recent studies have shown that activation of Src family kinases leads to tumor progression and metastasis by increasing cellular migration and invasion, promoting cell growth and survival, and deregulating expression of proangiogenic molecules. Therefore, selective inhibitors of Src are being developed for cancer therapy. In this study, we characterize the biological effects of the novel ATP-based Src family kinase inhibitor, AP23846, in tumor cells with high Src activity. As a lead compound, AP23846 is a potent c-Src kinase inhibitor (IC50 ∼0.5 nmol/L in vitro, ∼10-fold more potent than PP2, the most widely used commercially available Src family kinase inhibitor). At concentrations of 1 μmol/L, AP23846 led to complete Src inhibition for 48 hours in cells. No cytotoxicity was observed under these conditions, although proliferation rates were slower. Therefore, this was an excellent inhibitor to examine Src-regulated signaling pathways in tumor cells. AP23846 reduced cellular migration, vascular endothelial growth factor, and interleukin-8 in a dose-dependent fashion in pancreatic adenocarcinoma cells grown in vitro. Correspondingly, cell culture supernatants from L3.6pl pancreatic adenocarcinoma cells pretreated with AP23846 failed to promote migration of hepatic endothelial cells in vitro and failed to support angiogenesis into gel foams implanted s.c. in mice in vivo. These results suggest that Src inhibitors affect biological properties of tumor progression and may be useful as cancer therapeutic agents in more advanced disease. [Mol Cancer Ther 2005;4(12):1900–11]

Discovery of Brigatinib (AP26113), a Phosphine Oxide-Containing, Potent, Orally Active Inhibitor of Anaplastic Lymphoma Kinase

In the treatment of echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase positive (ALK+) non-small-cell lung cancer (NSCLC), secondary mutations within the ALK kinase domain have emerged as a major resistance mechanism to both first- and second-generation ALK inhibitors. This report describes the design and synthesis of a series of 2,4-diarylaminopyrimidine-based potent and selective ALK inhibitors culminating in identification of the investigational clinical candidate brigatinib. A unique structural feature of brigatinib is a phosphine oxide, an overlooked but novel hydrogen-bond acceptor that drives potency and selectivity in addition to favorable ADME properties. Brigatinib displayed low nanomolar IC50s against native ALK and all tested clinically relevant ALK mutants in both enzyme-based biochemical and cell-based viability assays and demonstrated efficacy in multiple ALK+ xenografts in mice, including Karpas-299 (anaplastic large-cell lymphomas [ALCL]) and H3122 (NSCLC). Brigatinib represents the most clinically advanced phosphine oxide-containing drug candidate to date and is currently being evaluated in a global phase 2 registration trial.

Abstract LB-298: AP26113, a potent ALK inhibitor, overcomes mutations in EML4-ALK that confer resistance to PF-02341066 (PF1066)

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC AP26113 is a potent and selective inhibitor of anaplastic lymphoma kinase (ALK) (AACR 2010; #3623). Activating gene rearrangements of ALK, such as EML4-ALK, have been identified as driver mutations in NSCLC and other cancers. There is strong precedence for the development of resistance to targeted therapies that inhibit driver mutations. Kinase domain mutations that confer resistance in patients have been successfully predicted by in vitro mutagenesis screens in BaF3 cells (e.g. BCR-ABL in CML). Here, the BaF3 system was used to identify mutations in ALK that confer resistance to PF1066, a clinically validated dual Met/ALK inhibitor (ASCO 2009; #3509), or AP26113. PF1066-resistant mutations were identified at all concentrations tested (up to 2000 nM). In contrast, 1000 nM AP26113 completely suppressed emergence of resistance. Six mutations, all in the kinase domain, were identified that confer some degree of resistance to 1 or both compounds (Table). AP26113 inhibited viability of BaF3 cells expressing these mutants with IC50s of 23 - 269 nM. PF1066 inhibited viability with IC50s of 311 -1419 nM, with 3 mutants having sensitivity indistinguishable from parental BaF3 cells, which lack EML4-ALK. The 2 mutations that confer the greatest resistance to PF1066 were examined in a BaF3 xenograft model in which compounds were administered daily by oral dosing. A 200 mg/kg dose of PF1066 induced regression of tumors expressing native EML4-ALK but was completely inactive against G1269S or L1196M (gatekeeper) mutants. In contrast, AP26113 induced regression of tumors expressing native EML4-ALK and the G1269S and L1196M mutants at 25, 50 and 50 mg/kg, respectively. Analysis of ALK phosphorylation in tumors demonstrated strong inhibition of the mutants by 50 mg/kg AP26113 but not 200 mg/kg PF1066. These results identify several mutations that may confer resistance to PF1066 in patients and suggest that more potent compounds such as AP26113 may be required to overcome such resistance. View this table: Sensitivity of BaF3 cells expressing native and mutant EML4-ALK to PF-02341066 and AP26113 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-298.

Bone-Targeted 2,6,9-Trisubstituted Purines: Novel Inhibitors of Src Tyrosine Kinase for the Treatment of Bone Diseases

Novel bone-targeted 2,6,9-trisubstituted purine template-based inhibitors of Src tyrosine kinase are described. Drug design studies of known purine compounds revealed that both positions-2 and -6 were suitable for incorporating bone-seeking moieties. A variety of bone-targeting groups with different affinity to hydroxyapatite were utilized in the study. Compound 3d was determined to be a potent Src inhibitor and was quite selective against a panel of other protein kinases.

Abstract 1794: AP26113 is a dual ALK/EGFR inhibitor: Characterization against EGFR T790M in cell and mouse models of NSCLC

Background: The EGFR T790M gatekeeper mutation accounts for ∼50% of resistance observed in patients treated with first generation inhibitors that target activated variants of EGFR. Multiple irreversible T790M inhibitors are in development but can exhibit skin and GI toxicity due to co-inhibition of native (endogenous) EGFR, suggesting that T790M-selective agents will be required. Previously, we have identified AP26113 as a potent ALK inhibitor that maintains activity against crizotinib-resistant variants, including the L1196M gatekeeper mutant. Here we further characterize AP261139s activity as a reversible inhibitor of activated and T790M-mutant EGFR. Methods: The activity of AP26113 against native EGFR, or activated forms (delE746_A750 [DEL]) with or without a T790M resistance mutation, was examined in NSCLC as well as engineered Ba/F3 cell lines. EGFR activity was assessed by measuring levels of phosphorylated EGFR, in vitro proliferation measured by MTS assay, and in vivo tumor growth measured in mouse xenografts following daily oral dosing. Results: AP26113 did not inhibit native EGFR phosphorylation in a NSCLC cell line (H358) or in engineered Ba/F3 cells (IC50s >3000 nM). In contrast, potent activity was observed against activated forms of EGFR, with or without the T790M mutation. In Ba/F3 cells expressing EGFR-DEL, AP26113 inhibited EGFR phosphorylation and viability with IC50s of 75 and 114 nM, respectively. In Ba/F3 cells expressing EGFR-DEL/T790M, AP26113 inhibited EGFR phosphorylation and viability with IC50s of 15 and 281 nM, respectively. In a NSCLC line expressing EGFR-DEL (HCC827), AP26113 inhibited EGFR phosphorylation with an IC50 of 62 nM and cell growth with a GI50 of 165 nM. In HCC827 cells expressing EGFR-DEL/T790M AP26113 inhibited EGFR phosphorylation with an IC50 of 59 nM and cell growth with a GI50 of 245 nM. AP26113 also exhibited similar potency against HCC827 cells expressing either EGFR-DEL or EGFR-DEL/T790M in a xenograft model, with daily oral doses of 25 mg/kg or greater leading to tumor regression in both models. Anti-tumor activity that was associated with inhibition of EGFR phosphorylation was also seen in a Ba/F3 EGFR-DEL/T790M tumor model. Conclusions: AP26113 is a potent, reversible inhibitor of activated and T790M-mutant EGFR that does not inhibit the native enzyme. Importantly, orally efficacious doses in mice against activated and T790M-mutant EGFR are similar to those active against crizotinib-resistant ALK variants, suggesting that AP26113 is a dual ALK/mutant EGFR inhibitor with potential to target these two well-defined and important subsets of NSCLC. Based on these data, we recently initiated a Phase 1/2 clinical trial of AP26113 (NCT01449461, www.clinicaltrials.gov) in ALK and EGFR+ NSCLC patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1794. doi:1538-7445.AM2012-1794

Abstract 3623: Efficacy and pharmacodynamic analysis of AP26113, a potent and selective orally active inhibitor of Anaplastic Lymphoma Kinase (ALK)

Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified in anaplastic large cell lymphoma (ALCL; NPM-ALK) and non-small cell lung cancer (NSCLC; EML4-ALK). The dual Met/ALK inhibitor PF-02341066 (PF1066) has demonstrated promising clinical activity against tumors carrying activating ALK gene rearrangements (Kwak ASCO 2009: #3509) validating ALK as a therapeutic target. Previously, AP26113 was identified as a novel, potent, orally bioavailable ALK inhibitor with demonstrated selectivity over related receptor tyrosine kinase family members IGF-1R and InsR and no inhibition of Met. Here the efficacy and exposure/activity relationship of AP26113 was further characterized in preclinical models and compared to that of PF1066. In a panel of 7 EML4-ALK or NPM-ALK positive NSCLC and ALCL cell lines, the concentration of AP26113 that inhibited growth by 50% (GI50) ranged from 4.2 - 30.8 nM. In each cell line the GI50 for PF1066 was ∼10-fold greater (range 62 - 309 nM). In 4 cells lines tested, the IC50 for inhibition of ALK phosphorylation tracked with potency in cell proliferation assays and was 10-fold greater for PF1066 than AP26113. Across 3 ALK-negative NSCLC and ALCL cell lines the GI50s for AP26113 (503 - 2387 nM) and PF1066 (928 - 1773 nM) were similar. Overall, AP26113 exhibited ∼100-fold selectivity for ALK-positive lines compared with a ∼10-fold selectivity for PF1066. The in vivo activities of daily oral dosing of AP26113 (10, 25 and 50 mg/kg) and PF1066 (25, 50 and 100 mg/kg) were examined in Karpas-299 ALCL (2 week dosing) and H3122 NSCLC (3 week dosing) xenograft models. At the highest doses tested, strong regressions were achieved with AP26113, but not PF1066. Tumor growth inhibition by 25 mg/kg and 10 mg/kg doses of AP26113 in the ALCL and NSCLC models, respectively, was similar to that of 100 mg/kg PF1066. In a PK/PD study in the ALCL model, inhibition of ALK phosphorylation after administration of 100 mg/kg PF1066 was intermediate between that observed after administration of 10 or 25 mg/kg AP26113. Results from the analysis of plasma levels of each drug showed that AP26113 had equivalent efficacy to PF1066 at 4- to 10-fold lower levels of exposure (AUC and 24 h trough plasma levels). AP26113 demonstrated favorable properties including moderate in vitro plasma protein binding (≤77% in mouse, rat, monkey, and human plasma), negligible inhibition of major CYP isoforms (IC50 > 10 μM for 3A4, 2C9, 2D6), and good oral bioavailability (multiple animal species). In animal models, AP26113 was well-tolerated at and above predicted clinically effective plasma levels. In conclusion, these data demonstrate that AP26113 is a highly potent and selective inhibitor of ALK and support the clinical evaluation of AP26113 in patients with ALK-driven tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3623.

Bone-Targeted Src kinase inhibitors: novel pyrrolo- and pyrazolopyrimidine analogues

Src tyrosine kinase is a therapeutic target for bone diseases that has been validated by gene knockout studies. Furthermore, in vitro cellular studies implicate that Src has a positive regulatory role in osteoclasts and a negative regulatory role in osteoblasts. The potential use of Src inhibitors for osteoporosis therapy has been previously shown by novel bone-targeted ligands of the Src SH2 (e.g., AP22408) and non-bone-targeted, ATP-based inhibitors of Src kinase. Significant to this study, compounds 2-12 exemplify novel analogues of known pyrrolopyrimidine and pyrazolopyrimidine template-based Src kinase inhibitors that incorporate bone-targeting group modifications designed to provide tissue (bone) selectivity and diminished side effects. Accordingly, we report here the structure-based design, synthetic chemistry and biological testing of these compounds and proof-of-concept studies thereof.

Novel N9-arenethenyl purines as potent dual Src/Abl tyrosine kinase inhibitors.

Novel N(9)-arenethenyl purines, optimized potent dual Src/Abl tyrosine kinase inhibitors, are described. The key structural feature is a trans vinyl linkage at N(9) on the purine core which projects hydrophobic substituents into the selectivity pocket at the rear of the ATP site. Their synthesis was achieved through a Horner-Wadsworth-Emmons reaction of N(9)-phosphorylmethylpurines and substituted benzaldehydes or Heck reactions between 9-vinyl purines and aryl halides. Most compounds are potent inhibitors of both Src and Abl kinase, and several possess good oral bioavailability.