Discovery of vimseltinib (DCC-3014), a highly selective CSF1R switch-control kinase inhibitor, in clinical development for the treatment of Tenosynovial Giant Cell Tumor (TGCT).

Based on knowledge of kinase switch-control inhibition and using a combination of structure-based drug design and standard medicinal chemistry principles, we identified a novel series of dihydropyrimidone-based CSF1R kinase inhibitors displaying exquisite selectivity for CSF1R versus a large panel of kinases and non-kinase protein targets. Starting with lead compound 3, an SAR optimization campaign led to the discovery of vimseltinib (DCC-3014; compound 20) currently undergoing clinical evaluation for the treatment of Tenosynovial Giant Cell Tumor (TGCT), a locally aggressive benign tumor associated with substantial morbidity. 2021 Elsevier ltd. All rights reserved.

Discovery of acyl ureas as highly selective small molecule CSF1R kinase inhibitors.

Based on the structure of an early lead identified in Deciphera's proprietary compound collection of switch control kinase inhibitors and using a combination of medicinal chemistry guided structure activity relationships and structure-based drug design, a novel series of potent acyl urea-based CSF1R inhibitors was identified displaying high selectivity for CSF1R versus the other members of the Type III receptor tyrosine kinase (RTK) family members (KIT, PDGFR-α, PDGFR-ß, and FLT3), VEGFR2 and MET. Based on in vitro biology, in vitro ADME and in vivo PK/PD studies, compound 10 was selected as an advanced lead for Deciphera's CSF1R research program.

Vimseltinib: A Precision CSF1R Therapy for Tenosynovial Giant Cell Tumors and Diseases Promoted by Macrophages.

Macrophages can be co-opted to contribute to neoplastic, neurologic, and inflammatory diseases. Colony-stimulating factor 1 receptor (CSF1R)-dependent macrophages and other inflammatory cells can suppress the adaptive immune system in cancer and contribute to angiogenesis, tumor growth, and metastasis. CSF1R-expressing osteoclasts mediate bone degradation in osteolytic cancers and cancers that metastasize to bone. In the rare disease tenosynovial giant cell tumor (TGCT), aberrant CSF1 expression and production driven by a gene translocation leads to the recruitment and growth of tumors formed by CSF1R-dependent inflammatory cells. Small molecules and antibodies targeting the CSF1/CSF1R axis have shown promise in the treatment of TGCT and cancer, with pexidartinib recently receiving FDA approval for treatment of TGCT. Many small-molecule kinase inhibitors of CSF1R also inhibit the closely related kinases KIT, PDGFRA, PDGFRB, and FLT3, thus CSF1R suppression may be limited by off-target activity and associated adverse events. Vimseltinib (DCC-3014) is an oral, switch control tyrosine kinase inhibitor specifically designed to selectively and potently inhibit CSF1R by exploiting unique features of the switch control region that regulates kinase conformational activation. In preclinical studies, vimseltinib durably suppressed CSF1R activity in vitro and in vivo, depleted macrophages and other CSF1R-dependent cells, and resulted in inhibition of tumor growth and bone degradation in mouse cancer models. Translationally, in a phase I clinical study, vimseltinib treatment led to modulation of biomarkers of CSF1R inhibition and reduction in tumor burden in TGCT patients.

Ripretinib (DCC-2618) Is a Switch Control Kinase Inhibitor of a Broad Spectrum of Oncogenic and Drug-Resistant KIT and PDGFRA Variants.

Ripretinib (DCC-2618) was designed to inhibit the full spectrum of mutant KIT and PDGFRA kinases found in cancers and myeloproliferative neoplasms, particularly in gastrointestinal stromal tumors (GISTs), in which the heterogeneity of drug-resistant KIT mutations is a major challenge. Ripretinib is a "switch-control" kinase inhibitor that forces the activation loop (or activation "switch") into an inactive conformation. Ripretinib inhibits all tested KIT and PDGFRA mutants, and notably is a type II kinase inhibitor demonstrated to broadly inhibit activation loop mutations in KIT and PDGFRA, previously thought only achievable with type I inhibitors. Ripretinib shows efficacy in preclinical cancer models, and preliminary clinical data provide proof-of-concept that ripretinib inhibits a wide range of KIT mutants in patients with drug-resistant GISTs.

The Selective Tie2 Inhibitor Rebastinib Blocks Recruitment and Function of Tie2Hi Macrophages in Breast Cancer and Pancreatic Neuroendocrine Tumors.

Tumor-infiltrating myeloid cells promote tumor progression by mediating angiogenesis, tumor cell intravasation, and metastasis, which can offset the effects of chemotherapy, radiation, and antiangiogenic therapy. Here, we show that the kinase switch control inhibitor rebastinib inhibits Tie2, a tyrosine kinase receptor expressed on endothelial cells and protumoral Tie2-expressing macrophages in mouse models of metastatic cancer. Rebastinib reduces tumor growth and metastasis in an orthotopic mouse model of metastatic mammary carcinoma through reduction of Tie2+ myeloid cell infiltration, antiangiogenic effects, and blockade of tumor cell intravasation mediated by perivascular Tie2Hi/Vegf-AHi macrophages in the tumor microenvironment of metastasis (TMEM). The antitumor effects of rebastinib enhance the efficacy of microtubule inhibiting chemotherapeutic agents, either eribulin or paclitaxel, by reducing tumor volume, metastasis, and improving overall survival. Rebastinib inhibition of angiopoietin/Tie2 signaling impairs multiple pathways in tumor progression mediated by protumoral Tie2+ macrophages, including TMEM-dependent dissemination and angiopoietin/Tie2-dependent angiogenesis. Rebastinib is a promising therapy for achieving Tie2 inhibition in cancer patients. Mol Cancer Ther; 16(11); 2486-501. ©2017 AACR.

A suspected delayed hemolytic transfusion reaction mediated by anti-Joa.

CONCLUSIONS: A 32-year-old African-American woman with a history of sickle cell disease presented for surgical evaluation of left total hip arthroplasty due to avascular necrosis of the femoral head. In anticipation of a complex orthopedic procedure, pre-surgical blood work was ordered. The patient's Fenwal blood sample typed as group O, D+. Although the patient had a history of anti-Fya, the antibody identification was inconclusive, so the workup was sent to a reference laboratory. The patient was last transfused with red blood cells (RBCs) 2 years earlier, but had no history of transfusion reactions. Due to surgery, the patient's hemoglobin (Hb) decreased from 10.2 g/dL (preoperative) to 8.6 g/dL (postoperative). One unit of weakly crossmatch-incompatible Fy(a-), C-, E-, K-, and sickle cell hemoglobin S (HbS)-negative RBCs was transfused without incident, and the patient was discharged. Several days later, the reference lab reported two new specificities, anti-Joa and anti-Jkb. Fortunately, the transfused RBC unit was Jk(b-). Therefore, the crossmatch incompatibility was attributed to anti-Joa, which targets a high-prevalence antigen found in 100 percent of most populations. Two weeks after discharge, the patient returned in sickle vaso-occlusive pain crisis. The patient was clinically stable, but her Hb was 6.7 g/dL. One unit of Fy(a-), Jk(b-), C-, E-, K-, HbS- RBCs, which was weakly crossmatch-incompatible, was transfused. The following day, her Hb was unchanged, lactic acid dehydrogenase increased from 951 to 2464 U/L, potassium increased from 3.7 to 4.6 mEq/L, creatinine increased from 0.60 to 0.98 mg/dL, and the patient developed a 38.4°C fever. These findings are consistent with a delayed hemolytic transfusion reaction (DHTR), mediated by anti-Joa, occurring 2 weeks after the first RBC transfusion. Further care could not be provided because the patient left the hospital against medical advice. The purpose of this case study is to report findings consistent with a DHTR attributed to anti-Joa, an antibody with relatively unknown clinical significance.

Phase 1 dose-finding study of rebastinib (DCC-2036) in patients with relapsed chronic myeloid leukemia and acute myeloid leukemia.

A vailable tyrosine kinase inhibitors for chronic myeloid leukemia bind in an adenosine 5'-triphosphate-binding pocket and are affected by evolving mutations that confer resistance. Rebastinib was identified as a switch control inhibitor of BCR-ABL1 and FLT3 and may be active against resistant mutations. A Phase 1, first-in-human, single-agent study investigated rebastinib in relapsed or refractory chronic or acute myeloid leukemia. The primary objectives were to investigate the safety of rebastinib and establish the maximum tolerated dose and recommended Phase 2 dose. Fifty-seven patients received treatment with rebastinib. Sixteen patients were treated using powder-in-capsule preparations at doses from 57 mg to 1200 mg daily, and 41 received tablet preparations at doses of 100 mg to 400 mg daily. Dose-limiting toxicities were dysarthria, muscle weakness, and peripheral neuropathy. The maximum tolerated dose was 150 mg tablets administered twice daily. Rebastinib was rapidly absorbed. Bioavailability was 3- to 4-fold greater with formulated tablets compared to unformulated capsules. Eight complete hematologic responses were achieved in 40 evaluable chronic myeloid leukemia patients, 4 of which had a T315I mutation. None of the 5 patients with acute myeloid leukemia responded. Pharmacodynamic analysis showed inhibition of phosphorylation of substrates of BCR-ABL1 or FLT3 by rebastinib. Although clinical activity was observed, clinical benefit was insufficient to justify continued development in chronic or acute myeloid leukemia. Pharmacodynamic analyses suggest that other kinases inhibited by rebastinib, such as TIE2, may be more relevant targets for the clinical development of rebastinib (clinicaltrials.gov Identifier:00827138).

Altiratinib Inhibits Tumor Growth, Invasion, Angiogenesis, and Microenvironment-Mediated Drug Resistance via Balanced Inhibition of MET, TIE2, and VEGFR2.

Altiratinib (DCC-2701) was designed based on the rationale of engineering a single therapeutic agent able to address multiple hallmarks of cancer (1). Specifically, altiratinib inhibits not only mechanisms of tumor initiation and progression, but also drug resistance mechanisms in the tumor and microenvironment through balanced inhibition of MET, TIE2 (TEK), and VEGFR2 (KDR) kinases. This profile was achieved by optimizing binding into the switch control pocket of all three kinases, inducing type II inactive conformations. Altiratinib durably inhibits MET, both wild-type and mutated forms, in vitro and in vivo. Through its balanced inhibitory potency versus MET, TIE2, and VEGFR2, altiratinib provides an agent that inhibits three major evasive (re)vascularization and resistance pathways (HGF, ANG, and VEGF) and blocks tumor invasion and metastasis. Altiratinib exhibits properties amenable to oral administration and exhibits substantial blood-brain barrier penetration, an attribute of significance for eventual treatment of brain cancers and brain metastases.

Discovery of 1-(3,3-dimethylbutyl)-3-(2-fluoro-4-methyl-5-(7-methyl-2-(methylamino)pyrido[2,3-d]pyrimidin-6-yl)phenyl)urea (LY3009120) as a pan-RAF inhibitor with minimal paradoxical activation and activity against BRAF or RAS mutant tumor cells.

The RAS-RAF-MEK-MAPK cascade is an essential signaling pathway, with activation typically mediated through cell surface receptors. The kinase inhibitors vemurafenib and dabrafenib, which target oncogenic BRAF V600E, have shown significant clinical efficacy in melanoma patients harboring this mutation. Because of paradoxical pathway activation, both agents were demonstrated to promote growth and metastasis of tumor cells with RAS mutations in preclinical models and are contraindicated for treatment of cancer patients with BRAF WT background, including patients with KRAS or NRAS mutations. In order to eliminate the issues associated with paradoxical MAPK pathway activation and to provide therapeutic benefit to patients with RAS mutant cancers, we sought to identify a compound not only active against BRAF V600E but also wild type BRAF and CRAF. On the basis of its superior in vitro and in vivo profile, compound 13 was selected for further development and is currently being evaluated in phase I clinical studies.

Conformational control inhibition of the BCR-ABL1 tyrosine kinase, including the gatekeeper T315I mutant, by the switch-control inhibitor DCC-2036.

Acquired resistance to ABL1 tyrosine kinase inhibitors (TKIs) through ABL1 kinase domain mutations, particularly the gatekeeper mutant T315I, is a significant problem for patients with chronic myeloid leukemia (CML). Using structure-based drug design, we developed compounds that bind to residues (Arg386/Glu282) ABL1 uses to switch between inactive and active conformations. The lead "switch-control" inhibitor, DCC-2036, potently inhibits both unphosphorylated and phosphorylated ABL1 by inducing a type II inactive conformation, and retains efficacy against the majority of clinically relevant CML-resistance mutants, including T315I. DCC-2036 inhibits BCR-ABL1(T315I)-expressing cell lines, prolongs survival in mouse models of T315I mutant CML and B-lymphoblastic leukemia, and inhibits primary patient leukemia cells expressing T315I in vitro and in vivo, supporting its clinical development in TKI-resistant Ph(+) leukemia.

The ABL switch control inhibitor DCC-2036 is active against the chronic myeloid leukemia mutant BCR-ABLT315I and exhibits a narrow resistance profile.

Acquired point mutations within the BCR-ABL kinase domain represent a common mechanism of resistance to ABL inhibitor therapy in patients with chronic myeloid leukemia (CML). The BCR-ABL(T315I) mutant is highly resistant to imatinib, nilotinib, and dasatinib, and is frequently detected in relapsed patients. This critical gap in resistance coverage drove development of DCC-2036, an ABL inhibitor that binds the switch control pocket involved in conformational regulation of the kinase domain. We evaluated the efficacy of DCC-2036 against BCR-ABL(T315I) and other mutants in cellular and biochemical assays and conducted cell-based mutagenesis screens. DCC-2036 inhibited autophosphorylation of ABL and ABL(T315I) enzymes, and this activity was consistent with selective efficacy against Ba/F3 cells expressing BCR-ABL (IC(50): 19 nmol/L), BCR-ABL(T315I) (IC(50): 63 nmol/L), and most kinase domain mutants. Ex vivo exposure of CML cells from patients harboring BCR-ABL or BCR-ABL(T315I) to DCC-2036 revealed marked inhibition of colony formation and reduced phosphorylation of the direct BCR-ABL target CrkL. Cell-based mutagenesis screens identified a resistance profile for DCC-2036 centered around select P-loop mutations (G250E, Q252H, Y253H, E255K/V), although a concentration of 750 nmol/L DCC-2036 suppressed the emergence of all resistant clones. A decreased concentration of DCC-2036 (160 nmol/L) in dual combination with either nilotinib or dasatinib achieved the same zero outgrowth result. Further screens for resistance due to BCR-ABL compound mutations (two mutations in the same clone) identified BCR-ABL(E255V / T315I) as the most resistant mutant. Taken together, these findings support continued evaluation of DCC-2036 as an important new agent for treatment-refractory CML.

Switch control pocket inhibitors of p38-MAP kinase. Durable type II inhibitors that do not require binding into the canonical ATP hinge region.

Switch control pocket inhibitors of p38-alpha kinase are described. Durable type II inhibitors were designed which bind to arginines (Arg67 or Arg70) that function as key residues for mediating phospho-threonine 180 dependant conformational fluxing of p38-alpha from an inactive type II state to an active type I state. Binding to Arg70 in particular led to potent inhibitors, exemplified by DP-802, which also exhibited high kinase selectivity. Binding to Arg70 obviated the requirement for binding into the ATP Hinge region. X-ray crystallography revealed that DP-802 and analogs induce an enhanced type II conformation upon binding to either the unphosphorylated or the doubly phosphorylated form of p38-alpha kinase.

Examination of edge effects with different storage conditions of preplated dimethyl sulfoxide nanospots in ChemLib 1,536- and 3,456-well assay-ready plates.

For ultra-high-throughput screening, 10-30 nl of compound dissolved in 75% dimethyl sulfoxide (DMSO)/25% water (vol/vol) is spotted into 1,536- and 3,456-well ChemLib plates (Aurora Biotechnologies, Carlsbad, CA) and stored appropriately for a short time before screening. Although this practice eliminates the compound plating bottleneck, plated volumes of DMSO slowly evaporate from assay wells if plates are not properly stored in the interim. Since many assays are sensitive to DMSO concentrations, even slight evaporation may cause intra-plate variation and thus decrease assay quality. Using a cytochrome P450 3A4 Vivid Blue assay (Invitrogen, Carlsbad), we investigated the rate, pattern, and quantity of evaporation over a 1-year time frame to identify best practices for long-term (i.e., 6 months or greater) storage of assay-ready compound plates. Our findings regarding evaporation at plate edges indicate that nanospots preplated in ChemLib 1,536- or 3,456-well plates are best stored at -80 degrees C, in a bag, with or without the outer evaporation wells filled or at -20 degrees C, in a bag, with evaporation wells filled.

Simultaneous screening of multiple bacterial tRNA synthetases using an Escherichia coli S30-based transcription and translation assay.

The search for novel antibiotics to combat the growing threat of resistance has led researchers to screen libraries with coupled transcription and translation systems. In these systems, a bacterial cell lysate supplies the proteins necessary for transcription and translation, a plasmid encoding a reporter protein is added as a template, and a complex mixture of amino acids and cofactors is added to supply building blocks and energy to the assay. Firefly luciferase is typically used as the reporter protein in high-throughput screens because the luminescent signal is strong and, since bacterial lysates contain no luciferase, the background is negligible. The typical coupled transcription and translation assay is sensitive to inhibitors of RNA polymerase and to compounds that bind tightly to the ribosome. We have found a way to increase the information content of the screen by making the assay more sensitive to inhibitors of tRNA synthetases. Restricting the concentration of amino acids added to the reaction mixture allows the simultaneous screening of multiple tRNA synthetase enzymes along with the classic transcription and translation targets. In addition, this assay can be used as a convenient way to determine if an antibacterial compound of unknown mechanism inhibits translation through inhibition of a tRNA synthetase, and to identify which synthetase is the target.