ABSTRACT
Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of atypical peptides. Our results suggest that generation of MHC class Iâ¢peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.
Subject(s)
Antigen Presentation , Proteasome Endopeptidase Complex , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Histocompatibility Antigens Class I/metabolism , Peptides/metabolism , Ubiquitin/metabolismABSTRACT
Inactive state-selective KRAS(G12C) inhibitors1-8 demonstrate a 30-40% response rate and result in approximately 6-month median progression-free survival in patients with lung cancer9. The genetic basis for resistance to these first-in-class mutant GTPase inhibitors remains under investigation. Here we evaluated matched pre-treatment and post-treatment specimens from 43 patients treated with the KRAS(G12C) inhibitor sotorasib. Multiple treatment-emergent alterations were observed across 27 patients, including alterations in KRAS, NRAS, BRAF, EGFR, FGFR2, MYC and other genes. In preclinical patient-derived xenograft and cell line models, resistance to KRAS(G12C) inhibition was associated with low allele frequency hotspot mutations in KRAS(G12V or G13D), NRAS(Q61K or G13R), MRAS(Q71R) and/or BRAF(G596R), mirroring observations in patients. Single-cell sequencing in an isogenic lineage identified secondary RAS and/or BRAF mutations in the same cells as KRAS(G12C), where they bypassed inhibition without affecting target inactivation. Genetic or pharmacological targeting of ERK signalling intermediates enhanced the antiproliferative effect of G12C inhibitor treatment in models with acquired RAS or BRAF mutations. Our study thus suggests a heterogenous pattern of resistance with multiple subclonal events emerging during G12C inhibitor treatment. A subset of patients in our cohort acquired oncogenic KRAS, NRAS or BRAF mutations, and resistance in this setting may be delayed by co-targeting of ERK signalling intermediates. These findings merit broader evaluation in prospective clinical trials.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Drug Resistance, Neoplasm/genetics , Mutation , Neoplasms/drug therapy , Neoplasms/genetics , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Proto-Oncogene Proteins p21(ras)/genetics , Acetonitriles/pharmacology , Animals , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Cell Line , Cohort Studies , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Humans , MAP Kinase Signaling System/drug effects , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Piperazines/pharmacology , Piperazines/therapeutic use , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Sotorasib is a first-in class KRASG12C covalent inhibitor in clinical development for the treatment of tumors with the KRAS p.G12C mutation. In the nonclinical toxicology studies of sotorasib, the kidney was identified as a target organ of toxicity in the rat but not the dog. Renal toxicity was characterized by degeneration and necrosis of the proximal tubular epithelium localized to the outer stripe of the outer medulla (OSOM), which suggested that renal metabolism was involved. Here, we describe an in vivo mechanistic rat study designed to investigate the time course of the renal toxicity and sotorasib metabolites. Renal toxicity was dose- and time-dependent, restricted to the OSOM, and the morphologic features progressed from vacuolation and necrosis to regeneration of tubular epithelium. The renal toxicity correlated with increases in renal biomarkers of tubular injury. Using mass spectrometry and matrix-assisted laser desorption/ionization, a strong temporal and spatial association between renal toxicity and mercapturate pathway metabolites was observed. The rat is reported to be particularly susceptible to the formation of nephrotoxic metabolites via this pathway. Taken together, the data presented here and the literature support the hypothesis that sotorasib-related renal toxicity is mediated by a toxic metabolite derived from the mercapturate and ß-lyase pathway. Our understanding of the etiology of the rat specific renal toxicity informs the translational risk assessment for patients.
Subject(s)
Acetylcysteine/metabolism , Acute Kidney Injury/metabolism , Piperazines/metabolism , Piperazines/toxicity , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/metabolism , Pyridines/toxicity , Pyrimidines/metabolism , Pyrimidines/toxicity , Signal Transduction/drug effects , Acute Kidney Injury/chemically induced , Acute Kidney Injury/pathology , Animals , Dose-Response Relationship, Drug , Male , Rats , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit predominantly CD4+ T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8+ and CD4+ T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP-Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4+ T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP-Lpx stimulated both CD4+ and CD8+ T cells and suppressed tumor growth in a CD8+ T cell-dependent manner. Combination of the SLP-Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4+ and CD8+ T cell responses that inhibit tumor growth.
ABSTRACT
BACKGROUND: No therapies for targeting KRAS mutations in cancer have been approved. The KRAS p.G12C mutation occurs in 13% of non-small-cell lung cancers (NSCLCs) and in 1 to 3% of colorectal cancers and other cancers. Sotorasib is a small molecule that selectively and irreversibly targets KRASG12C. METHODS: We conducted a phase 1 trial of sotorasib in patients with advanced solid tumors harboring the KRAS p.G12C mutation. Patients received sotorasib orally once daily. The primary end point was safety. Key secondary end points were pharmacokinetics and objective response, as assessed according to Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. RESULTS: A total of 129 patients (59 with NSCLC, 42 with colorectal cancer, and 28 with other tumors) were included in dose escalation and expansion cohorts. Patients had received a median of 3 (range, 0 to 11) previous lines of anticancer therapies for metastatic disease. No dose-limiting toxic effects or treatment-related deaths were observed. A total of 73 patients (56.6%) had treatment-related adverse events; 15 patients (11.6%) had grade 3 or 4 events. In the subgroup with NSCLC, 32.2% (19 patients) had a confirmed objective response (complete or partial response) and 88.1% (52 patients) had disease control (objective response or stable disease); the median progression-free survival was 6.3 months (range, 0.0+ to 14.9 [with + indicating that the value includes patient data that were censored at data cutoff]). In the subgroup with colorectal cancer, 7.1% (3 patients) had a confirmed response, and 73.8% (31 patients) had disease control; the median progression-free survival was 4.0 months (range, 0.0+ to 11.1+). Responses were also observed in patients with pancreatic, endometrial, and appendiceal cancers and melanoma. CONCLUSIONS: Sotorasib showed encouraging anticancer activity in patients with heavily pretreated advanced solid tumors harboring the KRAS p.G12C mutation. Grade 3 or 4 treatment-related toxic effects occurred in 11.6% of the patients. (Funded by Amgen and others; CodeBreaK100 ClinicalTrials.gov number, NCT03600883.).
Subject(s)
Antineoplastic Agents/administration & dosage , Carcinoma, Non-Small-Cell Lung/drug therapy , Colorectal Neoplasms/drug therapy , Lung Neoplasms/drug therapy , Mutation , Piperazines/administration & dosage , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/administration & dosage , Pyrimidines/administration & dosage , Aged , Antineoplastic Agents/adverse effects , Antineoplastic Agents/pharmacokinetics , Dose-Response Relationship, Drug , Female , Humans , Male , Middle Aged , Neoplasms/drug therapy , Piperazines/adverse effects , Piperazines/pharmacokinetics , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/adverse effects , Pyridines/pharmacokinetics , Pyrimidines/adverse effects , Pyrimidines/pharmacokineticsABSTRACT
KRASG12C has emerged as a promising target in the treatment of solid tumors. Covalent inhibitors targeting the mutant cysteine-12 residue have been shown to disrupt signaling by this long-"undruggable" target; however clinically viable inhibitors have yet to be identified. Here, we report efforts to exploit a cryptic pocket (H95/Y96/Q99) we identified in KRASG12C to identify inhibitors suitable for clinical development. Structure-based design efforts leading to the identification of a novel quinazolinone scaffold are described, along with optimization efforts that overcame a configurational stability issue arising from restricted rotation about an axially chiral biaryl bond. Biopharmaceutical optimization of the resulting leads culminated in the identification of AMG 510, a highly potent, selective, and well-tolerated KRASG12C inhibitor currently in phase I clinical trials (NCT03600883).
Subject(s)
Antineoplastic Agents/therapeutic use , Neoplasms/drug therapy , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/therapeutic use , Pyrimidines/therapeutic use , Pyrimidinones/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Clinical Trials as Topic , Dogs , Drug Discovery , Humans , Isomerism , Madin Darby Canine Kidney Cells , Mice, Inbred BALB C , Mice, Nude , Mutation , Piperazines/chemistry , Piperazines/pharmacology , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/chemistry , Pyridines/pharmacokinetics , Pyridines/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Pyrimidinones/chemistry , Pyrimidinones/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
KRAS is the most frequently mutated oncogene in cancer and encodes a key signalling protein in tumours1,2. The KRAS(G12C) mutant has a cysteine residue that has been exploited to design covalent inhibitors that have promising preclinical activity3-5. Here we optimized a series of inhibitors, using novel binding interactions to markedly enhance their potency and selectivity. Our efforts have led to the discovery of AMG 510, which is, to our knowledge, the first KRAS(G12C) inhibitor in clinical development. In preclinical analyses, treatment with AMG 510 led to the regression of KRASG12C tumours and improved the anti-tumour efficacy of chemotherapy and targeted agents. In immune-competent mice, treatment with AMG 510 resulted in a pro-inflammatory tumour microenvironment and produced durable cures alone as well as in combination with immune-checkpoint inhibitors. Cured mice rejected the growth of isogenic KRASG12D tumours, which suggests adaptive immunity against shared antigens. Furthermore, in clinical trials, AMG 510 demonstrated anti-tumour activity in the first dosing cohorts and represents a potentially transformative therapy for patients for whom effective treatments are lacking.
Subject(s)
Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Lung Neoplasms/drug therapy , Lung Neoplasms/immunology , Piperazines/pharmacology , Piperazines/therapeutic use , Proto-Oncogene Proteins p21(ras)/antagonists & inhibitors , Pyridines/pharmacology , Pyridines/therapeutic use , Pyrimidines/pharmacology , Pyrimidines/therapeutic use , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Drug Synergism , Humans , Immunotherapy , Inflammation/chemically induced , Inflammation/immunology , Inflammation/pathology , Lung Neoplasms/genetics , Lung Neoplasms/pathology , Mice , Phosphorylation/drug effects , Piperazines/administration & dosage , Piperazines/chemistry , Proto-Oncogene Proteins p21(ras)/genetics , Pyridines/administration & dosage , Pyridines/chemistry , Pyrimidines/administration & dosage , Pyrimidines/chemistry , Signal Transduction/drug effects , Treatment Outcome , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunologyABSTRACT
KRAS regulates many cellular processes including proliferation, survival, and differentiation. Point mutants of KRAS have long been known to be molecular drivers of cancer. KRAS p.G12C, which occurs in approximately 14% of lung adenocarcinomas, 3-5% of colorectal cancers, and low levels in other solid tumors, represents an attractive therapeutic target for covalent inhibitors. Herein, we disclose the discovery of a class of novel, potent, and selective covalent inhibitors of KRASG12C identified through a custom library synthesis and screening platform called Chemotype Evolution and structure-based design. Identification of a hidden surface groove bordered by H95/Y96/Q99 side chains was key to the optimization of this class of molecules. Best-in-series exemplars exhibit a rapid covalent reaction with cysteine 12 of GDP-KRASG12C with submicromolar inhibition of downstream signaling in a KRASG12C-specific manner.
ABSTRACT
Pim kinases are a family of constitutively active serine/threonine kinases that are partially redundant and regulate multiple pathways important for cell growth and survival. In human disease, high expression of the three Pim isoforms has been implicated in the progression of hematopoietic and solid tumor cancers, which suggests that Pim kinase inhibitors could provide patients with therapeutic benefit. Herein, we describe the structure-guided optimization of a series of quinazolinone-pyrrolodihydropyrrolone analogs leading to the identification of potent pan-Pim inhibitor 28 with improved potency, solubility, and drug-like properties. Compound 28 demonstrated on-target Pim activity in an in vivo pharmacodynamic assay with significant inhibition of BAD phosphorylation in KMS-12-BM multiple myeloma tumors for 16 h postdose. In a 2-week mouse xenograft model, daily dosing of compound 28 resulted in 33% tumor regression at 100 mg/kg.
Subject(s)
Antineoplastic Agents/therapeutic use , Hematologic Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrroles/therapeutic use , Quinazolinones/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Female , Humans , Mice, SCID , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Pyrroles/chemical synthesis , Pyrroles/pharmacokinetics , Quinazolinones/chemical synthesis , Quinazolinones/pharmacokinetics , Structure-Activity Relationship , Swine , Xenograft Model Antitumor AssaysABSTRACT
Proteolysis targeting chimeras (PROTACs) are bispecific molecules containing a target protein binder and an ubiquitin ligase binder connected by a linker. By recruiting an ubiquitin ligase to a target protein, PROTACs promote ubiquitination and proteasomal degradation of the target protein. The generation of effective PROTACs depends on the nature of the protein/ligase ligand pair, linkage site, linker length, and linker composition, all of which have been difficult to address in a systematic way. Herein, we describe a "click chemistry" approach for the synthesis of PROTACs. We demonstrate the utility of this approach with the bromodomain and extraterminal domain-4 (BRD4) ligand JQ-1 (3) and ligase binders targeting cereblon (CRBN) and Von Hippel-Lindau (VHL) proteins. An AlphaScreen proximity assay was used to determine the ability of PROTACs to form the ternary ligase-PROTAC-target protein complex and a MSD assay to measure cellular degradation of the target protein promoted by PROTACs.
Subject(s)
Click Chemistry , Drug Evaluation, Preclinical , Nuclear Proteins , Proteolysis , Transcription Factors , Humans , Adaptor Proteins, Signal Transducing , Cell Cycle Proteins , Click Chemistry/methods , Drug Evaluation, Preclinical/methods , Ligands , Nuclear Proteins/metabolism , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Peptides/pharmacology , Proteolysis/drug effects , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Transcription Factors/metabolism , Ubiquitin-Protein Ligases , Von Hippel-Lindau Tumor Suppressor Protein/metabolismABSTRACT
High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers, suggesting that inhibition of Pim signaling could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal using a screening hit (rac-1) as a starting point. Modification of the indazole ring resulted in the discovery of a series of imidazopyridazine-based Pim inhibitors exemplified by compound 22m, which was found to be a subnanomolar inhibitor of the Pim-1 and Pim-2 isoforms (IC50 values of 0.024nM and 0.095nM, respectively) and to potently inhibit the phosphorylation of BAD in a cell line that expresses high levels of all Pim isoforms, KMS-12-BM (IC50=28nM). Profiling of Pim-1 and Pim-2 expression levels in a panel of multiple myeloma cell lines and correlation of these data with the potency of compound 22m in a proliferation assay suggests that Pim-2 inhibition would be advantageous for this indication.
Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Pyridazines/chemistry , Pyridazines/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Models, Molecular , Multiple Myeloma/drug therapy , Multiple Myeloma/metabolism , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-pim-1/metabolism , Structure-Activity RelationshipABSTRACT
The high expression of proviral insertion site of Moloney murine leukemia virus kinases (Pim-1, -2, and -3) in cancers, particularly the hematopoietic malignancies, is believed to play a role in promoting cell survival and proliferation while suppressing apoptosis. The three isoforms of Pim protein appear largely redundant in their oncogenic functions. Thus, a pan-Pim kinase inhibitor is highly desirable. However, cell active pan-Pim inhibitors have proven difficult to develop because Pim-2 has a low Km for ATP and therefore requires a very potent inhibitor to effectively block the kinase activity at cellular ATP concentrations. Herein, we report a series of quinazolinone-pyrrolopyrrolones as potent and selective pan-Pim inhibitors. In particular, compound 17 is orally efficacious in a mouse xenograft model (KMS-12 BM) of multiple myeloma, with 93% tumor growth inhibition at 50 mg/kg QD upon oral dosing.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrroles/pharmacology , Quinazolinones/pharmacology , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Humans , Mice , Models, Molecular , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/pathology , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins c-pim-1/metabolism , Pyrroles/administration & dosage , Pyrroles/chemistry , Quinazolinones/administration & dosage , Quinazolinones/chemistry , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
The identification of Pim-1/2 kinase overexpression in B-cell malignancies suggests that Pim kinase inhibitors will have utility in the treatment of lymphoma, leukemia, and multiple myeloma. Starting from a moderately potent quinoxaline-dihydropyrrolopiperidinone lead, we recognized the potential for macrocyclization and developed a series of 13-membered macrocycles. The structure-activity relationships of the macrocyclic linker were systematically explored, leading to the identification of 9c as a potent, subnanomolar inhibitor of Pim-1 and -2. This molecule also potently inhibited Pim kinase activity in KMS-12-BM, a multiple myeloma cell line with relatively high endogenous levels of Pim-1/2, both in vitro (pBAD IC50 = 25 nM) and in vivo (pBAD EC50 = 30 nM, unbound), and a 100 mg/kg daily dose was found to completely arrest the growth of KMS-12-BM xenografts in mice.
ABSTRACT
High levels of Pim expression have been implicated in several hematopoietic and solid tumor cancers. These findings suggest that inhibition of Pim signaling by a small molecule Pim-1,2 inhibitor could provide patients with therapeutic benefit. Herein, we describe our progress towards this goal starting from the highly Pim-selective indole-thiadiazole compound (1), which was derived from a nonselective hit identified in a high throughput screening campaign. Optimization of this compound's potency and its pharmacokinetic properties resulted in the discovery of compound 29. Cyclopropane 29 was found to exhibit excellent enzymatic potency on the Pim-1 and Pim-2 isoforms (Ki values of 0.55nM and 0.28nM, respectively), and found to inhibit the phosphorylation of BAD in the Pim-overexpressing KMS-12 cell line (IC50=150nM). This compound had moderate clearance and bioavailability in rat (CL=2.42L/kg/h; %F=24) and exhibited a dose-dependent inhibition of p-BAD in KMS-12 tumor pharmacodynamic (PD) model with an EC50 value of 6.74µM (18µg/mL) when dosed at 10, 30, 100 and 200mg/kg po in mice.
Subject(s)
Oxadiazoles/pharmacology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Crystallography, X-Ray , Drug Discovery , Molecular Structure , Oxadiazoles/chemistryABSTRACT
The structure-based design and optimization of a novel series of selective PERK inhibitors are described resulting in the identification of 44 as a potent, highly selective, and orally active tool compound suitable for PERK pathway biology exploration both in vitro and in vivo.
Subject(s)
Drug Discovery , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , eIF-2 Kinase/antagonists & inhibitors , Animals , Cells, Cultured , Dose-Response Relationship, Drug , Humans , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Structure-Activity Relationship , eIF-2 Kinase/metabolismABSTRACT
PIM kinases are a family of Ser/Thr kinases that are implicated in tumorigenesis. The discovery of a new class of PIM inhibitors, 5-(1H-indol-5-yl)-1,3,4-thiadiazol-2-amines, is discussed with optimized compounds showing excellent potency against all three PIM isoforms.
Subject(s)
Amines/chemistry , Amines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Amines/chemical synthesis , Carbolines/chemical synthesis , Carbolines/chemistry , Carbolines/pharmacology , Cell Line, Tumor , Drug Discovery , Humans , Models, Molecular , Protein Kinase Inhibitors/chemical synthesis , Proto-Oncogene Proteins c-pim-1/chemistry , Structure-Activity RelationshipABSTRACT
The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to tumorigenesis. As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Herein, we describe our efforts toward the development of a potent, pan-Pim inhibitor. The synthesis and hit-to-lead SAR development from a 3-(pyrazin-2-yl)-1H-indazole derived hit 2 to the identification of a series of potent, pan-Pim inhibitors such as 13o are described.
Subject(s)
Indazoles/chemistry , Indazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Drug Discovery , Humans , Structure-Activity RelationshipABSTRACT
The kinase/endonuclease inositol requiring enzyme 1 (IRE1α), one of the sensors of unfolded protein accumulation in the endoplasmic reticulum that triggers the unfolded protein response (UPR), has been investigated as an anticancer target. We identified potent allosteric inhibitors of IRE1α endonuclease activity that bound to the kinase site on the enzyme. Structure-activity relationship (SAR) studies led to 16 and 18, which were selective in kinase screens and were potent against recombinant IRE1α endonuclease as well as cellular IRE1α. The first X-ray crystal structure of a kinase inhibitor (16) bound to hIRE1α was obtained. Screening of native tumor cell lines (>300) against selective IRE1α inhibitors failed to demonstrate any effect on cellular viability. These results suggest that IRE1α activity is not essential for viability in most tumor cell lines, in vitro, and that interfering with the survival functions of the UPR may not be an effective strategy to block tumorigenesis.
ABSTRACT
Growing evidence supports the notion that proteasome-mediated destruction of transcriptional activators can be intimately coupled to their function. Recently, Nalley et al. challenged this view by reporting that the prototypical yeast activator Gal4 does not dynamically associate with chromatin, but rather 'locks in' to stable promoter complexes that are resistant to competition. Here we present evidence that the assay used to reach this conclusion is unsuitable, and that promoter-bound, active Gal4 is indeed susceptible to competition in vivo. Our data challenge the key evidence that Nalley et al. used to reach their conclusion, and indicate that Gal4 functions in vivo within the context of dynamic promoter complexes.