ABSTRACT
The interleukin-1 receptor-activated kinase 4 (IRAK4) belongs to the IRAK family of serine/threonine kinases and plays a central role in the innate immune response. However, the function of IRAK4 in tumor growth and progression remains elusive. Here we sought to determine the enzymatic and scaffolding functions of IRAK4 in activated B-cell-like diffuse large B cell lymphoma (ABC DLBCL). We chose a highly selective IRAK4 kinase inhibitor to probe the biological effects of kinase inhibition and developed a series of IRAK4 degraders to evaluate the effects of protein degradation in ABC DLBCL cells. Interestingly, the results demonstrated that neither IRAK4 kinase inhibition nor protein degradation led to cell death or growth inhibition, suggesting a redundant role for IRAK4 in ABC DLBCL cell survival. IRAK4 degraders characterized in this study provide useful tools for understanding IRAK4 protein scaffolding function, which was previously unachievable using pharmacological perturbation.
Subject(s)
Interleukin-1 Receptor-Associated Kinases/antagonists & inhibitors , Interleukin-1 Receptor-Associated Kinases/metabolism , Lymphoma, Large B-Cell, Diffuse/pathology , Protein Kinase Inhibitors/pharmacology , Proteolysis/drug effects , Cell Line, Tumor , Drug Design , HumansABSTRACT
The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RASERK signalling pathway. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy. Here we report the discovery of a highly potent (IC50 = 0.071 µM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RASERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.
Subject(s)
Neoplasms/drug therapy , Neoplasms/enzymology , Piperidines/pharmacology , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrimidines/pharmacology , Receptor Protein-Tyrosine Kinases/metabolism , Allosteric Regulation/drug effects , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Female , Humans , Inhibitory Concentration 50 , MAP Kinase Signaling System/drug effects , Mice , Mice, Nude , Models, Molecular , Neoplasms/pathology , Oncogene Protein p21(ras)/metabolism , Piperidines/chemistry , Piperidines/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Stability/drug effects , Protein Structure, Tertiary/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Pyrimidines/chemistry , Pyrimidines/therapeutic use , Reproducibility of Results , Xenograft Model Antitumor AssaysABSTRACT
SHP2 is a nonreceptor protein tyrosine phosphatase (PTP) encoded by the PTPN11 gene involved in cell growth and differentiation via the MAPK signaling pathway. SHP2 also purportedly plays an important role in the programmed cell death pathway (PD-1/PD-L1). Because it is an oncoprotein associated with multiple cancer-related diseases, as well as a potential immunomodulator, controlling SHP2 activity is of significant therapeutic interest. Recently in our laboratories, a small molecule inhibitor of SHP2 was identified as an allosteric modulator that stabilizes the autoinhibited conformation of SHP2. A high throughput screen was performed to identify progressable chemical matter, and X-ray crystallography revealed the location of binding in a previously undisclosed allosteric binding pocket. Structure-based drug design was employed to optimize for SHP2 inhibition, and several new protein-ligand interactions were characterized. These studies culminated in the discovery of 6-(4-amino-4-methylpiperidin-1-yl)-3-(2,3-dichlorophenyl)pyrazin-2-amine (SHP099, 1), a potent, selective, orally bioavailable, and efficacious SHP2 inhibitor.
Subject(s)
Antineoplastic Agents/chemistry , Piperidines/chemistry , Protein Tyrosine Phosphatase, Non-Receptor Type 11/antagonists & inhibitors , Pyrazines/chemistry , Pyrimidines/chemistry , Administration, Oral , Allosteric Regulation , Allosteric Site , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Crystallography, X-Ray , Drug Design , Female , Heterografts , High-Throughput Screening Assays , Humans , Male , Mice, Inbred C57BL , Mice, Nude , Models, Molecular , Neoplasm Transplantation , Piperidines/chemical synthesis , Piperidines/pharmacokinetics , Piperidines/pharmacology , Protein Conformation , Protein Tyrosine Phosphatase, Non-Receptor Type 11/chemistry , Pyrazines/chemical synthesis , Pyrazines/pharmacokinetics , Pyrazines/pharmacology , Pyrimidines/chemical synthesis , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Structure-Activity RelationshipABSTRACT
The synthesis of three potent new antitumor agents is described: the A83586C-citropeptin hybrid (1), the A83586C-GE3 hybrid (2), and l-Pro-A83586C (3). Significantly, compounds 1 and 2 function as highly potent inhibitors of beta-catenin/TCF4 signaling within cancer cells, while simultaneously downregulating osteopontin (Opn) expression. A83586C antitumor cyclodepsipeptides also inhibit E2F-mediated transcription by downregulating E2F1 expression and inducing dephosphorylation of the oncogenic hyperphosphorylated retinoblastoma protein (pRb).
Subject(s)
Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , DNA-Binding Proteins/antagonists & inhibitors , Depsipeptides/chemical synthesis , Depsipeptides/pharmacology , E2F Transcription Factors/metabolism , Transcription Factors/antagonists & inhibitors , beta Catenin/antagonists & inhibitors , Antineoplastic Agents/chemistry , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Depsipeptides/chemistry , Drug Screening Assays, Antitumor , E2F1 Transcription Factor/metabolism , Humans , Inhibitory Concentration 50 , Transcription Factor 4ABSTRACT
A cancer drug target is only truly validated by demonstrating that a given therapeutic agent is clinically effective and acts through the target against which it was designed. Nevertheless, it is desirable to declare an early-stage drug target as 'validated' before investing in a full-scale drug discovery programme dedicated to it. Although the outcome of validation studies can guide cancer research programmes, strictly defined universal validation criteria have not been established.
Subject(s)
Drug Evaluation, Preclinical/methods , Neoplasms/drug therapy , Neoplasms/metabolism , Animals , Cells/drug effects , Cells/metabolism , Disease Models, Animal , Drug Evaluation, Preclinical/standards , Humans , Neoplasms/genetics , Neoplasms/pathology , Reproducibility of Results , Substrate SpecificityABSTRACT
Key molecular lesions in colorectal and other cancers cause beta-catenin-dependent transactivation of T cell factor (Tcf)-dependent genes. Disruption of this signal represents an opportunity for rational cancer therapy. To identify compounds that inhibit association between Tcf4 and beta-catenin, we screened libraries of natural compounds in a high-throughput assay for immunoenzymatic detection of the protein-protein interaction. Selected compounds disrupt Tcf/beta-catenin complexes in several independent in vitro assays and potently antagonize cellular effects of beta-catenin-dependent activities, including reporter gene activation, c-myc or cyclin D1 expression, cell proliferation, and duplication of the Xenopus embryonic dorsal axis. These compounds thus meet predicted criteria for disrupting Tcf/beta-catenin complexes and define a general standard to establish mechanism-based activity of small molecule inhibitors of this pathogenic protein-protein interaction.
