Development Of Next-generation Inhibitors Research Articles

Molecular pathway of anticancer effect of next-generation HSP90 inhibitors XL-888 and Debio0932 in neuroblastoma cell line

Neuroblastoma is a common nervous system tumor in childhood, and current treatments are not adequate. HSP90 is a molecular chaperone protein that plays a critical role in the regulation of cancer-related proteins. HSP90 inhibition may exert anticancer effects by targeting cancer-related processes such as tumor growth, cell proliferation, metastasis, and apoptosis. Therefore, HSP90 inhibition is a promising strategy in the treatment of various types of cancer, and the development of next-generation inhibitors could potentially lead to more effective and safer treatments. XL-888 and Debio0932 is a next-generation HSP90 inhibitor and can inhibit the correct folding and stabilization of client proteins that cancer-associated HSP90 helps to fold correctly. In this study, we aimed to investigate the comprehensive molecular pathways of the anticancer activity of XL-888 and Debio0932 in human neuroblastoma cells SH-SY5Y. The cytotoxic effects of XL-888 and Debio0932 on the neuroblastoma cell line SH-SY5Y cells were evaluated by MTT assay. Then, the effect of these HSP90 inhibitors on the expression of important genes in cancer was revealed by Quantitative Real Time Polymerase Chain Reaction (qRT-PCR) method. The qRT-PCR data were evaluated using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) biological process tools. Finally, the effect of HSP90 inhibitors on HSP27, HSP70 and HSP90 protein expression was investigated by Western blotting analysis. The results revealed that XL-888 and Debio0932 had a role in regulating many cancer-related pathways such as migration, invasion, metastasis, angiogenesis, and apoptosis in SH-SY5Y cells. In conclusion, it shows that HSP90 inhibitors can be considered as a promising candidate in the treatment of neuroblastoma and resistance to chemotherapy.

Identification of protein interactors of BRAF via Chemical Induced Dimerization‐BRAF and LC‐MS/MS

Rapidly Accelerated Fibrosarcoma (ARAF, BRAF, CRAF) Kinase is central to the MAPK Signaling Cascade (RAS‐RAF‐MEK‐ERK). Despite over three decades of scrutiny, the regulatory mechanisms of BRAF are not well understood. According to the accepted model, inactive RAF kinase is monomeric, autoinhibited, and cytosolic while activated RAF is membrane recruited via RAS‐GTP and dimerized; however—direct evidence and key details are still missing. Using a chemical biology approach to target this complex biological system, we seek to exert spatial control using the genetically encodable Chemically Induced Dimerization (CID)‐BRAF; CID‐BRAF exploits the FRB‐rapamycin‐FKBP system to membrane recruit BRAF. We hypothesize that membrane recruitment of BRAF enables diversification of PPI, which remain elusive due to the transient nature of these interactions. To capture transient protein‐protein interactions (PPI), cytosolic BRAF and membrane‐recruited BRAF cells were crosslinked, co‐immunoprecipitated, and identified via LC‐MS/MS. Current efforts are focused on the validation of putative MS identified PPI and identification of BRAF interaction sites. Identifying novel PPI will provide additional mechanistic insight to the regulation of BRAF and facilitate the development of next generation inhibitors of dysregulated BRAF.

Abstract C050: A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist

Abstract The microenvironment of solid tumors is known to be hypoxic and requires induction of genes associated with metabolism, growth, proliferation, and angiogenesis for tumor cells to survive and metastasize. The master transcriptional regulator of hypoxia-induced genes is the Hypoxia-Inducible Factor (HIF), consisting of an oxygen-regulated alpha monomer, of which there are three isoforms (HIF-1α, HIF-2α, and HIF-3α), that can heterodimerize with a constitutively-expressed beta monomer (HIF-1β/ARNT) using Per-ARNT-SIM (PAS) protein-protein interaction domains. In normoxia, proline residues present in the oxygen-dependent degradation domain of the HIF-α subunits are hydroxylated, allowing for recognition by the von Hippel-Lindau (pVHL) E3-ubiquitin ligase complex and subsequent proteasomal degradation. Upon exposure to low oxygen conditions or in the case of VHL mutation or silencing, HIF-α subunits accumulate in the cell and mediate transcription of various pro-tumorigenic gene sets. In patients, overexpression of HIF is associated with poor prognosis, and both preclinical and clinical evidence is mounting that suggests inhibiting HIF-2α is a valid approach to destroy tumor cells, particularly in clear cell renal carcinoma, warranting development of next-generation inhibitors. Using a suite of in vitro and in vivo assays designed to evaluate HIF-2α-specific effects, herein we describe pharmacological properties associated with novel, potent, and selective small-molecule antagonists of HIF-2α. These compounds inhibited HIF-dependent reporter gene transcription as well as VEGF protein secretion in a human renal cell adenocarcinoma line. Compounds that were confirmed to bind the HIF-2α PAS-B domain by Microscale thermophoresis (MST) and Thermal shift assay (TSA) also significantly inhibited HIF-2α, but not HIF-1α, target gene expression in a hepatocellular carcinoma cell line (P<0.05). Further, NanoString analyses revealed pathway signatures upregulated in hypoxic conditions, including angiogenesis, metabolism, and metastasis, that were significantly decreased with antagonist treatment. Selective antagonists were evaluated in vivo to determine efficacy and PK-PD relationships. Additionally, syngeneic tumor models were used to characterize the effects of HIF-2α inhibition (alone or in combination with anti-PD-1 and anti-adenosine agents) on tumor growth, immune cell infiltration, and expression of genes associated with adenosine production/signaling. Collectively, these data support the rationale to target HIF-2α for the treatment of cancers, particularly indications with a HIF-2α-driven hypoxia signature, and describe the properties of novel and selective HIF-2α antagonists. Citation Format: Kelsey E Sivick Gauthier, Kenneth V Lawson, Dana Piovesan, Matthew J Walters, Ada Chen, Xiaoning Zhao, Cesar Meleza, Nikki Kimura, Tim Park, Steve Young, Anh Tran, Samuel L Drew, Lixia Jin, Manmohan Leleti, Elaine Ginn, Jay P Powers. A novel, potent, and selective hypoxia-inducible factor (HIF)-2α antagonist [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C050. doi:10.1158/1535-7163.TARG-19-C050

Abstract 5115: Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies

Abstract The enzymes tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO1) catalyse oxidation of the essential amino acid tryptophan (Trp) leading to the formation of immunosuppressive kynurenine (Kyn) pathway metabolites that dampen the immune response in the tumor microenvironment. Both IDO1 and TDO have been shown to be up-regulated in a variety of cancers and blockade of their activity has been shown to stimulate the anti-tumour immune response in pre-clinical animal models. We have discovered and optimized multiple novel chemical series of both highly selective and dual-acting inhibitors of IDO1 and TDO. We describe the characterization of molecules that we are progressing into the clinic. We have characterized the IDO1 and TDO-selective inhibitors and dual-acting inhibitors which demonstrate nM potencies. In IFNγ-stimulated A172 glioblastoma cells expressing both enzymes, the dual-acting molecules fully inhibit Kyn production, whilst selective inhibitors yield only a partial response, demonstrating the utility of dual enzyme blockade in cancer cells. The compounds also relieve inhibition of T-cell proliferation in cancer cell conditioned media/T-cell co-culture assays and demonstrate highly favourable physico-chemical and pharmacokinetic properties. These properties translate to superior PK/PD effects following oral dosing in rodents with profound and sustained modulation of plasma Kyn levels, combined with an exceptional safety profile. In a mouse model of LPS-induced IDO1 activation, our IDO1 inhibitors fully ablate elevation of Kyn in both plasma and lung in a dose-dependent manner. Following oral administration, the IOmet IDO1 inhibitors provide excellent single-agent tumor growth control in PAN02 pancreatic adenocarcinoma syngeneic models. In addition, combining IOmet IDO1 inhibitors with standard-of-care chemotherapy (gemcitabine/abraxane) in the PAN02 model completely inhibits tumor growth. In summary, we describe the characterization of novel potent, selective and dual-acting IDO1 and TDO inhibitors. These candidate molecules demonstrate excellent rodent in vivo PK/PD, safety and anti-tumor profiles, supporting their progression into the clinic. Citation Format: Alan Wise, Barry E. McGuinness, Sarah C. Trewick, Thomas J. Brown, Phillip M. Cowley. Pre-clinical development of next generation inhibitors of the enzymes indoleamine 2,3-dioxygenase 1 and tryptophan 2,3-dioxygenase as cancer immunotherapies. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5115.