ABSTRACT
A major challenge of targeted cancer therapy is the selection for drug-resistant mutations in tumor cells leading to loss of treatment effectiveness. p97/VCP is central regulator of protein homeostasis and a promising anticancer target because of its vital role in cell growth and survival. One ATP-competitive p97 inhibitor, CB-5083, has entered clinical trials. Selective pressure on HCT116 cells dosed with CB-5083 identified five different resistant mutants. Identification of p97 inhibitors with different mechanisms of action would offer the potential to overcome this class of resistance mutations. Our results demonstrate that two CB-5083 resistant p97 mutants, N660â K and T688â A, were also resistant to several other ATP-competitive p97 inhibitors, whereas inhibition by two allosteric p97 inhibitors NMS-873 and UPCDC-30245 were unaffected by these mutations. We also established a CB-5083 resistant cell line that harbors a new p97 double mutation (D649â A/T688â A). While CB-5083, NMS-873, and UPCDC-30245 all effectively inhibited proliferation of the parental HCT116â cell line, NMS-873 and UPCDC-30245 were 30-fold more potent in inhibiting the CB-5083 resistant D649â A/T688â A double mutant than CB-5083. Our results suggest that allosteric p97 inhibitors are promising alternatives when resistance to ATP-competitive p97 inhibitors arises during anticancer treatment.
Subject(s)
Acetanilides/pharmacology , Adenosine Triphosphatases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Benzothiazoles/pharmacology , Enzyme Inhibitors/pharmacology , Indoles/pharmacology , Nuclear Proteins/antagonists & inhibitors , Pyrimidines/pharmacology , Acetanilides/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Allosteric Regulation/drug effects , Antineoplastic Agents/chemistry , Benzothiazoles/chemistry , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm/drug effects , Drug Screening Assays, Antitumor , Enzyme Inhibitors/chemistry , HCT116 Cells , Humans , Indoles/chemistry , Models, Molecular , Molecular Structure , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Pyrimidines/chemistryABSTRACT
Valosin-containing protein (VCP), together with several partner proteins, extracts ubiquitinated client proteins from E3 ligase complex and facilitates their degradation through ubiquitin-proteasome system. Therefore, it plays an important role in regulating protein quality control and various cellular pathways. Recent studies also identified VCP as a lineage-specific essential gene in ovarian cancer. An orally bioavailable VCP inhibitor, CB-5083, is currently in Phase I clinical trials because it shows therapeutic effects in multiple tumor xenograft models. However, the mechanism of resistance to CB-5083 is unknown. Here, we characterized molecular mechanism of resistance to CB-5083. Using incremental exposure to CB-5083, we established CB-5083-resistant ovarian cancer cells that showed five- to six-fold resistance in vitro compared with parental cells. Genomic and complementary DNA sequencing of the VCP coding region revealed a pattern of co-selected mutations: (1) missense mutations at codon 470 in one copy resulting in increased ATPase activity and (2) nonsense or frameshift mutations at codon 606 or codon 616 in another copy causing the loss of allele-specific expression. Unbiased molecular docking studies showed codon 470 as a putative binding site for CB-5083. Furthermore, the analysis of somatic mutations in cancer genomes from the Cancer Genome Atlas (TCGA) indicated that codon 616 contains hotspot mutations in VCP. Thus, identification of these mutations associated with in vitro resistance to VCP inhibitors may be useful as potential theranostic markers while screening for patients to enroll in clinical trials. VCP has emerged as a viable therapeutic target for several cancer types, and therefore targeting such hyperactive VCP mutants should aid in improving the therapeutic outcome in cancer patients.
ABSTRACT
Early metastasis is still the most recalcitrant factor in the treatment of lung cancer patients. By analyzing the structures and comparing the docking scores of the known pharmacophores, the authors of this paper designed 5-(bis(3-(2-hydroxyethyl)-1H-indol-2-yl)methyl)-2-hydroxybenzoic acid (BHIMHA) as a promising lead compound to develop metastasis inhibitors. In vitro 5, 10, and 20 µM of BHIMHA concentration dependently inhibited the migration and invasion of A549 cells. In vivo 0.4, 2.0, and 8.9 µmol/kg of BHIMHA dose dependently inhibited the metastasis of LLC (Lewis Lung Carcinoma) toward lung. In vivo, 2 µmol/kg of BHIMHA showed additional actions of slowing the growth of the primary tumor of C57BL/6 mice and S180 mice as well as inhibiting xylene-induced ear edema of the mice. Therefore, BHIMHA simultaneously blocked tumor metastasis toward lung, slowed the primary tumor growth, and limited the inflammation. These pharmacological actions were correlated with the inhibition of PKCα and NF-κB expression.
Subject(s)
Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Lung Neoplasms/drug therapy , Protein Kinase C-alpha/antagonists & inhibitors , Salicylates/chemical synthesis , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Design , Humans , Indoles/chemistry , Indoles/pharmacology , Lung Neoplasms/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Inbred ICR , NF-kappa B/physiology , Neoplasm Metastasis/prevention & control , Protein Kinase C-alpha/chemistry , Salicylates/chemistry , Salicylates/pharmacologyABSTRACT
Vascular thrombosis is a major risk of the onset of stroke and so novel therapeutic candidates have been attracting interest. In this context, here docking based computer assisted screening and mesoscale simulation were used to design N-[(S)-6,7-dihydroxy-1,1-dimethyl-1,2,3,4-tetrahydroisoquinoline-3-carbonyl]-Lys(Pro-Ala-Lys), DHDMIQK(KAP), for inhibiting P-selectin expression. In vitro, 1 nM of DHDMIQK(KAP) effectively down-regulated P-selectin expression. In water, in rat plasma and in the solid state DHDMIQK(KAP) formed nanoparticles of a size capable of suitable delivery in the blood circulation. FT-MS and NOESY 2D NMR spectra showed DHDMIQK(KAP) formed hexamers, identified the intermolecular interactions of the hexamer, and assigned the hexamer a butterfly like conformation. Transmission electron microscopy, scanning electron microscopy and atomic force microscopy (AFM) imaged DHDMIQK(KAP) forming size-suitable nanoparticles for safe delivery in the blood circulation. In particular, AFM images showed that the nanoparticles effectively adhered onto the surfaces of the platelets. In vivo DHDMIQK(KAP) lysed the thrombus and inhibited thrombosis with a minimal effective dose of 0.01 nmol kg-1. FT-MS spectrum analyses defined a specific distribution of DHDMIQK(KAP) in the thrombus, but not in the blood and vital organs. Therefore, DHDMIQK(KAP) should be a novel nano-delivery system of 6,7-dihydroxyl-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid and KPAK to target the thrombus.
