ABSTRACT
Replication Protein A (RPA), the major eukaryotic single stranded DNA-binding protein, binds to exposed ssDNA to protect it from nucleases, participates in a myriad of nucleic acid transactions and coordinates the recruitment of other important players. RPA is a heterotrimer and coats long stretches of single-stranded DNA (ssDNA). The precise molecular architecture of the RPA subunits and its DNA binding domains (DBDs) during assembly is poorly understood. Using cryo electron microscopy we obtained a 3D reconstruction of the RPA trimerisation core bound with ssDNA (â¼55 kDa) at â¼4.7 Å resolution and a dimeric RPA assembly on ssDNA. FRET-based solution studies reveal dynamic rearrangements of DBDs during coordinated RPA binding and this activity is regulated by phosphorylation at S178 in RPA70. We present a structural model on how dynamic DBDs promote the cooperative assembly of multiple RPAs on long ssDNA.
Subject(s)
DNA, Single-Stranded/metabolism , Models, Structural , Replication Protein A/metabolism , Cryoelectron Microscopy , Escherichia coli , Fluorescence Resonance Energy Transfer , Protein Domains , Protein Multimerization , Replication Protein A/genetics , Saccharomyces cerevisiaeABSTRACT
Ex vivo systems that incorporate features of the tumor microenvironment and model the dynamic response to immune checkpoint blockade (ICB) may facilitate efforts in precision immuno-oncology and the development of effective combination therapies. Here, we demonstrate the ability to interrogate ex vivo response to ICB using murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS). MDOTS/PDOTS isolated from mouse and human tumors retain autologous lymphoid and myeloid cell populations and respond to ICB in short-term three-dimensional microfluidic culture. Response and resistance to ICB was recapitulated using MDOTS derived from established immunocompetent mouse tumor models. MDOTS profiling demonstrated that TBK1/IKKε inhibition enhanced response to PD-1 blockade, which effectively predicted tumor response in vivo Systematic profiling of secreted cytokines in PDOTS captured key features associated with response and resistance to PD-1 blockade. Thus, MDOTS/PDOTS profiling represents a novel platform to evaluate ICB using established murine models as well as clinically relevant patient specimens.Significance: Resistance to PD-1 blockade remains a challenge for many patients, and biomarkers to guide treatment are lacking. Here, we demonstrate feasibility of ex vivo profiling of PD-1 blockade to interrogate the tumor immune microenvironment, develop therapeutic combinations, and facilitate precision immuno-oncology efforts. Cancer Discov; 8(2); 196-215. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Deng et al., p. 216This article is highlighted in the In This Issue feature, p. 127.
Subject(s)
Antineoplastic Agents, Immunological/pharmacology , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Animals , Cell Culture Techniques , Cell Line, Tumor , Cytokines/metabolism , Drug Resistance, Neoplasm , Flow Cytometry , Humans , Immunohistochemistry , Immunophenotyping , Mice , Microfluidic Analytical Techniques , Programmed Cell Death 1 Receptor/metabolism , Spheroids, Cellular , Time-Lapse Imaging , Tumor Cells, CulturedABSTRACT
Phosphoinositide 3-kinase (PI3K) ß signaling is required to sustain cancer cell growth in which the tumor suppressor phosphatase and tensin homolog (PTEN) has been deactivated. This manuscript describes the discovery, optimization, and in vivo evaluation of a novel series of PI3Kß/δ inhibitors in which PI3Kß potency was built in a PI3Kδ-selective template. This work led to the discovery of a highly selective PI3Kß/δ inhibitor displaying excellent pharmacokinetic profile and efficacy in a human PTEN-deficient LNCaP prostate carcinoma xenograft tumor model.
Subject(s)
PTEN Phosphohydrolase/genetics , Phosphoinositide-3 Kinase Inhibitors , Prostatic Neoplasms/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/therapeutic use , Animals , Cell Line, Tumor , Class Ia Phosphatidylinositol 3-Kinase/metabolism , Dogs , Haplorhini , Humans , Male , Mice , Models, Molecular , Prostate/drug effects , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Rats , Rats, Sprague-DawleyABSTRACT
Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) is an appealing target for several hematological malignancies and inflammatory diseases. Herein, we describe the discovery and optimization of a series of propeller shaped PI3Kδ inhibitors comprising a novel triaminopyrimidine hinge binder. Combinations of electronic and structural strategies were employed to mitigate aldehyde oxidase mediated metabolism. This medicinal chemistry effort culminated in the identification of 52, a potent and highly selective inhibitor of PI3Kδ that demonstrates efficacy in a rat model of arthritis.
Subject(s)
Arthritis, Experimental/drug therapy , Class I Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , Quinazolinones/pharmacology , Animals , Arthritis, Experimental/chemically induced , Arthritis, Experimental/enzymology , B-Lymphocytes/cytology , B-Lymphocytes/drug effects , B-Lymphocytes/enzymology , Cells, Cultured , Collagen/toxicity , Crystallography, X-Ray , Disease Models, Animal , Female , Hepatocytes/drug effects , Hepatocytes/enzymology , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Models, Molecular , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrimidines/pharmacokinetics , Quinazolinones/chemistry , Quinazolinones/pharmacokinetics , Rats , Rats, Inbred Lew , Tissue DistributionABSTRACT
Analogs of hemiasterlin (1) and HTI-286 (2), which contain various aromatic rings in the A segment, were synthesized as potential inhibitors of tubulin polymerization. The structure-activity relationships related to stereo- and regio-chemical effects of substituents on the aromatic ring in the A segment were studied. Analogs, which carry a meta-substituted phenyl ring in the A segment show comparable activity for inhibition of tubulin polymerization to 2, as well as in the cell proliferation assay using KB cells containing P-glycoprotein, compared to those of 1 and 2.