28 research outputs found
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Small-molecule and mutational analysis of allosteric Eg5 inhibition by monastrol
BACKGROUND: A recent crystal structure of monastrol in a ternary complex with the kinesin Eg5 motor domain highlights a novel, induced-fit drug binding site at atomic resolution. Mutational obliteration of the monastrol binding site results in a monastrol-resistant, but otherwise catalytically active Eg5 motor domain. However, considering the conformational changes at this site, it is unclear what specific interactions stabilize the interaction between monastrol and the Eg5 motor domain. RESULTS: To study the molecular complementarity of the monastrol-Eg5 interaction, we used a combination of synthetic chemistry and targeted mutations in Eg5 to measure the contribution of specific contacts to inhibition of Eg5 in vitro and in cultured cells. Structure-activity data on chemical derivatives, sequence analysis of Eg5 homologs from different species, and the effect of mutations near the drug binding site were consistent with the crystal structure. CONCLUSION: The mechanism of monastrol revealed by our data rationalizes its specificity for Eg5 over other kinesins and highlights a potential mechanism of drug resistance for anti-cancer therapy targeting this site in Eg5
Immunogenomic profiling determines responses to combined PARP and PD-1 inhibition in ovarian cancer
Combined PARP and immune checkpoint inhibition has yielded encouraging results in ovarian cancer, but predictive biomarkers are lacking. We performed immunogenomic profiling and highly multiplexed single-cell imaging on tumor samples from patients enrolled in a Phase I/II trial of niraparib and pembrolizumab in ovarian cancer (NCT02657889). We identify two determinants of response; mutational signature 3 reflecting defective homologous recombination DNA repair, and positive immune score as a surrogate of interferon-primed exhausted CD8+T-cells in the tumor microenvironment. Presence of one or both features associates with an improved outcome while concurrent absence yields no responses. Single-cell spatial analysis reveals prominent interactions of exhausted CD8+T-cells and PD-L1+macrophages and PD-L1+tumor cells as mechanistic determinants of response. Furthermore, spatial analysis of two extreme responders shows differential clustering of exhausted CD8+T-cells with PD-L1+macrophages in the first, and exhausted CD8+T-cells with cancer cells harboring genomic PD-L1 and PD-L2 amplification in the second. A Phase I/II trial previously revealed variable anti-tumor efficacy of the PARP inhibitor niraparib in combination with the PD-1 inhibitor pembrolizumab in platinum-resistant ovarian cancer patients. Here, the authors perform an integrated genomic and immunomics analysis of tumor samples from the same patients and find potential predictive biomarkers of response to such combination therapy.Peer reviewe
Phase Transitions Drive the Formation of Vesicular Stomatitis Virus Replication Compartments
RNA viruses compartmentalize their replication machinery to evade detection by host pattern recognition receptors and concentrate the machinery of RNA synthesis. For positive-strand RNA viruses, RNA replication occurs in a virus-induced membrane-associated replication organelle. For NNS RNA viruses, the replication compartment is a cytoplasmic inclusion that is not circumscribed by a cellular membrane. Such structures were first observed in the cell bodies of neurons from humans infected with rabies virus and were termed Negri bodies. How the replication machinery that forms this inclusion remains associated in the absence of a membrane has been an enduring mystery. In this article, we present evidence that the VSV replication compartments form through phase separation. Phase separation is increasingly recognized as responsible for cellular structures as diverse as processing bodies (P-bodies) and nucleoli and was recently demonstrated for rabies virus. This article further links the fields of host-pathogen interaction with that of phase separation.RNA viruses that replicate in the cell cytoplasm typically concentrate their replication machinery within specialized compartments. This concentration favors enzymatic reactions and shields viral RNA from detection by cytosolic pattern recognition receptors. Nonsegmented negative-strand (NNS) RNA viruses, which include some of the most significant human, animal, and plant pathogens extant, form inclusions that are sites of RNA synthesis and are not circumscribed by a membrane. These inclusions share similarities with cellular protein/RNA structures such as P granules and nucleoli, which are phase-separated liquid compartments. Here we show that replication compartments of vesicular stomatitis virus (VSV) have the properties of liquid-like compartments that form by phase separation. Expression of the individual viral components of the replication machinery in cells demonstrates that the 3 viral proteins required for replication are sufficient to drive cytoplasmic phase separation. Therefore, liquid-liquid phase separation, previously linked to organization of P granules, nucleolus homeostasis, and cell signaling, plays a key role in host-pathogen interactions. This work suggests novel therapeutic approaches to the problem of combating NNS RNA viral infections
Hydrolysis of 2′3′-cGAMP by ENPP1 and design of non-hydrolyzable analogs
Agonists of mouse STING (TMEM173) shrink and even cure solid tumor by activating innate immunity; human STING agonists are needed to test this therapeutic hypothesis in man. The endogenous STING agonist is 2′3′-cGAMP, a 2nd messenger that signals the presence of cytosolic dsDNA. We report activity-guided partial purification and identification of ENPP1 as the dominant 2′3′-cGAMP hydrolyzing activity in cultured cells. The hydrolysis activity of ENPP1 was confirmed using recombinant protein and was depleted in tissue extracts and plasma from Enpp1-/- mice. We synthesized a hydrolysis-resistant bis-phosphothioate analog of 2′3′-cGAMP (2′3′-cGsAsMP) with similar affinity for human STING in vitro and 10 times more potent at inducing IFN-β secretion from human THP1 monocytes. Studies in mouse Enpp1-/- lung fibroblasts indicate that resistance to hydrolysis contributes significantly to its higher potency. 2′3′-cGsAsMP is therefore improved over natural 2′3′-cGAMP as a model agonist, and has potential as a vaccine adjuvant and cancer therapeutic
Erratum: Corrigendum: Hydrolysis of 2′3′-cGAMP by ENPP1 and design of nonhydrolyzable analogs
Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI
Treatment of atherosclerotic disease often focuses
on reducing plasma LDL-cholesterol or increasing
plasma HDL-cholesterol. We examined in vitro the effects
on HDL receptor [scavenger receptor class B type I (SR-BI)]
activity of three classes of clinical and experimental plasma
HDL-cholesterol-elevating compounds: niacin, fibrates, and
HDL376. Fenofibrate (FF) and HDL376 were potent (IC50
? 1 mM), direct inhibitors of SR-BI-mediated lipid transport
in cells and in liposomes reconstituted with purified SR-BI.
FF, a prodrug, was a more potent inhibitor of SR-BI than an
activator of peroxisome proliferator-activated receptor a, a
target of its active fenofibric acid (FFA) derivative. Nevertheless,
FFA, four other fibrates (clofibrate, gemfibrozil,
ciprofibrate, and bezafibrate), and niacin had little, if any,
effect on SR-BI, suggesting that they do not directly target
SR-BI in vivo. However, similarities of HDL376 treatment
and SR-BI gene knockout on HDL metabolism in vivo
(increased HDL-cholesterol and HDL particle sizes) and
structure-activity relationship analysis suggest that SR-BI
may be a target of HDL376 in vivo. HDL376 and other
inhibitors may help elucidate SR-BI function in diverse
mammalian models and determine the therapeutic potential
of SR-BI-directed pharmaceuticals.—Nieland, T. J. F.,
J. T. Shaw, F. A. Jaipuri, Z. Maliga, J. L. Duffner, A. N.
Koehler, and M. Krieger. Influence of HDL-cholesterolelevating
drugs on the in vitro activity of the HDL receptor
SR-BI