Accurately predicting the binding affinities of small organic molecules to biological macro-molecules can greatly accelerate drug discovery by reducing the number of compounds that must be synthesized to realize desired potency and selectivity goals. Unfortunately, the process of assessing the accuracy of current computational approaches to affinity prediction against binding data to biological macro-molecules is frustrated by several challenges, such as slow conformational dynamics, multiple titratable groups, and the lack of high-quality blinded datasets. Over the last several SAMPL blind challenge exercises, host-guest systems have emerged as a practical and effective way to circumvent these challenges in assessing the predictive performance of current-generation quantitative modeling tools, while still providing systems capable of possessing tight binding affinities. Here, we present an overview of the SAMPL6 host-guest binding affinity prediction challenge, which featured three supramolecular hosts: octa-acid (OA), the closely related tetra-endo-methyl-octa-acid (TEMOA), and cucurbit[8]uril (CB8), along with 21 small organic guest molecules. A total of 119 entries were received from 10 participating groups employing a variety of methods that spanned from electronic structure and movable type calculations in implicit solvent to alchemical and potential of mean force strategies using empirical force fields with explicit solvent models. While empirical models tended to obtain better performance than first-principle methods, it was not possible to identify a single approach that consistently provided superior results across all host-guest systems and statistical metrics. Moreover, the accuracy of the methodologies generally displayed a substantial dependence on the system considered, emphasizing the need for host diversity in blind evaluations. Several entries exploited previous experimental measurements of similar host-guest systems in an effort to improve their physical-based predictions via some manner of rudimentary machine learning; while this strategy succeeded in reducing systematic errors, it did not correspond to an improvement in statistical correlation. Comparison to previous rounds of the host-guest binding free energy challenge highlights an overall improvement in the correlation obtained by the affinity predictions for OA and TEMOA systems, but a surprising lack of improvement regarding root mean square error over the past several challenge rounds. The data suggests that further refinement of force field parameters, as well as improved treatment of chemical effects (e.g., buffer salt conditions, protonation states) may be required to further enhance predictive accuracy.
Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca2+-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca2+ signals representing Ca2+ influx through single TRPA1 channels, which we called “TRPA1 sparklets.” TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca2+ entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca2+ channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca2+-sensitive K+ channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca2+ influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.
The analysis of damage products as biomarkers of inflammation has been hampered by a poor understanding of the chemical biology of inflammation, the lack of sensitive analytical methods, and a focus on single chemicals as surrogates for inflammation. To overcome these problems, we developed a general and sensitive liquid chromatographic tandem mass spectrometry (LC/MS-MS) method to quantify, in a single DNA sample, the nucleoside forms of seven DNA lesions reflecting the range of chemistries associated with inflammation: 2′-deoxyuridine, 2′-deoxyxanthosine, and 2′-deoxyinosine from nitrosative deamination; 8-oxo-2′-deoxyguanosine from oxidation; and 1,N 2 -etheno-2′-deoxyguanosine, 1,N 6 -etheno-2′-deoxyadenosine, and 3,N 4 -etheno-2′-deoxycytidine arising from reaction of DNA with lipid peroxidation products. Using DNA purified from cells or tissues under conditions that minimize artifacts, individual nucleosides are purified by HPLC and quantified by isotope-dilution, electrospray ionization LC/MS-MS. The method can be applied to other DNA damage products and requires 4-6 days to complete depending upon the number of samples. Search terms
The (pro)renin receptor, which binds both renin and prorenin, is a newly discovered component of the renin angiotensin system that is highly expressed in the central nervous system. The significance of brain PRRs in mediating local angiotensin II formation and regulating blood pressure remains unclear. The current study was performed to test the hypothesis that PRR-mediated, non-proteolytic activation of prorenin is the main source of angiotensin II in the brain. Thus, PRR knockout in the brain is expected to prevent angiotensin II formation and development of deoxycorticosterone acetate salt induced hypertension. A neuron-specific PRR (ATP6AP2) knockout mouse model was generated using the Cre-LoxP system. Physiological parameters were recorded by telemetry. (Pro)renin receptor expression, detected by immunostaining and RT-PCR, was significantly decreased in the brains of knockout compared with wide-type mice. Intracerebroventricular infusion of mouse prorenin increased blood pressure and angiotensin II formation in wild type mice. This hypertensive response was abolished in (pro)renin receptor knockout mice in association with a reduction in angiotensin II levels. Deoxycorticosterone acetate salt increased (pro)renin receptor expression and angiotensin II formation in the brains of wild-type mice, an effect that was attenuated in (pro)renin receptor knockout mice. (Pro)renin receptor knockout in neurons prevented the development of Deoxycorticosterone acetate salt-induced hypertension as well as activation of cardiac and vasomotor sympathetic tone. In conclusion, non-proteolytic activation of prorenin through binding to the PRR mediates angiotensin II formation in the brain. Neuron-specific PRR knockout prevents the development of deoxycorticosterone acetate salt-induced hypertension, possibly through diminished angiotensin II formation.
Maintaining constant blood flow in the face of fluctuations in blood pressure is a critical autoregulatory feature of cerebral arteries. An increase in pressure within the artery lumen causes the vessel to constrict through depolarization and contraction of the encircling smooth muscle cells. This pressure-sensing mechanism involves activation of two types of transient receptor potential (TRP) channels: TRPC6 and TRPM4. We provide evidence that the activation of the γ1 isoform of phospholipase C (PLCγ1) is critical for pressure sensing in cerebral arteries. Inositol 1,4,5-trisphosphate (IP3), generated by PLCγ1 in response to pressure, sensitized IP3 receptors (IP3Rs) to Ca2+ influx mediated by the mechanosensitive TRPC6 channel, synergistically increasing IP3R-mediated Ca2+ release to activate TRPM4 currents, leading to smooth muscle depolarization and constriction of isolated cerebral arteries. Proximity ligation assays demonstrated colocalization of PLCγ1 and TRPC6 with TRPM4, suggesting the presence of a force-sensitive, local signaling network comprising PLCγ1, TRPC6, TRPM4, and IP3Rs. Src tyrosine kinase activity was necessary for stretch-induced TRPM4 activation and myogenic constriction, consistent with the ability of Src to activate PLCγ isoforms. We conclude that contraction of cerebral artery smooth muscle cells requires the integration of pressure-sensing signaling pathways and their convergence on IP3Rs, which mediate localized Ca2+-dependent depolarization through the activation of TRPM4.
Deamination of nucleobases in DNA and RNA results in the formation of xanthine (X), hypoxanthine (I), oxanine, and uracil, all of which are miscoding and mutagenic in DNA and can interfere with RNA editing and function. Among many forms of nucleic acid damage, deamination arises from several unrelated mechanisms, including hydrolysis, nitrosative chemistry, and deaminase enzymes. Here we present a fourth mechanism contributing to the burden of nucleobase deamination: incorporation of hypoxanthine and xanthine into DNA and RNA caused by defects in purine nucleotide metabolism. Using Escherichia coli and Saccharomyces cerevisiae with defined mutations in purine metabolism in conjunction with analytical methods for quantifying deaminated nucleobases in DNA and RNA, we observed large increases (up to 600-fold) in hypoxanthine in both DNA and RNA in cells unable to convert IMP to XMP or AMP (IMP dehydrogenase, guaB; adenylosuccinate synthetase, purA, and ADE12), and unable to remove dITP/ITP and dXTP/XTP from the nucleotide pool (dITP/XTP pyrophosphohydrolase, rdgB and HAM1). Conversely, modest changes in xanthine levels were observed in RNA (but not DNA) from E. coli lacking purA and rdgB and the enzyme converting XMP to GMP (GMP synthetase, guaA). These observations suggest that disturbances in purine metabolism caused by known genetic polymorphisms could increase the burden of mutagenic deaminated nucleobases in DNA and interfere with gene expression and RNA function, a situation possibly exacerbated by the nitrosative stress of concurrent inflammation. The results also suggest a mechanistic basis for the pathophysiology of human inborn errors of purine nucleotide metabolism.DNA and RNA damage | mass spectrometry | nucleobase deamination | purine metabolism | DNA repair T he chemical modification of nucleobases in DNA and RNA can arise from both physiological and adventitious mechanisms at all stages of nucleic acid metabolism. This is particularly true for deaminated versions of the nucleobases. As shown in Fig. 1 for purines, nucleobase deamination in DNA and RNA leads to the formation of 2′-deoxy-and ribonucleoside forms of hypoxanthine (2′-deoxyinosine, dI; inosine, Ino) from adenine, xanthine (2′-deoxyxanthosine, dX; xanthosine, Xao) and oxanine (2′-deoxyoxanosine, dO; oxanosine, Oxo) from guanine, and uracil (2′-deoxyuridine, dU; uridine, Urd) from cytosine (1). All of these products are miscoding and mutagenic in DNA (2-4) and can interfere with RNA editing (5) and the function of noncoding RNAs (6).There are three recognized mechanisms that contribute to nucleobase deamination in DNA and RNA, the simplest of which is hydrolysis (7). A second source of nucleobase deamination is associated with the nitrosative stress caused by increases in nitric oxide-derived nitrous anhydride during inflammation (1). A third mechanism is associated with deaminase enzymes acting on RNA and DNA, with activation-induced cytidine deaminase converting cytidine to uridine during immunoglobulin diversification in B lymphocytes an...
We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT1 receptor–dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension.
A complete understanding of the transcriptional regulation of developmental lineages requires that all relevant factors be identified. Here, we have taken a proteomic approach to identify novel proteins associated with GATA-1, a lineage-restricted zinc finger transcription factor required for terminal erythroid and megakaryocytic maturation. We identify the Krüppel-type zinc finger transcription factor ZBP-89 as being a component of multiprotein complexes involving GATA-1 and its essential cofactor Friend of GATA-1 (FOG-1). Using chromatin immunoprecipitation assays, we show that GATA-1 and ZBP-89 cooccupy cis-regulatory elements of certain erythroid and megakaryocyte-specific genes, including an enhancer of the GATA-1 gene itself. Loss-of-function studies in zebrafish and mice demonstrate an in vivo requirement for ZBP-89 in megakaryopoiesis and definitive erythropoiesis but not primitive erythropoiesis, phenocopying aspects of FOG-1-and GATA-1-deficient animals. These findings identify ZBP-89 as being a novel transcription factor involved in erythroid and megakaryocytic development and suggest that it serves a cooperative function with GATA-1 and/or FOG-1 in a developmental stage-specific manner.Lineage-specific transcription factors play essential roles in development. However, most of these factors have relatively small consensus DNA binding motifs, and by themselves, they are not likely to account for high-fidelity lineage-specific gene expression in higher organisms. Indeed, recent studies employing chromatin immunoprecipitation (IP) (ChIP) across extended loci or entire genomes show that only a small
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.