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Endogenous Digitalis-Like Factors: Past Progress and Future Prospects
Published in Antonio Coca, Ricardo P. Garay, Ionic Transport in Hypertension: New Perspectives, 2019
A unique approach to delineating a role for a digitalis-like factor in blood pressure regulation was presented by Graves et al.77 A potential complication of bone marrow transplants and alkylating agent therapy is severe hypertension. This is a unique model because patients can be followed from normo- to hypertension. The changes in mean arterial pressure produced by the treatment could be significantly and positively correlated with the changes in circulating digoxin-like im-munoactivity. In addition, the immunoassay in patients was confirmed by a Na,K-ATPase assay, with a highly significant correlation. These data comprise the first longitudinal analysis of digitalis-like activity in hypertension, and strongly suggest that a causal relationship is present.
Muscle Fiber Types
Published in Charles Paul Lambert, Physiology and Nutrition for Amateur Wrestling, 2020
There are three major fiber types: Slow Twitch (Type I), Fast Twitch Oxidative Glycolytic (FOG) (Type IIa), and Fast Twitch Glycolytic (Type IIb) fibers. This is via staining by way of the Myosin ATPase assay (Padykula and Herman 1955; Dubowitz and Neville 1973). The myosin corresponding to these fiber types is actually on a continuum meaning that the Myosin ATPase assay is a way to force them into categories. More recent analyses include gel electrophoresis, and clearly there is a continuum from slow to fast rather than distinct delineations. Nonetheless, the Myosin ATPase method of classification has stood the test time. Slow twitch fibers have low contraction velocity, low force, and generally not very fatigable. FOG fibers have high contraction velocity, high force, and low to moderate fatigability. Fast twitch glycolytic fibers have a high contraction velocity, high force, and high fatigability. Typically, during exercise of increasing intensity, slow twitch fibers are recruited first, followed by FOG, and by fast twitch glycolytic fibers. Training can result in a fast twitch glycolytic fiber becoming a FOG fiber. Under situations of extreme endurance training a FOG fiber can become a slow twitch fiber. Based on the sum toto of all the data it does not appear that a slow twitch fiber can become a FOG fiber or a fast twitch glycolytic fiber although there have been scant reports of this occurrence (Jansson et al. 1990). Generally speaking, endurance training will increase the number of capillaries in skeletal muscle capillary beds that are used, increase the number and function of mitochondria, and increase the amount of oxidative enzymes in the skeletal muscle. These adaptations will improve arteriovenous oxygen difference one of the components of VO2max/peak (Wilmore and Costill 1994) (Table 8.1).
The Application of Fragment-based Approaches to the Discovery of Drugs for Neglected Tropical Diseases
Published in Venkatesan Jayaprakash, Daniele Castagnolo, Yusuf Özkay, Medicinal Chemistry of Neglected and Tropical Diseases, 2019
Christina Spry, Anthony G. Coyne
In an attempt to identify novel binding sites and/or starting points for inhibitor development, a library of 500 rule-of-three compliant fragments was screened against each of the target proteins using DSF. In this primary screen, in which fragments were tested at a concentration of 2 mM, 36 and 32 fragments increased the melting temperature of Hel (from DENV-4) and MTase (from DENV-3) by > 0.5°C, respectively, consistent with binding. These fragments were therefore progressed to X-ray crystallography experiments for hit confirmation and binding mode determination. Unfortunately, in the case of Hel, crystal-soaking experiments did not produce a single fragment co-crystal structure and as such none of the fragment hits were confirmed by X-ray crystallography. By contrast, a total of seven fragments were observed by X-ray crystallography to bind S-adenosyl-l-methionine (SAM)-bound DENV-3 MTase. Furthermore, the seven fragments were found to bind at four distinct sites—one fragment was observed to bind in a site overlapping with the GTP binding site, while the remaining six fragments bind at one of three novel binding sites. The inhibitory effect of the MTase fragment hits on DENV-3 MTase activity was also determined and five of the seven X-ray crystallography-confirmed hits were found to inhibit 2’-O-MTase activity with IC50 values between 180 μM and 9 mM, with the most active 2’-O-MTase inhibitor also inhibiting N7-MTase activity (IC50 = 2 mM). Notably, the fragment binding at the GTP-binding site showed little-to-no inhibition of MTase activity. Hel and MTase inhibitors were also found among Hel and MTase fragment hits that did not yield crystal structures in soaking experiments (or, where attempted, co-crystallization experiments); however, because detailed knowledge of a fragment’s binding mode is important for fragment elaboration, these fragments were not progressed. The failure to obtain any Hel-fragment co-crystal structures despite some of the DSF fragment hits possessing weak inhibitory activity against the enzyme in an ATPase assay, was hypothesized to be a consequence of the fragment hits inducing a conformational change in the protein that cannot occur in the crystal; previous studies had shown the protein to be highly dynamic (Luo et al. 2008).
Discovery of a novel Aurora B inhibitor GSK650394 with potent anticancer and anti-aspergillus fumigatus dual efficacies in vitro
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2022
Yuhua He, Wei Fu, Liyang Du, Huiqiao Yao, Zhengkang Hua, Jinyu Li, Zhonghui Lin
To obtain active enzyme, the Hs.Aurora B kinase domain (aa.70–344) was co-expressed in E. coli cells with a fragment of Hs.INCENP (aa.835–904). The human Aurora B/INCENP complex, hereafter referred to as Aurora B for short, was purified and its kinase activity was confirmed towards the phosphorylation of histone H3 at Ser10 (Figure S1). Importantly, we found that Aurora B can hydrolyse ATP in the absence of peptide or protein substrates by luciferase-based luminescence assay (Figure 1(a)), suggesting that it also possesses an intrinsic ATPase activity. With this ATPase assay, we next carried out a small-molecule inhibitor screen with the bioactive compound library purchased from TopScience (Shanghai China). Among about 8,000 compounds, we identified a compound GSK650394 (Figure 1(b)) that selectively inhibits the ATPase activity of Aurora B, with an IC50 of 5.68 µM (Figure 1(c)), but only weakly inhibits another important mitotic kinase Haspin (IC50=41.4 µM, Figure S2). The results of in vitro kinase assay shows that this compound can also diminish the phosphorylation of histone H3 Ser10 by Aurora B (Figure 2(d)). Together, these results suggest that GSK650394 is a novel Aurora B inhibitor.
Challenges with the precise prediction of ABC-transporter interactions for improved drug discovery
Published in Expert Opinion on Drug Discovery, 2018
In vitro assays are based on cell membranes (ATPase, vesicles) or intact cells (accumulation, bidirectional) expressing the transporter protein of interest [32,33]. ATPase assays are performed with isolated inverted vesicles or membranes from cells expressing the transporter. Since the efflux of compounds by ABC-efflux transporters requires ATP, the rate of ATP cleavage to ADP is applied to determine whether a drug interacts with ATP. The amount of ATP cleaved is quantified by measuring the amount of inorganic phosphate generated in the reaction. Positive results in the assay only indicate that a drug interacts with the transporter without determining whether the drug is binding to the transporter or is a substrate or inhibitor of the transporter. The ATPase assay is suitable for early discovery stages to screen a large number of compounds as it is relatively inexpensive, high throughput, and less labor intensive than other assays. However, this model experiences high intra- and inter-assay variability, lack of concentration-dependent effect, and high rates of false-positives [32,33].
M2 macrophage-derived exosomes suppress tumor intrinsic immunogenicity to confer immunotherapy resistance
Published in OncoImmunology, 2023
Naisheng Zheng, Tingting Wang, Qin Luo, Yi Liu, Junyao Yang, Yunlan Zhou, Guohua Xie, Yanhui Ma, Xiangliang Yuan, Lisong Shen
ATPase Assay Colorimetric kits were used for experiments (ab234055, Abcam). Briefly, reaction samples were prepared for each 10 min time point between 0 and 60 min. BiP (ab78432, Abcam) at 10 μM concentration was used in all experiments, to which 10 μM of ApoE (ab55210, Abcam) or 50 μM EZ-482 was added for 4°C overnight incubation. Then add ATPase Assay Developer to all samples and measure OD at 650 nm in endpoint mode according to the manufacturer’s protocol.