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Mechanisms of Cholestasis
Published in Robert G. Meeks, Steadman D. Harrison, Richard J. Bull, Hepatotoxicology, 2020
Early studies in the dog showed competitive inhibition of taurocholate uptake by the co-administration of other bile acids (Glasinovic, 1975b). Coadministration of non-bile acid organic anions such as indocyanine green in the rat, however, did not show inhibition (Paumgartner and Reichen, 1975). These data led to the suggestion that distinct carrier systems were responsible for the uptake of the bile acids and the non-bile organic anions. More recent data have shown that the substrate specificities of these carriers is much more complicated. Photoaffinity labeling studies utilizing a photoreactive diazirine derivative of taurocholate, (7,7-azo-3α,12α-dihydroxy-5β-cholan-24-oyl)-2-aminoethanesulfonic acid, identified two bile acid-binding proteins in the basolateral plasma membrane with molecular weights of 54,000 and 48,000 Da (von Dippe and Levy, 1983; Kramer et al., 1982; Wieland et al., 1984). The 48-49-kDa protein has been shown to represent the Na+-dependent bile acid carrier (Ananthanarayanan et al., 1988), whereas the 54-kDa protein is postulated to represent a multispecific Na+-independent organic anion carrier which is shared by taurocholate and organic anions such as bromosulfophthalein (BSP) and bilirubin (Reichen and Berk, 1979; Stremmel et al., 1983; Wolkoff and Chung, 1980; Stremmel and Berk, 1986), the nonconjugated bile acid, cholate, and other inorganic and organic anions such as sulfate, thiosulfate, oxalate, and succinate (Cheng and Levy, 1980; Ziegler et al., 1984; Hugentobler and Meier, 1986).
Affinity Labeling
Published in Roger L. Lundblad, Chemical Reagents for Protein Modification, 2020
The variety of photoactivated reagents is limited only by the imagination of the investigator. The criteria for a suitable photoaffinity reagent are discussed in detail elsewhere85,86 and will only be summarized here. First, the synthesis of the precursor azide, diazo compound, or diazirine should be reasonable and a stable product obtained which can be stored until use. The precursor must be stable under the solution conditions to be used in the photolysis reaction. For example, a reducing environment should be avoided with aryl azides as such a compound can be reduced by dithiothreitol to the corresponding amine.91 Other possibilities with thiol groups include the oxidation of cysteine to cystine during photolysis of an aromatic azide, such as that observed with cytosolic phosphoenolpyruvate carboxykinase and 8-azidoguanosine 5’-triphosphate.92 The active species, either a nitrene or a carbene, must be generated under conditions where the absorption characteristics of the biologic system under study does not present difficulties. An excitation wavelength less than 300 nm should be avoided when working with either proteins or nucleic acids. Photolysis properties of the precursor can be altered by changing the chemistry of the reagent. This is illustrated by the examples in Figure 19 (adapted from Reference 86). The parent compound, phenyl azide (I) absorbs at 250 nm. Changing the chemistry of the ring by addition of a carboxylate function increases the wavelength of maximum absorption to 280. Placement of even strongly electron-withdrawing component, a nitro function, further increases the absorption maximum to approximately 330 nm. The photolysis process should be efficient in that the precursor has a high extinction coefficient with generation of the active species with a reasonably high quantum yield. Finally, the reactive species should have a short lifetime (highly reactive species).
Different chemical proteomic approaches to identify the targets of lapatinib
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2023
Tatjana Kovačević, Krunoslav Nujić, Mario Cindrić, Snježana Dragojević, Adrijana Vinter, Amela Hozić, Milan Mesić
Based on protein-ligand X-ray crystallography data of lapatinib bound to the EGFR kinase domain (PDB: 1xkk)14 we performed computational docking of proposed lapatinib analogues using Glide from the Schrodinger software suite (Figure 1). This demonstrated that all of the proposed lapatinib analogues could bind to EGFR, preserving the original lapatinib binding mode. The compounds 2 and 3 (amine and alkyne bearing analogues) are oriented towards the solvent exposed region of EGFR (Figures 1(a–d)) and we do not expect that they will interfere significantly with the protein. Also, compound 4, which carries a large phenyl group onto which are attached dual tag motifs (diazirine and alkyne at the phenyl ring), could possibly by accommodated in the “solvent-exposed” region (Figures 1(e,f)). Nevertheless, we anticipated that the largest influence on in vitro affinity towards EGFR would be from compound 4.
A patent and literature review of CDK12 inhibitors
Published in Expert Opinion on Therapeutic Patents, 2022
Ruijun Tang, Jing Liu, Shuyao Li, Junjie Zhang, Chunhong Yu, Honglu Liu, Fang Chen, Lu Lv, Qian Zhang, Kai Yuan, Hao Shao
Small-molecule degraders typically function by hijacking CRL E3 ligases. Winter et al. hypothesized that parallel screening of small molecules against cells with wildtype and inactive CRLs could obtain small molecules that functionally depend on CRLs [59]. Most CRLs are activated by the attachment of the ubiquitin-like protein NEDD8 to Cullins by sequential actions of E1 enzyme (NAE), E2s (UBE2M and UBE2F) and E3 enzymes [64]. A KBM7 cell line with inactive CRLs was generated by mutating UBE2M. By screening approximately 2,000 cytostatic/cytotoxic small molecules, several chemical compounds showed differential sensitivities to these two cell lines: 29 (dCeMM2, Figure 5), 30 (dCeMM3) and the close analog 31 (dCeMM3-1), as well as 32 (dCeMM4). Using quantitative proteome-wide mass spectrometry and loss of function CRISPR-cas9 screen, it was found that these molecules shared a similar mechanism of action with (R)-CR8. To validate if the drug-induced degradation of cyclin K via the CRL4B–DDB1 complex was mediated via direct drug engagement, the complex formation was confirmed through affinity proteomics using two dCeMM3 analogs: 33 (Figure 5) with free amine, which can be immobilized on sepharose beads, and 34 (Figure 5) containing a photoactive diazirine moiety and an alkyne group. This conclusion was further supported by enzyme-catalyzed proximity labeling using the biotin ligase miniTurbo.
Off-target identification by chemical proteomics for the understanding of drug side effects
Published in Expert Review of Proteomics, 2020
ABPP uses activity-based probes (ABPs) to covalently react with the catalytic residues of specific enzyme families from complex proteomes. A functional ABP usually consists of three components, a reactive group (also called warhead) that can covalently bind to the active sites of target proteins, a reporter tag (such as fluorophore, biotin, or alkyne) for the downstream purification and detection of target proteins, a linker (such as PEG, alkyl, or peptide) which connects the reactive group and tag as well as avoids steric hindrance [4]. According to the different characteristics of reactive groups, ABPs can be used to target different types of enzymes. For example, the fluorophosphonate (FP) probe has been well developed to capture serine hydrolases, while the epoxide electrophile probe has been designed for the binding of cysteine proteases [5]. However, the commonly used biotin or fluorophore tags are bulky and membrane impermeable, these ABPP approaches are unable to detect the small molecule–target interactions in situ. To this end, a two-step tag-then-capture approach called click chemistry-ABPP (CC-ABPP) has been developed, which is based on the copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC), a typical bioorthogonal ligation. Using this two-step post-labeling CC-ABPP, target proteins can be probed in living cells, the CuAAC reaction can then be carried out in cell lysates [6]. In addition to covalent binding, ABPP can also investigate noncovalent interactions between small molecule and their targets through photo-affinity labeling (PAL), in which benzophenone, aryl azide, or diazirine is applied as a photocrosslinker [7].