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Intracellular Peptide Turnover: Properties and Physiological Significance of the Major Peptide Hydrolases of Brain Cytosol
Published in Gerard O’Cuinn, Metabolism of Brain Peptides, 2020
Prolyl oligopeptidase is inactivated by inhibitors of both cysteine and serine proteinases. Early studies clearly established this enzyme to be a member of the family of serine proteinases. It is quite sensitive to inactivation by diisopropyl fluorophosphate (DFP) and radiolabelled DFP is incorporated into the enzyme in a 1:1 molar ratio60. The rat brain enzyme is inactivated by DFP at a rate constant greater than that for trypsin56. The titration curve of kcat/Km exhibits a pKa of 6.2 consistent with a serine proteinase57. It is of interest that the enzyme is not inhibited by the serine proteinase inhibitor phenylmethylsulfonyl fluoride48. This likely reflects inaccessibility of this bulky reagent to the active site. Chloromethyl ketones known to alkylate active site histidine residues of serine proteinases also inhibit prolyl oligopeptidase60.
Examination of sulfonamide-based inhibitors of MMP3 using the conditioned media of invasive glioma cells
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Alisha T. Poole, Christopher A. Sitko, Caitlin Le, Christian C. Naus, Bryan M. Hill, Eric A. C. Bushnell, Vincent C. Chen
The leucine-tryptophan (Leu-Trp) backbone, and AP-1 were synthesised. All synthesised compounds were analysed by mass spectrometry (Figure S2). The NFF-3 assay was used to determine the biological inhibition of MMP-3 activity for ilomastat and synthesised compounds, Leu-Trp, and AP-1 relative to untreated controls. NFF-3 is a substrate selective for MMP3 (kcat/Km = 218,000 s−1 M−1), and to a much lesser degree, MMP9 (kcat/Km = 10,000 s−1 M−1)54. Consistent with our MMP3 SDS-PAGE/westernblot datum, the conditioned medium of C6-Cx43 demonstrated robust fluorescence activity compared to low motility C6 cells and unconditioned DMEM18. Linear responses of MMP3 activity was robustly measured from 30 to 300 min. All compounds were tested at a concentration of 50 μM and 100 μM, and compared to determine MMP3 suppression. From Figure 4 concentration dependent inhibition was observed. Here it can be seen that for all compounds the 100 μM was better at inhibiting MMP-3 than the 50 μM concentration. This analysis also demonstrates Leu-Trp backbone can act as a competitive inhibitor. Consistent with this interpretation, as discussed above in the docking study, Leu-Trp was found to ligate the Zn2+ via the Trp and Leu backbone carbonyl O-atoms. Demonstrating the relative contributions of the ZBG in AP-1 and ilomastat, both compounds perform better than Leu-Trp at inhibiting MMP-3.
Advances in detection of hazardous organophosphorus compounds using organophosphorus hydrolase based biosensors
Published in Critical Reviews in Toxicology, 2019
Monika Jain, Priyanka Yadav, Abhijeet Joshi, Prashant Kodgire
Cherny et al. used iterative computational modeling to design variants of OPH which resulted in a 5000-fold increase in catalytic efficiency for detoxification of V-series of nerve agents (VE, VG, VM, VR, and VX) and also was effective for detoxification of G-series of nerve agents (GA, GB, GD, GF, and GV) (Cherny et al. 2013). In another study, diSioudi et al. replaced the histidine at position 254 with arginine (H254R) and the one at position 257 with leucine (H257L) independently, as well as double mutation (H254R/H257L) in OPH enzyme. Interestingly, these mutants of OPH possessed only two metals per dimer, as compared to four per dimer in native OPH. All three of these altered enzymes demonstrated a 2–30-fold increase in substrate specificity (kcat/Km) for demeton S (P─S bond), an analog for the chemical warfare agent VX. Furthermore, two of the altered enzymes, H257L and H254R/H257L, showed an 11- and 18-fold increase, respectively, in specificity for NPPMP, the analog for the chemical warfare agent soman (diSioudi et al. 1999).
Bispecific antibodies for the treatment of hemophilia A
Published in Expert Opinion on Biological Therapy, 2022
Emicizumab (originally designated as ACE910) is a fully humanized, IgG4, recombinant bispecific antibody recognizing factor IX(IXa) and factor X(Xa). The antibody was developed after a vast number of cross-species experiments that have been well described [17–21]. In outline, the studies involved screening a total of 40,000 clones and optimized by analyzing the biological properties of amino acid substitutions spanning 2400 patterns. Immunogenicity was also maximally controlled by in silico prediction [20]. The antibody places FIXa and FX in a spatially suitable position, and thereby accelerates FIXa-catalyzed FX activation. This is based on the fact that the distance between the FIXa- and FX-binding sites in FVIIIa molecule is similar to that between the two antigen-binding sites of human IgG. In addition, antibody binding to the appropriate epitope of FIXa would mimic the allosteric properties of FVIIIa and enhance the catalytic activity of FIXa [17]. Although this mode of action is similar to that of FVIIIa, emicizumab is unlike the natural procoagulant in that it does not depend on activation by thrombin or FXa. Emicizumab exerts cofactor activity at the beginning of coagulation cascade involving FVIIa/TF derived FIXa. This early reaction is maintained and propagated by FXIa-derived FIXa, and results in acceleration of FX activation. In addition, emicizumab also improves thrombin generation in FXI-deficient plasma, utilizing FIXa triggered by FVIIa/TF. Emicizumab activity in FVIII deficient plasma was shown to be remarkably depressed by FXIa depletion, however [22]. The antigen binding affinity of emicizumab is also different from that of FVIII, and emicizumab binds to FIX/FIXa and FX/FXa with moderate strength. The calculated KD values of 1.58/1.52 and 1.85/0.978 µM, respectively, indicated that the antigen-binding affinities of emicizumab were much greater than those of other antibody therapeutics [21]. Hence, generated FXa is released quickly from the FIXa–emicizumab–FX ternary complex, and is consequently supplied to downstream mechanisms of the coagulation cascade. Studies in this context also indicated that amount of the ternary complex in plasma correlated with thrombin generation, and suggested that the cofactor function of emicizumab reflected the plasma concentration of the ternary complex. Emicizumab-derived cofactor function is completely dependent on the presence of phospholipid. The enzymatic kinetic potentials of emicizumab in FIXa-catalyzed FXa generation, Kcat (/min) and Kcat/Km(/min/μM) are 2.88 and 570, respectively. These values were 1/44 and 1/11 when compared with those of FVIIIa [21]. Moreover, emicizumab potential is not affected by von Willebrand factor (VWF).