Carfilzomib [(2S)-N-[(S)-1-[(S)-4-methyl-1-[(R)-2-methyloxiran-2-yl]-1-oxopentan-2-ylcarbamoyl]-2-phenylethyl]-2-[(S)-
Mitochondria serve a pivotal role in the regulation of apoptosis or programmed cell death. Recent studies have demonstrated that reactive electrophiles induce mitochondrion-dependent apoptosis. We hypothesize that covalent modification of specific mitochondrial proteins by reactive electrophiles serves as a trigger leading to the initiation of apoptosis. In this study, we identified protein targets of the model biotin-tagged electrophile probes N-iodoacetyl-N-biotinylhexylene-diamine (IAB) and 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]carboxamido)butane (BMCC) in HEK293 cell mitochondrial fractions by liquid chromatography-tandem mass spectrometry (LC-MS-MS). These electrophiles reproducibly adducted a total of 1693 cysteine residues that mapped to 809 proteins. Protein modifications were selective in that only 438 cysteine sites in 1255 cysteinyl peptide adducts (35%) and 362 of the 809 identified protein targets (45%) were adducted by both electrophiles. Of these, approximately one-third were annotated to the mitochondria following protein database analysis. IAB initiated apoptotic events including cytochrome c release, caspase-3 activation, and poly(ADP-ribose)polymerase (PARP) cleavage, whereas BMCC did not. Of the identified targets of IAB and BMCC, 44 were apoptosis-related proteins, and adduction site specificity on these targets differed between the two probes. Differences in sites of modification between these two electrophiles may reveal alkylation sites whose modification triggers apoptosis.
N'-Nitrosonornicotine (NNN) and N-nitrosopiperidine (NPIP) are potent esophageal and nasal cavity carcinogens in rats and pulmonary carcinogens in mice. N-Nitrosopyrrolidine (NPYR) induces mainly liver tumors in rats and is a weak pulmonary carcinogen in mice. These nitrosamines may be causative agents in human cancer. alpha-Hydroxylation is believed to be the key activation pathway in their carcinogenesis. P450 2As are important enzymes of nitrosamine alpha-hydroxylation. Therefore, a structure-activity relationship study of rat P450 2A3, mouse P450 2A4 and 2A5, and human P450 2A6 and 2A13 was undertaken to compare the catalytic activities of these enzymes for alpha-hydroxylation of (R)-NNN, (S)-NNN, NPIP, and NPYR. Kinetic parameters differed significantly among the P450 2As although their amino acid sequence identities were 83% or greater. For NNN, alpha-hydroxylation can occur at the 2'- or 5'-carbon. P450 2As catalyzed 5'-hydroxylation of (R)- or (S)-NNN with Km values of 0.74-69 microM. All of the P450 2As except P450 2A6 catalyzed (R)-NNN 2'-hydroxylation with Km values of 0.73-66 microM. (S)-NNN 2'-hydroxylation was not observed. Although P450 2A4 and 2A5 differ by only 11 amino acids, they were the least and most efficient catalysts of NNN 5'-hydroxylation, respectively. The catalytic efficiencies (kcat/Km) for (R)-NNN differed by 170-fold whereas there was a 46-fold difference for (S)-NNN. In general, P450 2As catalyzed (R)- and (S)-NNN 5'-hydroxylation with significantly lower Km and higher kcat/Km values than NPIP or NPYR alpha-hydroxylation (p <0.05). Furthermore, P450 2As were better catalysts of NPIP alpha-hydroxylation than NPYR. P450 2A4, 2A5, 2A6, and 2A13 exhibited significantly lower Km and higher kcat/Km values for NPIP than NPYR alpha-hydroxylation (p <0.05), similar to previous reports with P450 2A3. Taken together, these data indicate that critical P450 2A residues determine the catalytic activities of NNN, NPIP, and NPYR alpha-hydroxylation.
BackgroundTo determine the safety and efficacy of the anti-colony-stimulating factor 1 receptor (anti-CSF1R) monoclonal antibody AMG 820 in combination with pembrolizumab in patients with select solid tumors.Patients and methodsPatients had advanced, refractory mismatch repair-proficient colorectal cancer, pancreatic cancer, or non-small cell lung cancer (NSCLC) with low (<50%) programmed cell death-ligand 1 (PD-L1) expression and were naïve to anti-programmed cell death-1 (PD-1)/PD-L1 or had relapsed/refractory NSCLC after anti-PD-1/PD-L1 treatment with low or high (≥50%) PD-L1 expression; all were anti-CSF1/CSF1R naïve. Patients received 1100 mg or 1400 mg AMG 820 plus 200 mg pembrolizumab intravenously every 3 weeks. The primary endpoints were incidence of dose-limiting toxicities (DLTs) and adverse events (AEs) and objective response rate per immune-related Response Evaluation Criteria in Solid Tumours at the recommended combination dose.ResultsOverall, 116 patients received ≥1 dose of AMG 820 plus pembrolizumab (18 at 1400 mg AMG 820; 98 at 1100 mg AMG 820). Most patients (64%) were male; the median age was 64 (range 30–86) years. Seven patients had DLTs (1 at 1400 mg AMG 820; 6 at 1100 mg AMG 820). Almost all patients (99.1%) had AEs, 87.9% with grade ≥3 AEs. The most common AEs were increased aspartate aminotransferase (59.5%), fatigue (48.3%), periorbital/face edema (48.3%), and rash/maculopapular rash (37.1%). The best response was immune-related partial response in 3 patients (3%; duration of response 9.2, 10.0, 12.5 months) and immune-related stable disease in 39 patients (34%). None of the completed phase II cohorts met the predefined threshold for efficacy. Post-treatment there was accumulation of serum colony-stimulating factor 1 (CSF1) and interleukin-34, reduction in CSF1-dependent CD16-expressing monocytes, and increased PD-L1 expression and CD4 and CD8 cell numbers in tumor biopsies.ConclusionsThe recommended combination dose of 1100 mg AMG 820 plus 200 mg pembrolizumab had an acceptable safety profile. Although pharmacodynamic effects were observed, antitumor activity was insufficient for further evaluation of this combination in selected patient populations.Trial registration numberNCT02713529
Among human P450s studied to date, P450 2A13 is the most efficient catalyst of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) alpha-hydroxylation. This reaction is a key bioactivation pathway in NNK-induced carcinogenesis. P450 2A13 mRNA has been detected in human tissues, but it is unknown whether the enzyme is functional in vivo. Therefore, we studied NNK alpha-hydroxylation in human fetal nasal mucosal microsomes, which have been shown to contain high levels of P450 2A protein, presumed to be a mixture of P450 2A6 and 2A13. The microsomes efficiently catalyzed NNK alpha-hydroxylation at the methylene and methyl carbons, as well as carbonyl reduction. Antibodies against mouse P450 2A5 inhibited alpha-hydroxylation by these microsomes greater than 90%. K(m) and V(max) values for alpha-methylene hydroxylation were 6.5 +/- 1.1 muM and 3.0 +/- 0.1 pmol/min/mg; for alpha-methyl hydroxylation, they were 6.7 +/- 0.8 microM and 0.85 +/- 0.03 pmol/min/mg. The K(m) values agree closely with those for NNK metabolism by P450 2A13. Using a new technique, we separated P450 2A13 from P450 2A6 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Quantitative immunoblot analysis indicated that the level of P450 2A13 in the pooled fetal nasal microsome sample used for kinetic analysis was approximately 1.6 pmol/mg protein. In the same sample, P450 2A6 was not detected (detection limit, 67 fmol/mg protein). These kinetic, immunoinhibition, and immunoblot data confirm that P450 2A13 is a functional enzyme and the catalyst of NNK alpha-hydroxylation in human fetal nasal mucosa. The results are also the first to demonstrate high efficiency NNK alpha-hydroxylation in a human tissue.
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