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PURPOSE - Cardiovascular adverse events (CVAEs) can occur during proteasome inhibitor (PI) therapy. We conducted a prospective, observational, multi-institutional study to define risk factors and outcomes in patients with multiple myeloma (MM) receiving PIs.
PATIENTS AND METHODS - Patients with relapsed MM initiating carfilzomib- or bortezomib-based therapy underwent baseline assessments and repeated assessments at regular intervals over 6 months, including cardiac biomarkers (troponin I or T, brain natriuretic peptide [BNP], and N-terminal proBNP), ECG, and echocardiography. Monitoring occurred over 18 months for development of CVAEs.
RESULTS - Of 95 patients enrolled, 65 received carfilzomib and 30 received bortezomib, with median 25 months of follow-up. Sixty-four CVAEs occurred, with 55% grade 3 or greater in severity. CVAEs occurred in 51% of patients treated with carfilzomib and 17% of those treated with bortezomib ( = .002). Median time to first CVAE from treatment start was 31 days, and 86% occurred within the first 3 months. Patients receiving carfilzomib-based therapy with a baseline elevated BNP level higher than 100 pg/mL or N-terminal proBNP level higher than 125 pg/mL had increased risk for CVAE (odds ratio, 10.8; < .001). Elevated natriuretic peptides occurring mid-first cycle of treatment with carfilzomib were associated with a substantially higher risk of CVAEs (odds ratio, 36.0; < .001). Patients who experienced a CVAE had inferior progression-free survival (log-rank = .01) and overall survival (log-rank < .001). PI therapy was safely resumed in 89% of patients, although 41% required chemotherapy modifications.
CONCLUSION - CVAEs are common during PI therapy for relapsed MM, especially with carfilzomib, particularly within the first 3 months of therapy. CVAEs were associated with worse overall outcomes, but usually, discontinuation of therapy was not required. Natriuretic peptides were highly predictive of CVAEs; however, validation of this finding is necessary before uniform incorporation into the routine management of patients receiving carfilzomib.
A convergent total synthesis of the siderophore coelichelin is described. The synthetic route also provided access to acetyl coelichelin and other congeners of the parent siderophore. The synthetic products were evaluated for their ability to bind ferric iron and promote growth of a siderophore-deficient strain of the Gram-negative bacterium Pseudomonas aeruginosa under iron restriction conditions. The results of these studies indicate coelichelin and several derivatives serve as ferric iron delivery vehicles for P. aeruginosa.
Over time, the long-lived proteins that are present throughout the human body deteriorate. Typically, they become racemized, truncated, and covalently cross-linked. One reaction responsible for age-related protein cross-linking in the lens was elucidated recently and shown to involve spontaneous formation of dehydroalanine (DHA) intermediates from phosphoserine. Cys residues are another potential source of DHA, and evidence for this was found in many lens crystallins. In the human lens, some sites were more prone to forming non-disulfide covalent cross-links than others. Foremost among them was Cys5 in βA4 crystallin. The reason for this enhanced reactivity was investigated using peptides. Oxidation of Cys to cystine was a prerequisite for DHA formation, and DHA production was accelerated markedly by the presence of a Lys, one residue separated from Cys5. Modeling and direct investigation of the N-terminal sequence of βA4 crystallin, as well as a variety of homologous peptides, showed that the epsilon amino group of Lys can promote DHA production by nucleophilic attack on the alpha proton of cystine. Once a DHA residue was generated, it could form intermolecular cross-links with Lys and Cys. In the lens, the most abundant cross-link involved Cys5 of βA4 crystallin attached via a thioether bond to glutathione. These findings illustrate the potential of Cys and disulfide bonds to act as precursors for irreversible covalent cross-links and the role of nearby amino acids in creating 'hotpsots' for the spontaneous processes responsible for protein degradation in aged tissues.
© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.
Carfilzomib (CFZ) is a second-generation proteasome inhibitor that is Food and Drug Administration and European Commission approved for the treatment of relapsed or refractory multiple myeloma. CFZ is an epoxomicin derivative with an epoxyketone electrophilic warhead that irreversibly adducts the catalytic threonine residue of the β5 subunit of the proteasome. Although CFZ produces a highly potent, sustained inactivation of the proteasome, the electrophilic nature of the drug could potentially produce off-target protein adduction. To address this possibility, we synthesized an alkynyl analog of CFZ and investigated protein adduction by this analog in HepG2 cells. Using click chemistry coupled with streptavidin based IP and shotgun tandem mass spectrometry (MS/MS), we identified two off-target proteins, cytochrome P450 27A1 (CYP27A1) and glutathione S-transferase omega 1 (GSTO1), as targets of the alkynyl CFZ probe. We confirmed the adduction of CYP27A1 and GSTO1 by streptavidin capture and immunoblotting methodology and then site-specifically mapped the adducts with targeted MS/MS methods. Although CFZ adduction of CYP27A1 and GSTO1 in vitro decreased the activities of these enzymes, the small fraction of these proteins modified by CFZ in intact cells should limit the impact of these off-target modifications. The data support the high selectivity of CFZ for covalent modification of its therapeutic targets, despite the presence of a reactive electrophile. The approach we describe offers a generalizable method to evaluate the safety profile of covalent protein-modifying therapeutics.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
With a newer, more selective and efficacious cytosolic phospholipase A2α (cPLA2α) inhibitor available, we revisited the role of cPLA2α activity in platelet activation and discovered that a component of platelet signaling, even larger than previously appreciated, relies on this enzyme. In a whole blood shear-based flow chamber assay, giripladib, a cPLA2α inhibitor, reduced platelet adhesion and accumulation on collagen. Moreover, giripladib differentially affected P-selectin expression and GPIIbIIIa activation depending on the agonist employed. While protease-activated receptor 1 (PAR1)-mediated platelet activation was unaffected by giripladib, the levels of PAR4- and GPVI-mediated platelet activation were significantly reduced. Meanwhile, the thromboxane A2 receptor antagonist SQ29548 had no effect on PAR-, GPVI-, or puriniergic receptor-mediated platelet activation, suggesting that another eicosanoid produced downstream of arachidonic acid liberation by cPLA2α was responsible for this large component of PAR4- and GPVI-mediated platelet activation. In parallel, we profiled PAR-mediated changes in glycerophospholipid (GPL) mass with and without giripladib to better understand cPLA2α-mediated lipid metabolism. Phosphatidylcholine and phosphatidylethanolamine (PE) demonstrated the largest consumption of mass during thrombin stimulation. Additionally, we confirm phosphatidylinositol as a major substrate of cPLA2α. A comparison of PAR1- and PAR4-induced metabolism revealed the consumption of more putative arachidonyl-PE species downstream of PAR1 activation. Instead of enhanced cPLA2α activity and therefore more arachidonic acid liberation downstream of PAR4, these results indicate the major role that cPLA2α activity plays in platelet function and suggest that a novel eicosanoid is produced in response to platelet activation that represents a large component of PAR4- and GPVI-mediated responses.
A platform technology has been developed and tested for delivery of intracellular-acting peptides through electrostatically complexed nanoparticles, or nano-polyplexes, formulated from an anionic endosomolytic polymer and cationic therapeutic peptides. This delivery platform has been initially tested and optimized for delivery of two unique vasoactive peptides, a phosphomimetic of heat shock protein 20 and an inhibitor of MAPKAP kinase II, to prevent pathological vasoconstriction (i.e., vasospasm) in human vascular tissue. These peptides inhibit vasoconstriction and promote vasorelaxation by modulating actin dynamics in vascular smooth muscle cells. Formulating these peptides into nano-polyplexes significantly enhances peptide uptake and retention, facilitates cytosolic delivery through a pH-dependent endosomal escape mechanism, and enhances peptide bioactivity in vitro as measured by inhibition of F-actin stress fiber formation. In comparison to treatment with the free peptides, which were endowed with cell-penetrating sequences, the nano-polyplexes significantly increased vasorelaxation, inhibited vasoconstriction, and decreased F-actin formation in the human saphenous vein ex vivo. These results suggest that these formulations have significant potential for treatment of conditions such as cerebral vasospasm following subarachnoid hemorrhage. Furthermore, because many therapeutic peptides include cationic cell-penetrating segments, this simple and modular platform technology may have broad applicability as a cost-effective approach for enhancing the efficacy of cytosolically active peptides.
In regions of the circulation where vessels are straight and unbranched, blood flow is laminar and unidirectional. In contrast, at sites of curvature, branch points, and regions distal to stenoses, blood flow becomes disturbed. Atherosclerosis preferentially develops in these regions of disturbed blood flow. Current therapies for atherosclerosis are systemic and may not sufficiently target these atheroprone regions. In this study, we sought to leverage the alterations on the luminal surface of endothelial cells caused by this atheroprone flow for nanocarrier targeting. In vivo phage display was used to discover unique peptides that selectively bind to atheroprone regions in the mouse partial carotid artery ligation model. The peptide GSPREYTSYMPH (PREY) was found to bind 4.5-fold more avidly to the region of disturbed flow and was used to form targeted liposomes. When administered intravenously, PREY-targeted liposomes preferentially accumulated in endothelial cells in the partially occluded carotid artery and other areas of disturbed flow. Proteomic analysis and immunoblotting indicated that fibronectin and Filamin-A were preferentially bound by PREY nanocarriers in vessels with disturbed flow. In additional experiments, PREY nanocarriers were used therapeutically to deliver the nitric oxide synthase cofactor tetrahydrobiopterin (BH4), which we have previously shown to be deficient in regions of disturbed flow. This intervention increased vascular BH4 and reduced vascular superoxide in the partially ligated artery in wild-type mice and reduced plaque burden in the partially ligated left carotid artery of fat fed atheroprone mice (ApoE(-/-)). Targeting atheroprone sites of the circulation with functionalized nanocarriers provides a promising approach for prevention of early atherosclerotic lesion formation.
The use of ion/ion reactions to effect gas-phase alkylation is demonstrated. Commonly used fixed-charge "onium" cations are well-suited for ion/ion reactions with multiply deprotonated analytes because of their tendency to form long-lived electrostatic complexes. Activation of these complexes results in an SN2 reaction that yields an alkylated anion with the loss of a neutral remnant of the reagent. This alkylation process forms the basis of a general method for alkylation of deprotonated analytes generated via electrospray, and is demonstrated on a variety of anionic sites. SN2 reactions of this nature are demonstrated empirically and characterized using density functional theory (DFT). This method for modification in the gas phase is extended to the transfer of larger and more complex R groups that can be used in later gas-phase synthesis steps. For example, N-cyclohexyl-N'-(2-morpholinoethyl)carbodiimide (CMC) is used to transfer a carbodiimide functionality to a peptide anion containing a carboxylic acid. Subsequent activation yields a selective reaction between the transferred carbodiimide group and a carboxylic acid, suggesting the carbodiimide functionality is retained through the transfer process. Many different R groups are transferable using this method, allowing for new possibilities for charge manipulation and derivatization in the gas phase.
BACKGROUND - Nonnucleoside reverse transcriptase inhibitor-based antiretroviral therapy is not suitable for all treatment-naive HIV-infected persons.
OBJECTIVE - To evaluate 3 nonnucleoside reverse transcriptase inhibitor-sparing initial antiretroviral regimens to show equivalence for virologic efficacy and tolerability.
DESIGN - A phase 3, open-label study randomized in a 1:1:1 ratio with follow-up for at least 96 weeks. (ClinicalTrials.gov: NCT00811954).
SETTING - 57 sites in the United States and Puerto Rico.
PATIENTS - Treatment-naive persons aged 18 years or older with HIV-1 RNA levels greater than 1000 copies/mL without resistance to nucleoside reverse transcriptase inhibitors or protease inhibitors.
INTERVENTION - Atazanavir, 300 mg/d, with ritonavir, 100 mg/d; raltegravir, 400 mg twice daily; or darunavir, 800 mg/d, with ritonavir, 100 mg/d, plus combination emtricitabine, 200 mg/d, and tenofovir disoproxil fumarate, 300 mg/d.
MEASUREMENTS - Virologic failure, defined as a confirmed HIV-1 RNA level greater than 1000 copies/mL at or after 16 weeks and before 24 weeks or greater than 200 copies/mL at or after 24 weeks, and tolerability failure, defined as discontinuation of atazanavir, raltegravir, or darunavir for toxicity. A secondary end point was a combination of virologic efficacy and tolerability.
RESULTS - Among 1809 participants, all pairwise comparisons of incidence of virologic failure over 96 weeks showed equivalence within a margin of equivalence defined as -10% to 10%. Raltegravir and ritonavir-boosted darunavir were equivalent for tolerability, whereas ritonavir-boosted atazanavir resulted in a 12.7% and 9.2% higher incidence of tolerability discontinuation than raltegravir and ritonavir-boosted darunavir, respectively, primarily because of hyperbilirubinemia. For combined virologic efficacy and tolerability, ritonavir-boosted darunavir was superior to ritonavir-boosted atazanavir, and raltegravir was superior to both protease inhibitors. Antiretroviral resistance at the time of virologic failure was rare but more frequent with raltegravir.
LIMITATION - The trial was open-label, and ritonavir was not provided.
CONCLUSION - Over 2 years, all 3 regimens attained high and equivalent rates of virologic control. Tolerability of regimens containing raltegravir or ritonavir-boosted darunavir was superior to that of the ritonavir-boosted atazanavir regimen.
PRIMARY FUNDING SOURCE - National Institute of Allergy and Infectious Diseases.
Peripheral artery disease (PAD) is characterized by vessel occlusion and ischemia in the limbs. Treatment for PAD with surgical interventions has been showing limited success. Moreover, recent clinical trials with treatment of angiogenic growth factors proved ineffective as increased angiogenesis triggered severe inflammation in a proportionally coupled fashion. Hence, the overarching goal of this research was to address this issue by developing a biomaterial system that enables controlled, dual delivery of pro-angiogenic C16 and anti-inflammatory Ac-SDKP peptides in a minimally-invasive way. To achieve the goal, a peptide-loaded injectable microgel system was developed and tested in a mouse model of PAD. When delivered through multiple, low volume injections, the combination of C16 and Ac-SDKP peptides promoted angiogenesis, muscle regeneration, and perfusion recovery, while minimizing detrimental inflammation. Additionally, this peptide combination regulated inflammatory TNF-α pathways independently of MMP-9 mediated pathways of angiogenesis in vitro, suggesting a potential mechanism by which angiogenic and inflammatory responses can be uncoupled in the context of PAD. This study demonstrates a translatable potential of the dual peptide-loaded injectable microgel system for PAD treatment.
Copyright © 2014 Elsevier Ltd. All rights reserved.