Landscaping macrocyclic peptides: stapling hDM2-binding peptides for helicity, protein affinity, proteolytic stability and cell uptake.

Cyclic peptides that modulate protein-protein interactions can be valuable therapeutic candidates if they can be delivered intact to their target proteins in cells. Here we systematically compare the effects of different helix-inducing cyclization constraints on the capacity of a macrocyclic peptide component to confer α-helicity, protein-binding affinity, resistance to degradative proteases and cell uptake to a 12-residue peptide fragment of tumor suppressor protein p53. We varied the helix-inducing constraint (hydrocarbon, lactam, aliphatic or aromatic thioether, etc.) and the position of the cyclization linker (i to i + 4 or i to i + 7 bridges) in order to sculpt the macrocyclic size, stabilize its structure, and promote cell uptake. We find that rigidifying the macrocycle leads to higher alpha helicity, target affinity and proteolytic stability to different extents, whereas cell uptake of compounds shown here is mostly driven by hydrophobicity and aromaticity of the macrocycle.

Investigation of Carboxylic Acid Isosteres and Prodrugs for Inhibition of the Human SIRT5 Lysine Deacylase Enzyme.

Sirtuin 5 (SIRT5) is a protein lysine deacylase enzyme that regulates diverse biology by hydrolyzing ϵ-N-carboxyacyllysine posttranslational modifications in the cell. Inhibition of SIRT5 has been linked to potential treatment of several cancers but potent compounds with activity in cells have been lacking. Here we developed mechanism-based inhibitors that incorporate isosteres of a carboxylic acid residue that is important for high-affinity binding to the enzyme active site. By masking of the tetrazole moiety of the most potent candidate from our initial SAR study, we achieved potent and cytoselective growth inhibition for the treatment of SIRT5-dependent leukemic cancer cell lines in culture. Thus, we provide an efficient, cellularly active small molecule that targets SIRT5, which can help elucidate its function and potential as a future drug target. This work shows that masked isosteres of carboxylic acids are viable chemical motifs for the development of inhibitors that target mitochondrial enzymes, which may have applications beyond the sirtuin field.

Novel methodology comparing two assay formats for the assessment of immunogenicity from clinical trial samples.

Aim: We present a novel methodology to compare results between distinct immunogenicity assays, performed by two laboratories, for the same biotherapeutic. Materials & methods: Human serum pools from clinical trials were generated to provide representative immunogenicity titers. Pools were evaluated at two laboratories in a blinded fashion to assess the effect of assay format and laboratory change on clinical interpretation of immunogenicity results. Results: The laboratories validated two different assay formats and demonstrated comparable sensitivity and drug tolerance. Overall, the comparisons in assay format and laboratory ensured a comparable ability to detect treatment-emergent antidrug antibodies for a biotherapeutic. Conclusion: We have established an approach, using pooling of patient samples, that allows for the interlaboratory comparisons without creating duplicative results.

Lay abstract Measuring immunogenicity, an immune response to a drug, is important in understanding the benefits and risks associated with a drug. Immunogenicity is measured by specific tests within a laboratory; however, these tests and laboratories may change over time. This paper proposes a method to determine if a change in test and laboratory will impact the interpretation of immunogenicity for a drug. Blood samples from clinical trial patients were combined in order to provide representative samples for the immunogenicity tests. The samples were tested at two laboratories with two tests to measure if any interpretation of immunogenicity results would change due to the different tests and laboratories. Laboratories and tests demonstrated similar and reliable results of the samples. This study has established a method which allows for the comparison of immunogenicity results when tests and/or laboratories are changed.

Late-Stage Hydrocarbon Conjugation and Cyclisation in Synthetic Peptides and Proteins.

The conventional S-alkylation of cysteine relies upon using activated electrophiles. Here we demonstrate high-yielding and selective S-alkylation and S-lipidation of cysteines in unprotected synthetic peptides and proteins by using weak electrophiles and a Zn2+ promoter. Linear or branched iodoalkanes can S-alkylate cysteine in an unprotected 38-residue Myc peptide fragment and in a 91-residue miniprotein Omomyc, thus highlighting selective late-stage synthetic modifications. Metal-assisted cysteine alkylation is also effective for incorporating dehydroalanine into unprotected peptides and for peptide cyclisation via aliphatic thioether crosslinks, including customising macrocycles to stabilise helical peptides for enhanced uptake and delivery to proteins inside cells. Chemoselective and efficient late-stage Zn2+ -promoted cysteine alkylation in unprotected peptides and proteins promises many useful applications.

The method history report: an adaptable tool for communicating immunogenicity assay development and validation.

Over the developmental lifetime of a therapeutic protein, the immunogenicity assay validation history can become substantial, frustrating review of clinical immunogenicity within the biologics license application. In our experience, this can lead to questions by regulators, resulting in numerous information requests during the review process. To address this, we propose a new document, the method history report (MHR), which can comprehensively present the history of the immunogenicity assay for regulators, including assay development and validation. The flexibility of the MHR allows for adaptation to the specific needs of each therapeutic program, while maintaining a consistent template. Here, we detail the rationale, general outline and template for the MHR and recommend others consider adopting it for their biologics license application-related activities.

The Impella Recover 2.5 and TandemHeart ventricular assist devices are safe and associated with equivalent clinical outcomes in patients undergoing high-risk percutaneous coronary intervention.

OBJECTIVES: To compare the practical use, safety, and clinical outcomes associated with the TandemHeart (TH) versus Impella Recover 2.5 (IR2.5) devices when used for circulatory support during high-risk percutaneous coronary intervention (PCI). BACKGROUND: Small studies and registries suggest safety and efficacy for the TH and IR2.5 percutaneous-left ventricular assist devices (P-LVADs). However, these P-LVADs differ markedly in their insertion, operation, and manner of circulatory augmentation. To date, no study has compared these devices. METHODS: We identified 68 patients (49 males, 19 females; age 71.1 ± 12.1 years) from our single-center database that underwent "high-risk" PCI with P-LVAD support from April 2005 to June 2010 (32 with TH, 36 with IR2.5). Relevant data were extracted for analysis. RESULTS: Baseline demographics were similar, including low LVEF (overall mean 31.0 ± 13.7%) and elevated STS mortality risk score (4.2 ± 3.7%). Angiographic characteristics were also similar, with a mean of 2.4 ± 1.0 lesions treated per patient, and 29% undergoing left main PCI. PCI success rates were 99% in both groups, with similar in-hospital outcomes and a combined 7% major vascular access site complication rate. A single episode of left atrial perforation occurred during TH use. No patient required emergent CABG and no in-hospital deaths occurred. The 30-day MACE rate (death, myocardial infarction, target lesion revascularization) was 5.8%. There were no differences between the IR2.5 and TH groups with respect to short- or long-term clinical outcomes. CONCLUSIONS: The IR2.5 and TH assist devices are safe, equally effective, and associated with acceptable short- and long-term clinical outcomes in patients undergoing "high-risk" PCI.

A novel, high-sensitivity and drug-tolerant sandwich immunoassay for the quantitative measurement of circulating proteins.

BACKGROUND: Accurate measurement of a total protein target (free plus bound) is essential to optimize dose selection for monoclonal antibody drugs. Herein, we describe a novel sandwich immunoassay format in which the biotherapeutic antibody itself serves as the primary detection antibody. A signal is then generated through the addition of a labeled secondary antibody that recognizes the biotherapeutic antibody. The secondary antibody is conjugated with ruthenium to facilitate electrochemiluminescent analysis. RESULTS: Data are presented from the analysis of two protein biomarkers having disparate size and structure; a 4.5 kDa peptide and a 60 kDa protein. In both cases, validated, highly specific assays were developed and shown to be tolerant to elevated levels of the therapeutic monoclonal antibody in question. CONCLUSION: Our novel format allows drug-tolerant measurement of soluble protein biomarkers targeted by monoclonal antibodies when only two independent epitopes for antibody binding are available and one is recognized by the therapeutic antibody.

Evaluation of bifenthrin applications in tires to prevent Aedes mosquito breeding.

The efficacy of maximum label rates of bifenthrin applications to dry tires to prevent Aedes mosquito breeding was investigated by field colonization and bioassay trials in shaded and unshaded locations. Aedes notoscriptus and Culex quinquefasciatus larvae were the most abundant species present in the field colonization trial. Colonization and survival of Ae. notoscriptus larvae to the late instar occurred significantly earlier in treated tires in shaded compared with unshaded locations (P = 0.002). Bifenthrin applications in shaded tires only prevented early instar survival for approximately 2.6 wk. Aedes notoscriptus late instars did not appear in the treated unshaded tires. Culex quinquefasciatus colonized treated tires from the 2nd wk in both shaded and unshaded treatments. In the bioassay, water from bifenthrin-treated tires, through extrapolation, was found to kill approximately 100% of late instar Ae. notoscriptus for only approximately 2.0-2.2 wk in shaded and unshaded tires. Under conditions optimal for Aedes breeding, such as shaded locations, high ambient temperatures, high relative humidity, and high amounts of leaf/organic matter accumulations, bifenthrin may not be effective as a larval control measure in tires for greater than 2.0-2.6 wk.

Recurrent ibuprofen-induced aseptic meningitis.

OBJECTIVE: To report a case of recurrent aseptic meningitis temporally associated with the use of ibuprofen. CASE SUMMARY: A previously well 51-year-old white man presented with acute confusion and aphasia 7 days after taking a variety of nonprescription medications, including ibuprofen. Imaging of the brain was unremarkable, and lumbar puncture revealed lymphocytic pleocytosis with an elevated protein level. The symptoms improved shortly after admission, and no infectious cause was identified. Two weeks later, the patient was readmitted with similar symptoms beginning immediately after resumption of ibuprofen. His symptoms resolved promptly after ibuprofen was discontinued. DISCUSSION: Drug-induced aseptic meningitis is an unusual complication of drug therapy. Nonsteroidal antiinflammatory drugs (NSAIDs), particularly ibuprofen, are among the most commonly implicated agents, but rechallenge with the suspected agent is uncommon. Use of an objective causality tool indicated a probable relationship between ibuprofen and development of aseptic meningitis in our patient. CONCLUSIONS: Clinicians should consider NSAIDs as potential causes of aseptic meningitis, especially in patients with recurrent illness and no obvious infectious cause. A detailed drug history is invaluable in the assessment of such patients, with particular attention to nonprescription medications such as ibuprofen.