288 results on '"Wade JD"'
Search Results
2. The principles and in vivo performance of the Edinburgh pivoted aerofoil-disc prosthetic heart valve
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Turina M, Wheatley Dj, Macleod N, and Wade Jd
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medicine.medical_specialty ,Materials science ,medicine.medical_treatment ,Biomedical Engineering ,Biophysics ,Thrombogenicity ,Bioengineering ,Prosthesis ,Canine experiments ,Biomaterials ,Dogs ,Heart Rate ,Coronary Circulation ,medicine ,Pressure ,Animals ,Ventricular Function ,Thrombus ,Cardiac Output ,Prosthetic heart ,General Medicine ,Forward flow ,medicine.disease ,Atrial Function ,Surgery ,Heart Valve Prosthesis ,Cineangiography ,Mitral Valve ,Cattle ,Tricuspid Valve - Abstract
Prototypes of the Edinburgh prosthetic heart valve, known from previous experiments in vitro to promote exceptionally smooth and undisturbed forward flow, have been shown here in canine experiments to possess hemodynamic characteristics not inferior to those of the Bjork-Shiley valve (a present standard of excellence). In addition, we report a low incidence of valve thrombus deposition among calves in which this prototype valve, fabricated from the conventional materials, Delrin and stainless steel, has been implanted in the tricuspid site for up to 100 days without use of anticoagulants. This suggests that the design of the valve is such as to render it of low thrombogenicity. The new valve is designed for fabrication throughout in vitreous or pyro-coated carbon, materials of very high inherent athrombogenicity. It appears from the results discussed here that the developed form of the prosthesis, composed wholly of these athrombogenic materials, is likely to have both acceptable hemodynamic characteristics and an exceptionally low tendency to form thrombus. The introduction of such valves promises to be very advantageous and the results given here appear to warrant their development as the next stage of this program.
- Published
- 1977
3. The effects of potassium citrate arrest on the dog's heart
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S. Zellos, Wade Jd, Davies Sh, A. H. B. Masson, Williams Aw, and McKENZIE Gc
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business.industry ,Potassium ,chemistry.chemical_element ,General Medicine ,Heart, Artificial ,030204 cardiovascular system & hematology ,Pharmacology ,Heart Arrest ,03 medical and health sciences ,0302 clinical medicine ,Dogs ,chemistry ,Potassium Citrate ,Medicine ,Animals ,030212 general & internal medicine ,Citrates ,business - Published
- 1962
4. Engineering of a Biologically Active Glycosylated Glucagon-Like Peptide-1 Analogue.
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Chandrashekar C, Lin F, Nishiuchi Y, Mohammed SF, White BF, Arsenakis Y, Yuliantie E, Zhao P, van Dun S, Koijen A, Kajihara Y, Wootten D, Dodd GT, van den Bos LJ, Wade JD, and Hossain MA
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- Glycosylation, Humans, Animals, Glucagon-Like Peptides pharmacology, Glucagon-Like Peptides chemistry, Glucagon-Like Peptides analogs & derivatives, Glucagon-Like Peptide-1 Receptor agonists, Glucagon-Like Peptide-1 Receptor metabolism, Hypoglycemic Agents pharmacology, Hypoglycemic Agents chemistry, Hypoglycemic Agents chemical synthesis, Male, Blood Glucose drug effects, Blood Glucose metabolism, Protein Engineering, Mice, Glucagon-Like Peptide 1 metabolism, Glucagon-Like Peptide 1 chemistry
- Abstract
Glucagon-like peptide receptor (GLP-1R) agonists (e.g., semaglutide, liraglutide, etc.) are efficient treatment options for people with type 2 diabetes and obesity. The manufacturing method to produce semaglutide, a blockbuster GLP-1 drug on the market, involves multistep synthesis. The large peptide has a hydrophobic fatty acid side chain that makes it sparingly soluble, and its handling, purification, and large-scale production difficult. The growing demand for semaglutide that the manufacturer is not capable of addressing immediately triggered a worldwide shortage. Thus, we have developed a potential alternative analogue to semaglutide by replacing the hydrophobic fatty acid with a hydrophilic human complex-type biantennary oligosaccharide. Our novel glycoGLP-1 analogue was isolated in an ∼10-fold higher yield compared with semaglutide. Importantly, our glycoGLP-1 analogue possessed a similar GLP-1R activation potency to semaglutide and was biologically active in vivo in reducing glucose levels to a similar degree as semaglutide.
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- 2024
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5. Practical approaches for supporting informed consent in neonatal clinical trials.
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Wootton SH, Rysavy M, Davis P, Thio M, Romero-Lopez M, Holzapfel LF, Thrasher T, Wade JD, and Owen LS
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- Infant, Newborn, Humans, Critical Illness, Informed Consent, Checklist
- Abstract
The survival and health of preterm and critically ill infants have markedly improved over the past 50 years, supported by well-conducted neonatal research. However, newborn research is difficult to undertake for many reasons, and obtaining informed consent for research in this population presents several unique ethical and logistical challenges. In this article, we explore methods to facilitate the consent process, including the role of checklists to support meaningful informed consent for neonatal clinical trials. CONCLUSION: The authors provide practical guidance on the design and implementation of an effective consent checklist tailored for use in neonatal clinical research., (© 2024 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.)
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- 2024
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6. Big peptide drugs in a small molecule world.
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Otvos L Jr and Wade JD
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A quarter of a century ago, designer peptide drugs finally broke through the glass ceiling. Despite the resistance by big pharma, biotechnology companies managed to develop injectable peptide-based drugs, first against orphan or other small volume diseases, and later for conditions affecting large patient populations such as type 2 diabetes. Even their lack of gastrointestinal absorption could be utilized to enable successful oral dosing against chronic constipation. The preference of peptide therapeutics over small molecule competitors against identical medical conditions can be achieved by careful target selection, intrachain and terminal amino acid modifications, appropriate conjugation to stability enhancers and chemical space expansion, innovative delivery and administration techniques and patient-focused marketing strategies. Unfortunately, however, pharmacoeconomical considerations, including the strength of big pharma to develop competing small molecule drugs, have somewhat limited the success of otherwise smart peptide-based therapeutics. Yet, with increasing improvement in peptide drug modification and formulation, these are continuing to gain significant, and growing, acceptance as desirable alternatives to small molecule compounds., Competing Interests: Author LO was employed by OLPE Pharmaceutical Consultants. The remaining author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declared that they were editorial board members of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Otvos and Wade.)
- Published
- 2023
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7. COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging.
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Vijaykumar K, Leung HM, Barrios A, Fernandez-Petty CM, Solomon GM, Hathorne HY, Wade JD, Monroe K, Slaten KB, Li Q, Leal SM Jr, Moates DB, Pierce HM, Olson KR, Currier P, Foster S, Marsden D, Tearney GJ, and Rowe SM
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- Humans, Tomography, Optical Coherence, COVID-19
- Published
- 2023
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8. Glycosylation Improves the Proteolytic Stability of Exenatide.
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Chandrashekar C, Nishiuchi Y, White BF, Arsenakis Y, Lin F, McNeill SM, Zhao P, van Dun S, Koijen A, Kajihara Y, Wootten D, van den Bos LJ, Wade JD, and Hossain MA
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- Humans, Exenatide, Hypoglycemic Agents, Glycosylation, Peptide Hydrolases, Glucagon-Like Peptide-1 Receptor agonists, Venoms, Diabetes Mellitus, Type 2 drug therapy
- Abstract
Exenatide was the first marketed GLP-1 receptor agonist for the treatment of type 2 diabetes. Modification to the chemical structure or the formulation has the potential to increase the stability of exenatide. We introduced human complex-type sialyloligosaccharide to exenatide at the native Asn28 position. The synthesis was achieved using both solid phase peptide synthesis (SPPS) and Omniligase-1-mediated chemoenzymatic ligation. The results demonstrate that glycosylation increases the proteolytic stability of exenatide while retaining its full biological activity.
- Published
- 2023
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9. Editorial: Innovators in chemical biology.
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Wade JD, Renaudet O, and Coleman MA
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Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2022
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10. Editorial: Editors' showcase: Chemical biology.
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Wade JD
- Abstract
Competing Interests: The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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- 2022
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11. COVID-19 Causes Ciliary Dysfunction as Demonstrated by Human Intranasal Micro-Optical Coherence Tomography Imaging.
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Vijaykumar K, Leung HM, Barrios A, Fernandez-Petty CM, Solomon GM, Hathorne HY, Wade JD, Monroe K, Slaten KB, Li Q, Leal SM, Moates DB, Pierce HM, Olson KR, Currier P, Foster S, Marsden D, Tearney GJ, and Rowe SM
- Abstract
Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causative agent of coronavirus disease 2019 (COVID-19), binds via ACE2 receptors, highly expressed in ciliated cells of the nasal epithelium. Micro-optical coherence tomography (μOCT) is a minimally invasive intranasal imaging technique that can determine cellular and functional dynamics of respiratory epithelia at 1-μm resolution, enabling real time visualization and quantification of epithelial anatomy, ciliary motion, and mucus transport. We hypothesized that respiratory epithelial cell dysfunction in COVID-19 will manifest as reduced ciliated cell function and mucociliary abnormalities, features readily visualized by μOCT. Symptomatic outpatients with SARS-CoV-2 aged ≥ 18 years were recruited within 14 days of symptom onset. Data was interpreted for subjects with COVID-19 (n=13) in comparison to healthy controls (n=8). Significant reduction in functional cilia, diminished ciliary beat frequency, and abnormal ciliary activity were evident. Other abnormalities included denuded epithelium, presence of mucus rafts, and increased inflammatory cells. Our results indicate that subjects with mild but symptomatic COVID-19 exhibit functional abnormalities of the respiratory mucosa underscoring the importance of mucociliary health in viral illness and disease transmission. Ciliary imaging enables investigation of early pathogenic mechanisms of COVID-19 and may be useful for evaluating disease progression and therapeutic response.
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- 2022
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12. Evaluation of Potential DnaK Modulating Proline-Rich Antimicrobial Peptides Identified by Computational Screening.
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Handley TNG, Li W, Welch NG, O'Brien-Simpson NM, Hossain MA, and Wade JD
- Abstract
The day is rapidly approaching where current antibiotic therapies will no longer be effective due to the development of multi-drug resistant bacteria. Antimicrobial peptides (AMPs) are a promising class of therapeutic agents which have the potential to help address this burgeoning problem. Proline-rich AMPs (PrAMPs) are a sub-class of AMPs, that have multiple modes of action including modulation of the bacterial protein folding chaperone, DnaK. They are highly effective against Gram-negative bacteria and have low toxicity to mammalian cells. Previously we used an in silico approach to identify new potential PrAMPs from the DRAMP database. Four of these peptides, antibacterial napin, attacin-C, P9, and PP30, were each chemically assembled and characterized. Together with synthetic oncocin as a reference, each peptide was then assessed for antibacterial activity against Gram-negative/Gram-positive bacteria and for in vitro DnaK modulation activity. We observed that these peptides directly modulate DnaK activity independently of eliciting or otherwise an antibiotic effect. Based on our findings, we propose a change to our previously established PrAMP definition to remove the requirement for antimicrobial activity in isolation, leaving the following classifiers: >25% proline, modulation of DnaK AND/OR the 70S ribosome, net charge of +1 or more, produced in response to bacterial infection AND/OR with pronounced antimicrobial activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Handley, Li, Welch, O’Brien-Simpson, Hossain and Wade.)
- Published
- 2022
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13. Systematic comparison of activity and mechanism of antimicrobial peptides against nosocomial pathogens.
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Lin B, Hung A, Li R, Barlow A, Singleton W, Matthyssen T, Sani MA, Hossain MA, Wade JD, O'Brien-Simpson NM, and Li W
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- Anti-Bacterial Agents chemistry, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial, Gram-Positive Bacteria, Humans, Microbial Sensitivity Tests, Antimicrobial Peptides, Cross Infection drug therapy
- Abstract
The World Health Organisation has deemed several multi-drug resistant (MDR) nosocomial bacterial pathogens to be of significant threat to human health. A stark increase in morbidity, mortality and the burden to healthcare systems around the world can be attributed to the development of resistance in these bacteria. Accordingly, alternative antimicrobial agents have been sought as an attractive means to combat MDR pathogens, with one such example being antimicrobial peptides (AMPs). Given the reported activity of AMPs, including Pardaxin, MSI-78, dermaseptin-PC (DMPC) and Cecropin B, it is important to understand their activities and modes of action against bacteria for further AMP design. In this study, we compared these AMPs against a panel of nosocomial bacterial pathogens, followed by detailed mechanistic studies. It was found that Pardaxin (1-22) and MSI-78 (4-20) displayed the most pronounced antimicrobial activity against the tested bacteria. The mechanistic studies by membrane permeability and molecular dynamics simulation further confirmed the strong membrane interaction and structure of Pardaxin (1-22) and MSI-78 (4-20), which contributed to their potent activity. This study demonstrated a structure and activity guidance for further design of Pardaxin (1-22) and MSI-78 (4-20) as therapeutics against MDR pathogens. The different effects of DMPC (1-19) and Cecropin B (1-21) on membrane integrity and phospholipid membrane interactions provided critical information for the rational design of next-generation analogues with specificity against either Gram-negative or Gram-positive bacteria., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)
- Published
- 2022
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14. The life and work of Robert (Bob) C. Sheppard.
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Wade JD and Jones JH
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- Solid-Phase Synthesis Techniques
- Abstract
The life and work of Robert Charles Sheppard (1932-2019), Bob Sheppard informally among friends, is outlined. He was a leading pioneer of solid phase peptide synthesis and made the most significant and fundamental European contribution to the art of peptide synthesis since Emil Fischer., (© 2021 European Peptide Society and John Wiley & Sons, Ltd.)
- Published
- 2022
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15. Enhancing proline-rich antimicrobial peptide action by homodimerization: influence of bifunctional linker.
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Li W, Lin F, Hung A, Barlow A, Sani MA, Paolini R, Singleton W, Holden J, Hossain MA, Separovic F, O'Brien-Simpson NM, and Wade JD
- Abstract
Antimicrobial peptides (AMPs) are host defense peptides, and unlike conventional antibiotics, they possess potent broad spectrum activities and, induce little or no antimicrobial resistance. They are attractive lead molecules for rational development to improve their therapeutic index. Our current studies examined dimerization of the de novo designed proline-rich AMP (PrAMP), Chex1-Arg20 hydrazide, via C-terminal thiol addition to a series of bifunctional benzene or phenyl tethers to determine the effect of orientation of the peptides and linker length on antimicrobial activity. Antibacterial assays confirmed that dimerization per se significantly enhances Chex1-Arg20 hydrazide action. Greatest advantage was conferred using perfluoroaromatic linkers (tetrafluorobenzene and octofluorobiphenyl) with the resulting dimeric peptides 6 and 7 exhibiting potent action against Gram-negative bacteria, especially the World Health Organization's critical priority-listed multidrug-resistant (MDR)/extensively drug-resistant (XDR) Acinetobacter baumannii as well as preformed biofilms. Mode of action studies indicated these lead PrAMPs can interact with both outer and inner bacterial membranes to affect the membrane potential and stress response. Additionally, 6 and 7 possess potent immunomodulatory activity and neutralise inflammation via nitric oxide production in macrophages. Molecular dynamics simulations of adsorption and permeation mechanisms of the PrAMP on a mixed lipid membrane bilayer showed that a rigid, planar tethered dimer orientation, together with the presence of fluorine atoms that provide increased bacterial membrane interaction, is critical for enhanced dimer activity. These findings highlight the advantages of use of such bifunctional tethers to produce first-in-class, potent PrAMP dimers against MDR/XDR bacterial infections., Competing Interests: The authors declare no conflict of interest., (This journal is © The Royal Society of Chemistry.)
- Published
- 2022
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16. Engineering of a Biologically Active Insulin Dimer.
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Liu M, White BF, Praveen P, Li W, Lin F, Wu H, Li R, Delaine C, Forbes BE, Wade JD, and Hossain MA
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- Animals, Binding, Competitive, Humans, Kinetics, Male, Mice, Mice, Inbred C57BL, Insulin chemistry
- Abstract
The growing epidemic of diabetes means that there is a need for therapies that are more efficacious, safe, and convenient. Here, we report the efficient synthesis of a novel disulfide dimer of human insulin tethered at the N-terminus of its B-chain through placement of a cysteine residue. The resulting peptide was shown to bind to both the insulin receptor isoform B and insulin-like growth factor-1 receptor with comparable affinity to native insulin. In in vivo insulin tolerance tests, the dimer was equipotent to Actrapid insulin and possessed a sustained duration of action greater than that of Actrapid and Glargine. While the secondary structure of our dimeric insulin was similar to that of insulin, it was more resistant to proteolysis. More importantly, our analogue was produced in quantitative yield from a monomeric thiol insulin scaffold. Our results suggest that this dimer has significant potential to address the clinical needs in the treatment of diabetes.
- Published
- 2021
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17. Identification, Synthesis, Conformation and Activity of an Insulin-like Peptide from a Sea Anemone.
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Mitchell ML, Hossain MA, Lin F, Pinheiro-Junior EL, Peigneur S, Wai DCC, Delaine C, Blyth AJ, Forbes BE, Tytgat J, Wade JD, and Norton RS
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- Amino Acid Sequence, Animals, Circular Dichroism, Gene Expression Profiling methods, Gene Expression Regulation, Insulin metabolism, Peptides chemistry, Peptides genetics, Peptides metabolism, Insulin chemistry, Insulin genetics, Sea Anemones metabolism
- Abstract
The role of insulin and insulin-like peptides (ILPs) in vertebrate animals is well studied. Numerous ILPs are also found in invertebrates, although there is uncertainty as to the function and role of many of these peptides. We have identified transcripts with similarity to the insulin family in the tentacle transcriptomes of the sea anemone Oulactis sp. (Actiniaria: Actiniidae). The translated transcripts showed that these insulin-like peptides have highly conserved A- and B-chains among individuals of this species, as well as other Anthozoa. An Oulactis sp. ILP sequence (IlO1_i1) was synthesized using Fmoc solid-phase peptide synthesis of the individual chains, followed by regioselective disulfide bond formation of the intra-A and two interchain disulfide bonds. Bioactivity studies of IlO1_i1 were conducted on human insulin and insulin-like growth factor receptors, and on voltage-gated potassium, sodium, and calcium channels. IlO1_i1 did not bind to the insulin or insulin-like growth factor receptors, but showed weak activity against K
V 1.2, 1.3, 3.1, and 11.1 (hERG) channels, as well as NaV 1.4 channels. Further functional studies are required to determine the role of this peptide in the sea anemone.- Published
- 2021
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18. Cationic Antimicrobial Peptides Are Leading the Way to Combat Oropathogenic Infections.
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Lin B, Li R, Handley TNG, Wade JD, Li W, and O'Brien-Simpson NM
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- Anti-Bacterial Agents pharmacology, Bacteria, Humans, Antimicrobial Cationic Peptides pharmacology, Microbiota
- Abstract
Oral dental infections are one of the most common diseases affecting humans, with caries and periodontal disease having the highest incidence. Caries and periodontal disease arise from infections caused by oral bacterial pathogens. Current misuse and overuse of antibiotic treatments have led to the development of antimicrobial resistance. However, recent studies have shown that cationic antimicrobial peptides are a promising family of antibacterial agents that are active against oral pathogenic bacteria and also possess less propensity for development of antimicrobial resistance. This timely Review has a focus on two primary subjects: (i) the oral bacterial pathogens associated with dental infections and (ii) the current development of antimicrobial peptides targeting oral pathogens.
- Published
- 2021
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19. Chemical Synthesis and Characterization of a Nonfibrillating Glycoglucagon.
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Liu M, Zhao P, Uddin MH, Li W, Lin F, Chandrashekar C, Nishiuchi Y, Kajihara Y, Forbes BE, Wootten D, Wade JD, and Hossain MA
- Abstract
The current commercially available glucagon formulations for the treatment of severe hypoglycemia must be reconstituted immediately prior to use, owing to the susceptibility of glucagon to fibrillation and aggregation in an aqueous solution. This results in the inconvenience of handling, misuse, and wastage of this drug. To address these issues, we synthesized a glycosylated glucagon analogue in which the 25th residue (Trp) was replaced with a cysteine (Cys) and a Br-disialyloligosaccharide was conjugated at the Cys thiol moiety. The resulting analogue, glycoglucagon, is a highly potent full agonist at the glucagon receptor. Importantly, glycoglucagon exhibits markedly reduced propensity for fibrillation and enhanced thermal and metabolic stability. This novel analogue is thus a valuable lead for producing stable liquid glucagon formulations that will improve patient compliance and minimize wastage.
- Published
- 2021
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20. Leptin antagonism inhibits prostate cancer xenograft growth and progression.
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Philp LK, Rockstroh A, Sadowski MC, Taherian Fard A, Lehman M, Tevz G, Libério MS, Bidgood CL, Gunter JH, McPherson S, Bartonicek N, Wade JD, Otvos L, and Nelson CC
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- Androgen Antagonists therapeutic use, Androgens metabolism, Animals, Cell Line, Tumor, Heterografts, Humans, Leptin, Male, Mice, Receptors, Androgen metabolism, Prostatic Neoplasms metabolism, Prostatic Neoplasms, Castration-Resistant metabolism
- Abstract
Hyperleptinaemia is a well-established therapeutic side effect of drugs inhibiting the androgen axis in prostate cancer (PCa), including main stay androgen deprivation therapy (ADT) and androgen targeted therapies (ATT). Given significant crossover between the adipokine hormone signalling of leptin and multiple cancer-promoting hallmark pathways, including growth, proliferation, migration, angiogenesis, metabolism and inflammation, targeting the leptin axis is therapeutically appealing, especially in advanced PCa where current therapies fail to be curative. In this study, we uncover leptin as a novel universal target in PCa and are the first to highlight increased intratumoural leptin and leptin receptor (LEPR) expression in PCa cells and patients' tumours exposed to androgen deprivation, as is observed in patients' tumours of metastatic and castrate resistant (CRPC) PCa. We also reveal the world-first preclinical evidence that demonstrates marked efficacy of targeted leptin-signalling blockade, using Allo-aca, a potent, specific, and safe LEPR peptide antagonist. Allo-aca-suppressed tumour growth and delayed progression to CRPC in mice bearing LNCaP xenografts, with reduced tumour vascularity and altered pathways of apoptosis, transcription/translation, and energetics in tumours determined as potential mechanisms underpinning anti-tumour efficacy. We highlight LEPR blockade in combination with androgen axis inhibition represents a promising new therapeutic strategy vital in advanced PCa treatment.
- Published
- 2021
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21. The Chemical Synthesis of Insulin: An Enduring Challenge.
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Karas JA, Wade JD, and Hossain MA
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- Amino Acid Sequence, Animals, Humans, Insulin analogs & derivatives, Insulin chemistry, Protein Conformation, Chemistry Techniques, Synthetic methods, Insulin chemical synthesis
- Abstract
The pancreatic peptide hormone insulin, first discovered exactly 100 years ago, is essential for glycemic control and is used as a therapeutic for the treatment of type 1 and, increasingly, type 2 diabetes. With a worsening global diabetes epidemic and its significant health budget imposition, there is a great demand for new analogues possessing improved physical and functional properties. However, the chemical synthesis of insulin's intricate 51-amino acid, two-chain, three-disulfide bond structure, together with the poor physicochemical properties of both the individual chains and the hormone itself, has long represented a major challenge to organic chemists. This review provides a timely overview of the past efforts to chemically assemble this fascinating hormone using an array of strategies to enable both correct folding of the two chains and selective formation of disulfide bonds. These methods not only have contributed to general peptide synthesis chemistry and enabled access to the greatly growing numbers of insulin-like and cystine-rich peptides but also, today, enable the production of insulin at the synthetic efficiency levels of recombinant DNA expression methods. They have led to the production of a myriad of novel analogues with optimized structural and functional features and of the feasibility for their industrial manufacture.
- Published
- 2021
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22. Chemically modified and conjugated antimicrobial peptides against superbugs.
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Li W, Separovic F, O'Brien-Simpson NM, and Wade JD
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- Anti-Bacterial Agents chemistry, Humans, Molecular Structure, Pore Forming Cytotoxic Proteins chemistry, Anti-Bacterial Agents pharmacology, Bacteria drug effects, Pore Forming Cytotoxic Proteins pharmacology
- Abstract
Antimicrobial resistance (AMR) is one of the greatest threats to human health that, by 2050, will lead to more deaths from bacterial infections than cancer. New antimicrobial agents, both broad-spectrum and selective, that do not induce AMR are urgently required. Antimicrobial peptides (AMPs) are a novel class of alternatives that possess potent activity against a wide range of Gram-negative and positive bacteria with little or no capacity to induce AMR. This has stimulated substantial chemical development of novel peptide-based antibiotics possessing improved therapeutic index. This review summarises recent synthetic efforts and their impact on analogue design as well as their various applications in AMP development. It includes modifications that have been reported to enhance antimicrobial activity including lipidation, glycosylation and multimerization through to the broad application of novel bio-orthogonal chemistry, as well as perspectives on the direction of future research. The subject area is primarily the development of next-generation antimicrobial agents through selective, rational chemical modification of AMPs. The review further serves as a guide toward the most promising directions in this field to stimulate broad scientific attention, and will lead to new, effective and selective solutions for the several biomedical challenges to which antimicrobial peptidomimetics are being applied.
- Published
- 2021
- Full Text
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23. Chemical Glycosylation and Its Application to Glucose Homeostasis-Regulating Peptides.
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Chandrashekar C, Hossain MA, and Wade JD
- Abstract
Peptides and proteins are attractive targets for therapeutic drug development due to their exquisite target specificity and low toxicity profiles. However, their complex structures give rise to several challenges including solubility, stability, aggregation, low bioavailability, and poor pharmacokinetics. Numerous chemical strategies to address these have been developed including the introduction of several natural and non-natural modifications such as glycosylation, lipidation, cyclization and PEGylation. Glycosylation is considered to be one of the most useful modifications as it is known to contribute to increasing the stability, to improve solubility, and increase the circulating half-lifves of these biomolecules. However, cellular glycosylation is a highly complex process that generally results in heterogenous glycan structures which confounds quality control and chemical and biological assays. For this reason, much effort has been expended on the development of chemical methods, including by solid phase peptide synthesis or chemoenzymatic processes, to enable the acquisition of homogenous glycopeptides to greatly expand possibilities in drug development. In this mini-review, we highlight the importance of such chemical glycosylation methods for improving the biophysical properties of naturally non-glycosylated peptides as applied to the therapeutically essential insulin and related peptides that are used in the treatment of diabetes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Chandrashekar, Hossain and Wade.)
- Published
- 2021
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24. Human glucose-dependent insulinotropic polypeptide (GIP) is an antimicrobial adjuvant re-sensitising multidrug-resistant Gram-negative bacteria.
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Jaradat DMM, Al-Karablieh N, Zaarer BHM, Li W, Saleh KKY, Rasras AJ, Abu-Romman S, O'Brien-Simpson NM, and Wade JD
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- Anti-Bacterial Agents chemistry, Glucagon-Like Peptides chemistry, Humans, Microbial Sensitivity Tests, Anti-Bacterial Agents pharmacology, Drug Resistance, Multiple, Bacterial drug effects, Enterobacteriaceae drug effects, Erwinia amylovora drug effects, Escherichia coli drug effects, Glucagon-Like Peptides pharmacology
- Abstract
Increasing antibiotic resistance in Gram-negative bacteria has mandated the development of both novel antibiotics and alternative therapeutic strategies. Evidence of interplay between several gastrointestinal peptides and the gut microbiota led us to investigate potential and broad-spectrum roles for the incretin hormone, human glucose-dependent insulinotropic polypeptide (GIP) against the Enterobacteriaceae bacteria, Escherichia coli and Erwinia amylovora . GIP had a potent disruptive action on drug efflux pumps of the multidrug resistant bacteria E. coli TG1 and E. amylovora 1189 strains. The effect was comparable to bacterial mutants lacking the inner and outer membrane efflux pump factor proteins AcrB and TolC. While GIP was devoid of direct antimicrobial activity, it has a potent membrane depolarizing effect, and at low concentrations, it significantly potentiated the activity of eight antibiotics and bile salt by reducing MICs by 4-8-fold in E. coli TG1 and 4-20-fold in E. amylovora 1189 . GIP can thus be regarded as an antimicrobial adjuvant with potential for augmenting the available antibiotic arsenal., (© 2020 Walter de Gruyter GmbH, Berlin/Boston.)
- Published
- 2021
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25. De Novo Design of Flavonoid-Based Mimetics of Cationic Antimicrobial Peptides: Discovery, Development, and Applications.
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Lin S, Wade JD, and Liu S
- Subjects
- Antimicrobial Cationic Peptides pharmacology, Biomimetic Materials chemistry, Biomimetic Materials pharmacology, Gram-Positive Bacteria drug effects, Hemolysis drug effects, Microbial Sensitivity Tests, Peptidomimetics chemical synthesis, Peptidomimetics chemistry, Structure-Activity Relationship, Xanthones chemistry, Antimicrobial Cationic Peptides chemistry, Drug Design, Flavonoids chemistry
- Abstract
The alarming rate at which pathogens are developing resistance to conventional antibiotics represents one of the major global challenges to public health care. The prevalence of multidrug-resistant microorganisms is a major impetus for the discovery and development of new antimicrobials. Nature has, to date, been the source of most of the antibiotics discovered and used, including cationic antimicrobial peptides (CAMPs). CAMPs are key components of the innate immune system that are widely found in humans, animals, plants, and microorganisms and that serve as a first line of defense for the host. The attractive features of CAMPs have led to their recognition as potential new antimicrobials. However, they possess several inherent flaws that limit their clinical application including low stability, poor oral bioavailability, poor in vivo efficacy, and a high production cost. To address these issues, small molecule-based peptidomimetic antimicrobials have been designed to biomimic the structural features and biological function of CAMPs. Plant-derived flavonoids (e.g., xanthones and flavones) are active components in traditional herbal medicines and have been reported to contain a variety of significant pharmacological actions including antibacterial, antiviral, antioxidant, and anticancer activities. Over the past decade, we have developed a new chemical strategy to design, discover, and develop xanthone- or flavone-based peptidomimetics and have designed, synthesized, and biologically evaluated a library of approximately 450 new xanthone or flavone derivatives. The designed, structurally diverse compounds can be generally classified into two subfamilies, namely, peptidic and nonpeptidic amphiphilic xanthone or flavone derivatives. In this Account, we describe our efforts on the design, synthesis, biological property evaluation, and mechanism of action model studies of synthetic mimics of CAMPs. The flavonoid compounds are an important component of these rationally designed mimics because they function as hydrophobic aromatic moieties conjugated with different length lipid moieties, behave like an unnatural hydrophobic residue, and provide a rigid scaffold, with the reduced conformational flexibility more likely to provide an active conformation. The mimics can effectively disrupt the integrity of the bacterial membranes. Our endeavors encompass design principles, chemical synthesis, in vitro screening, structural optimization, extensive structural-activity relationship analysis, and a mechanism of action study through biophysical technologies including NMR spectroscopy techniques and computer dynamics simulations, drug resistance assays, in vivo pharmaceutical kinetics (PK) analyses, and in vivo efficacy evaluations of selected promising compounds against drug-resistant bacteria and fungi. Our major contributions to the discovery and development of flavonoid-based mimics as antimicrobials include effectively addressing several limitations associated with CAMPs and have led to promising compounds with a notable potential for further development as new therapeutic antimicrobial agents for the treatment of drug-resistant bacteria- or fungi-induced infections.
- Published
- 2021
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26. Design, synthesis and characterization of a fluorescently labeled functional analog of full-length human ghrelin.
- Author
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Liu M, Whitfield EA, Fothergill LJ, Furness JB, Wade JD, Furness SGB, and Hossain MA
- Subjects
- Fluorescence, HEK293 Cells, Humans, Luminescent Proteins chemistry, Receptors, Ghrelin metabolism, Ghrelin analogs & derivatives, Ghrelin chemical synthesis, Luminescent Proteins chemical synthesis, Luminescent Proteins metabolism
- Abstract
Human ghrelin receptor (GHSR) is a recognized prospective target in the diagnosis and therapy of multiple cancer types. To gain a better understanding of this receptor signaling system, we have synthesized a novel full-length ghrelin analog that is fluorescently labeled at the side-chain of a C-terminal cysteine extension. This analog exhibited nanomolar affinity and potency for the ghrelin receptor. It shows comparable efficacy with that of endogenous ghrelin. The fluorescently-labeled ghrelin analog is a valuable tool for in vitro imaging of cell lines that express ghrelin receptor., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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27. Adiponectin receptor activation inhibits prostate cancer xenograft growth.
- Author
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Philp LK, Rockstroh A, Lehman M, Sadowski MC, Bartonicek N, Wade JD, Otvos L, and Nelson CC
- Subjects
- Animals, Disease Models, Animal, Humans, Male, Mice, Mice, Nude, Prostatic Neoplasms therapy, Receptors, Adiponectin therapeutic use
- Abstract
Adiponectin is an adipokine originally identified as dysregulated in obesity, with a key role in insulin sensitisation and in maintaining systemic energy balance. However, adiponectin is progressively emerging as having aberrant signalling in multiple disease states, including prostate cancer (PCa). Circulating adiponectin is lower in patients with PCa than in non-malignant disease, and inversely correlates with cancer severity. More severe hypoadiponectinemia is observed in advanced PCa than in organ-confined disease. Given the crossover between adiponectin signalling and several cancer hallmark pathways that influence PCa growth and progression, we hypothesised that targeting dysregulated adiponectin signalling may inhibit tumour growth and progression. We, therefore, aimed to test the efficacy of correcting the hypoadiponectinemia and dysregulated adiponectin signalling observed in PCa, a world-first PCa therapeutic approach, using peptide adiponectin receptor (ADIPOR) agonist ADP355 in mice bearing subcutaneous LNCaP xenografts. We demonstrate significant evidence for PCa growth inhibition by ADP355, which slowed tumour growth and delayed progression of serum PCa biomarker, prostate-specific antigen (PSA), compared to vehicle. ADP355 conferred a significant advantage by increasing time on treatment with a delayed ethical endpoint. mRNA sequencing and protein expression analyses of tumours revealed ADP355 PCa growth inhibition may be through altered cellular energetics, cellular stress and protein synthesis, which may culminate in apoptosis, as evidenced by the increased apoptotic marker in ADP355-treated tumours. Our findings highlight the efficacy of ADP355 in targeting classical adiponectin-associated signalling pathways in vivo and provide insights into the promising future for modulating adiponectin signalling through ADIPOR agonism as a novel anti-tumour treatment modality.
- Published
- 2020
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28. (Re)Defining the Proline-Rich Antimicrobial Peptide Family and the Identification of Putative New Members.
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Welch NG, Li W, Hossain MA, Separovic F, O'Brien-Simpson NM, and Wade JD
- Abstract
As we rapidly approach a post-antibiotic era in which multi-drug resistant bacteria are ever-pervasive, antimicrobial peptides (AMPs) represent a promising class of compounds to help address this global issue. AMPs are best-known for their membrane-disruptive mode of action leading to bacteria cell lysis and death. However, many AMPs are also known to be non-lytic and have intracellular modes of action. Proline-rich AMPs (PrAMPs) are one such class, that are generally membrane permeable and inhibit protein synthesis leading to a bactericidal outcome. PrAMPs are highly effective against Gram-negative bacteria and yet show very low toxicity against eukaryotic cells. Here, we review both the PrAMP family and the past and current definitions for this class of peptides. Computational analysis of known AMPs within the DRAMP database (http://dramp.cpu-bioinfor.org/) and assessment of their PrAMP-like properties have led us to develop a revised definition of the PrAMP class. As a result, we subsequently identified a number of unknown and unclassified peptides containing motifs of striking similarity to known PrAMP-based DnaK inhibitors and propose a series of new sequences for experimental evaluation and subsequent addition to the PrAMP family., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Welch, Li, Hossain, Separovic, O'Brien-Simpson and Wade.)
- Published
- 2020
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29. Mechanistic Model of Signaling Dynamics Across an Epithelial Mesenchymal Transition.
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Wade JD, Lun XK, Zivanovic N, Voit EO, and Bodenmiller B
- Abstract
Intracellular signaling pathways are at the core of cellular information processing. The states of these pathways and their inputs determine signaling dynamics and drive cell function. Within a cancerous tumor, many combinations of cell states and microenvironments can lead to dramatic variations in responses to treatment. Network rewiring has been thought to underlie these context-dependent differences in signaling; however, from a biochemical standpoint, rewiring of signaling networks should not be a prerequisite for heterogeneity in responses to stimuli. Here we address this conundrum by analyzing an in vitro model of the epithelial mesenchymal transition (EMT), a biological program implicated in increased tumor invasiveness, heterogeneity, and drug resistance. We used mass cytometry to measure EGF signaling dynamics in the ERK and AKT signaling pathways before and after induction of EMT in Py2T murine breast cancer cells. Analysis of the data with standard network inference methods suggested EMT-dependent network rewiring. In contrast, use of a modeling approach that adequately accounts for single-cell variation demonstrated that a single reaction-based pathway model with constant structure and near-constant parameters is sufficient to represent differences in EGF signaling across EMT. This result indicates that rewiring of the signaling network is not necessary for heterogeneous responses to a signal and that unifying reaction-based models should be employed for characterization of signaling in heterogeneous environments, such as cancer., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Wade, Lun, Zivanovic, Voit and Bodenmiller.)
- Published
- 2020
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30. Effects of C-Terminal B-Chain Modifications in a Relaxin 3 Agonist Analogue.
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Praveen P, Tailhades J, Rosengren KJ, Liu M, Wade JD, Bathgate RAD, and Hossain MA
- Abstract
The receptor for the neuropeptide relaxin 3, relaxin family peptide 3 (RXFP3) receptor, is an attractive pharmacological target for the control of eating, addictive, and psychiatric behaviors. Several structure-activity relationship studies on both human relaxin 3 (containing 3 disulfide bonds) and its analogue A2 (two disulfide bonds) suggest that the C-terminal carboxylic acid of the tryptophan residue in the B-chain is important for RXFP3 activity. In this study, we have added amide, alcohol, carbamate, and ester functionalities to the C-terminus of A2 and compared their structures and functions. As expected, the C-terminal amide form of A2 showed lower binding affinity for RXFP3 while ester and alcohol substitutions also demonstrated lower affinity. However, while these analogues showed slightly lower binding affinity, there was no significant difference in activation of RXFP3 compared to A2 bearing a C-terminal carboxylic acid, suggesting the binding pocket is able to accommodate additional atoms., Competing Interests: The authors declare no competing financial interest., (© 2020 American Chemical Society.)
- Published
- 2020
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31. Total Chemical Synthesis of a Nonfibrillating Human Glycoinsulin.
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Hossain MA, Okamoto R, Karas JA, Praveen P, Liu M, Forbes BE, Wade JD, and Kajihara Y
- Subjects
- Glycosylation, Humans, Insulin chemistry, Microscopy, Atomic Force, Glucose chemistry, Insulin chemical synthesis
- Abstract
Glycosylation is an accepted strategy to improve the therapeutic value of peptide and protein drugs. Insulin and its analogues are life-saving drugs for all type I and 30% of type II diabetic patients. However, they can readily form fibrils which is a significant problem especially for their use in insulin pumps. Because of the solubilizing and hydration effects of sugars, it was thought that glycosylation of insulin could inhibit fibril formation and lead to a more stable formulation. Since enzymatic glycosylation results in heterogeneous products, we developed a novel chemical strategy to produce a homogeneous glycoinsulin (disialo-glycoinsulin) in excellent yield (∼60%). It showed a near-native binding affinity for insulin receptors A and B in vitro and high glucose-lowering effects in vivo , irrespective of the route of administration ( s.c. vs i.p. ). The glycoinsulin retained insulin-like helical structure and exhibited improved stability in human serum. Importantly, our disialo-glycoinsulin analogue does not form fibrils at both high concentration and temperature. Therefore, it is an excellent candidate for clinical use in insulin pumps.
- Published
- 2020
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32. Coatings Releasing the Relaxin Peptide Analogue B7-33 Reduce Fibrotic Encapsulation.
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Welch NG, Mukherjee S, Hossain MA, Praveen P, Werkmeister JA, Wade JD, Bathgate RAD, Winkler DA, and Thissen H
- Subjects
- Animals, Coated Materials, Biocompatible chemistry, Humans, Mice, Peptide Fragments chemistry, Peptides chemistry, Peptides pharmacology, Polylactic Acid-Polyglycolic Acid Copolymer chemistry, Polylactic Acid-Polyglycolic Acid Copolymer pharmacology, Prostheses and Implants adverse effects, Receptors, G-Protein-Coupled antagonists & inhibitors, Relaxin chemistry, Coated Materials, Biocompatible pharmacology, Fibrosis prevention & control, Foreign-Body Reaction prevention & control, Peptide Fragments pharmacology, Relaxin pharmacology
- Abstract
The development of antifibrotic materials and coatings that can resist the foreign body response (FBR) continues to present a major hurdle in the advancement of current and next-generation implantable medical devices, biosensors, and cell therapies. From an implant perspective, the most important issue associated with the FBR is the prolonged inflammatory response leading to a collagenous capsule that ultimately blocks mass transport and communication between the implant and the surrounding tissue. Up to now, most attempts to reduce the capsule thickness have focused on providing surface coatings that reduce protein fouling and cell attachment. Here, we present an approach that is based on the sustained release of a peptide drug interfering with the FBR. In this study, the biodegradable polymer poly(lactic- co -glycolic) acid (PLGA) was used as a coating releasing the relaxin peptide analogue B7-33, which has been demonstrated to reduce organ fibrosis in animal models. While in vitro protein quantification was used to demonstrate controlled release of the antifibrotic peptide B7-33 from PLGA coatings, an in vitro reporter cell assay was used to demonstrate that B7-33 retains activity against the relaxin family peptide receptor 1 (RXFP1). Subcutaneous implantation of PLGA-coated polypropylene samples in mice with and without the peptide demonstrated a marked reduction in capsule thickness (49.2%) over a 6 week period. It is expected that this novel approach will open the door to a range of new and improved implantable medical devices.
- Published
- 2019
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33. Rapid Photolysis-Mediated Folding of Disulfide-Rich Peptides.
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Patil NA, Karas JA, Wade JD, Hossain MA, and Tailhades J
- Subjects
- Conotoxins chemistry, Conotoxins metabolism, Humans, Insulin chemistry, Insulin metabolism, Oxidation-Reduction, Peptides chemistry, Photolysis, Protein Folding, Stereoisomerism, Structure-Activity Relationship, Disulfides chemistry, Peptides metabolism, Ultraviolet Rays
- Abstract
Structure-activity relationship studies are a highly time-consuming aspect of peptide-based drug development, particularly in the assembly of disulfide-rich peptides, which often requires multiple synthetic steps and purifications. Therefore, it is vital to develop rapid and efficient chemical methods to readily access the desired peptides. We have developed a photolysis-mediated "one-pot" strategy for regioselective disulfide bond formation. The new pairing system utilises two ortho-nitroveratryl protected cysteines to generate two disulfide bridges in less than one hour in good yield. This strategy was applied to the synthesis of complex disulfide-rich peptides such as Rho-conotoxin ρ-TIA and native human insulin., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2019
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34. Robert C. Sheppard (1932-2019).
- Author
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Atherton E and Wade JD
- Subjects
- Fluorenes chemistry, History, 20th Century, Molecular Biology, Peptides chemistry, United Kingdom, Peptides chemical synthesis
- Published
- 2019
- Full Text
- View/download PDF
35. Structures of insect Imp-L2 suggest an alternative strategy for regulating the bioavailability of insulin-like hormones.
- Author
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Roed NK, Viola CM, Kristensen O, Schluckebier G, Norrman M, Sajid W, Wade JD, Andersen AS, Kristensen C, Ganderton TR, Turkenburg JP, De Meyts P, and Brzozowski AM
- Subjects
- Animals, Biological Availability, Drosophila, Humans, Insulin pharmacokinetics, Insulin-Like Growth Factor I chemistry, Protein Conformation, Drosophila Proteins chemistry, Insulin-Like Growth Factor Binding Proteins chemistry
- Abstract
The insulin/insulin-like growth factor signalling axis is an evolutionary ancient and highly conserved hormonal system involved in the regulation of metabolism, growth and lifespan in animals. Human insulin is stored in the pancreas, while insulin-like growth factor-1 (IGF-1) is maintained in blood in complexes with IGF-binding proteins (IGFBP1-6). Insect insulin-like polypeptide binding proteins (IBPs) have been considered as IGFBP-like structural and functional homologues. Here, we report structures of the Drosophila IBP Imp-L2 in its free form and bound to Drosophila insulin-like peptide 5 and human IGF-1. Imp-L2 contains two immunoglobulin-like fold domains and its architecture is unrelated to human IGFBPs, suggesting a distinct strategy for bioavailability regulation of insulin-like hormones. Similar hormone binding modes may exist in other insect vectors, as the IBP sequences are highly conserved. Therefore, these findings may open research routes towards a rational interference of transmission of diseases such as malaria, dengue and yellow fevers.
- Published
- 2018
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36. Advantage of a Narrow Spectrum Host Defense (Antimicrobial) Peptide Over a Broad Spectrum Analog in Preclinical Drug Development.
- Author
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Ostorhazi E, Hoffmann R, Herth N, Wade JD, Kraus CN, and Otvos L Jr
- Abstract
The APO-type proline-arginine-rich host defense peptides exhibit potent in vitro killing parameters against Enterobacteriaceae but not to other bacteria. Because of the excellent in vivo properties against systemic and local infections, attempts are regularly made to further improve the activity spectrum. A C-terminal hydrazide analog of the Chex1-Arg20 amide (ARV-1502) shows somewhat improved minimal inhibitory concentration against Moraxellaceae . Here we compared the activity of the two peptides as well as an inactive dimeric reverse amide analog in a systemic Acinetobacter baumannii infection. Only the narrow spectrum amide derivative reduced the 6-h blood bacterial burden by >2 log
10 units reaching statistical significance ( p = 0.03 at 5 mg/kg and 0.031 at 2 mg/kg administered intramuscularly). The hydrazide derivative, probably due to stronger activity on cell membranes, lysed erythrocytes at lower concentrations, and caused toxic effects at lower doses (10 mg/kg vs. 25 mg/kg). In a limited study, the amide induced a >5-fold production of the anti-inflammatory cytokine IL-10 over untreated naïve mice and minor increases in the anti-inflammatory IL-4 and pro-inflammatory cytokines TNF-α and IL-6, in blood. The blood of hydrazide-treated mice exhibited significantly lowered levels of IL-10 and slightly decreased IL-4 and TNF-α. These results suggest that the improved efficacy of the narrow-spectrum amide analog is likely associated with increased anti-inflammatory cytokine production and better stimulation of the immune system. Although blood IL-6 and TNF-α levels are frequently used as markers of potential toxicity in drug development, we did not observe any notable increase in mice receiving the toxic polyamide antibiotic colistin.- Published
- 2018
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37. Drosophila Insulin-Like Peptides DILP2 and DILP5 Differentially Stimulate Cell Signaling and Glycogen Phosphorylase to Regulate Longevity.
- Author
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Post S, Karashchuk G, Wade JD, Sajid W, De Meyts P, and Tatar M
- Abstract
Insulin and IGF signaling (IIS) is a complex system that controls diverse processes including growth, development, metabolism, stress responses, and aging. Drosophila melanogaster IIS is propagated by eight Drosophila insulin-like peptides (DILPs), homologs of both mammalian insulin and IGFs, with various spatiotemporal expression patterns and functions. DILPs 1-7 are thought to act through a single Drosophila insulin/IGF receptor, InR, but it is unclear how the DILPs thereby mediate a range of physiological phenotypes. We determined the distinct cell signaling effects of DILP2 and DILP5 stimulation upon Drosophila S2 cells. DILP2 and DILP5 induced similar transcriptional patterns but differed in signal transduction kinetics. DILP5 induced sustained phosphorylation of Akt, while DILP2 produced acute, transient Akt phosphorylation. Accordingly, we used phosphoproteomic analysis to identify distinct patterns of non-genomic signaling induced by DILP2 and DILP5. Across all treatments and replicates, 5,250 unique phosphopeptides were identified, representing 1,575 proteins. Among these peptides, DILP2, but not DILP5, dephosphorylated Ser15 on glycogen phosphorylase (GlyP), and DILP2, but not DILP5, was subsequently shown to repress enzymatic GlyP activity in S2 cells. The functional consequences of this difference were evaluated in adult Drosophila dilp mutants: dilp2 null adults have elevated GlyP enzymatic activity relative to wild type, while dilp5 mutants have reduced GlyP activity. In flies with intact insulin genes, GlyP overexpression extended lifespan in a Ser15 phosphorylation-dependent manner. In dilp2 mutants, that are otherwise long-lived, longevity was repressed by expression of phosphonull GlyP that is enzymatically inactive. Overall, DILP2, unlike DILP5, signals to affect longevity in part through its control of phosphorylation to deactivate glycogen phosphorylase, a central modulator of glycogen storage and gluconeogenesis.
- Published
- 2018
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38. Editorial: Antimicrobial and Anticancer Peptides.
- Author
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O'Brien-Simpson NM, Hoffmann R, Chia CSB, and Wade JD
- Published
- 2018
- Full Text
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39. Solid-Phase Synthesis of Difficult Purine-Rich PNAs through Selective Hmb Incorporation: Application to the Total Synthesis of Cell Penetrating Peptide-PNAs.
- Author
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Tailhades J, Takizawa H, Gait MJ, Wellings DA, Wade JD, Aoki Y, and Shabanpoor F
- Abstract
Antisense oligonucleotide (ASO)-based drug development is gaining significant momentum following the recent FDA approval of Eteplirsen (an ASO based on phosphorodiamidate morpholino) and Spinraza (2'- O -methoxyethyl-phosphorothioate) in late 2016. Their attractiveness is mainly due to the backbone modifications which have improved the in vivo characteristics of oligonucleotide drugs. Another class of ASO, based on peptide nucleic acid (PNA) chemistry, is also gaining popularity as a platform for development of gene-specific therapy for various disorders. However, the chemical synthesis of long PNAs, which are more target-specific, remains an ongoing challenge. Most of the reported methodology for the solid-phase synthesis of PNA suffer from poor coupling efficiency which limits production to short PNA sequences of less than 15 residues. Here, we have studied the effect of backbone modifications with Hmb (2-hydroxy-4-methoxybenzyl) and Dmb (2,4-dimethoxybenzyl) to ameliorate difficult couplings and reduce "on-resin" aggregation. We firstly synthesized a library of PNA dimers incorporating either Hmb or Dmb and identified that Hmb is superior to Dmb in terms of its ease of removal. Subsequently, we used Hmb backbone modification to synthesize a 22-mer purine-rich PNA, targeting dystrophin RNA splicing, which could not be synthesized by standard coupling methodology. Hmb backbone modification allowed this difficult PNA to be synthesized as well as to be continued to include a cell-penetrating peptide on the same solid support. This approach provides a novel and straightforward strategy for facile solid-phase synthesis of difficult purine-rich PNA sequences.
- Published
- 2017
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40. Novel Methods for the Chemical Synthesis of Insulin Superfamily Peptides and of Analogues Containing Disulfide Isosteres.
- Author
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Hossain MA and Wade JD
- Abstract
The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides. After initially using and subsequently identifying significant limitations of the approach of simultaneous random chain combination and oxidative folding, our laboratory undertook to develop robust chemical synthesis strategies in concert with orthogonal cysteine S-protecting groups and corresponding regioselective disulfide bond formation. These have included the separate synthesis of each of the two chains or of the two chains linked by an artificial C-peptide that is removed following postoxidative folding. These, in turn, have enabled an increased ease of acquisition in a good yield of not only members of human insulin superfamily but other insulin-like peptides. Importantly, these successful methods have enabled, for the first time, a detailed analysis of the role that the disulfide bonds play in the structure and function of such peptides. This was achieved by selective removal of the disulfide bonds or by the judicious insertion of disulfide isosteres that possess structurally subtle variations in bond length, hydrophobicity, and angle. These include lactam, dicarba, and cystathionine, each of which has required modifications to the peptide synthesis protocols for their successful placement within the peptides. Together, these synthesis improvements and the novel chemical developments of cysteine/cystine analogues have greatly aided in the development of novel insulin-like peptide (INSL) analogues, principally with intra-A-chain disulfide isosteres, possessing not only improved functional properties such as increased receptor selectivity but also, with one important and unexpected exception, greater in vivo half-lives due to stability against disulfide reductases. Such analogues greatly will aid further biochemical and pharmacological analyses to delineate the structure-function relationships of INSLs and also future potential drug development.
- Published
- 2017
- Full Text
- View/download PDF
41. Relaxin family peptides: structure-activity relationship studies.
- Author
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Patil NA, Rosengren KJ, Separovic F, Wade JD, Bathgate RAD, and Hossain MA
- Subjects
- Humans, Models, Molecular, Relaxin chemistry, Structure-Activity Relationship, Insulin chemistry, Proteins chemistry, Relaxin analogs & derivatives
- Abstract
The human relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through relaxin family peptide receptors, RXFP1-4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure-activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5)., Linked Articles: This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc., (© 2016 The British Pharmacological Society.)
- Published
- 2017
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42. Influence of node abundance on signaling network state and dynamics analyzed by mass cytometry.
- Author
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Lun XK, Zanotelli VR, Wade JD, Schapiro D, Tognetti M, Dobberstein N, and Bodenmiller B
- Subjects
- Computer Simulation, Gene Expression Profiling methods, HEK293 Cells, Humans, Signal Transduction, Epidermal Growth Factor metabolism, ErbB Receptors metabolism, Flow Cytometry methods, Gene Expression Regulation physiology, Models, Biological, Proteome metabolism
- Abstract
Signaling networks are key regulators of cellular function. Although the concentrations of signaling proteins are perturbed in disease states, such as cancer, and are modulated by drug therapies, our understanding of how such changes shape the properties of signaling networks is limited. Here we couple mass-cytometry-based single-cell analysis with overexpression of tagged signaling proteins to study the dependence of signaling relationships and dynamics on protein node abundance. Focusing on the epidermal growth factor receptor (EGFR) signaling network in HEK293T cells, we analyze 20 signaling proteins during a 1-h EGF stimulation time course using a panel of 35 antibodies. Data analysis with BP-R
2 , a measure that quantifies complex signaling relationships, reveals abundance-dependent network states and identifies novel signaling relationships. Further, we show that upstream signaling proteins have abundance-dependent effects on downstream signaling dynamics. Our approach elucidates the influence of node abundance on signal transduction networks and will further our understanding of signaling in health and disease.- Published
- 2017
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43. The Effect of Selective D- or N α -Methyl Arginine Substitution on the Activity of the Proline-Rich Antimicrobial Peptide, Chex1-Arg20.
- Author
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Li W, Sun Z, O'Brien-Simpson NM, Otvos L, Reynolds EC, Hossain MA, Separovic F, and Wade JD
- Abstract
In vivo pharmacokinetics studies have shown that the proline-rich antimicrobial peptide, A3-APO, which is a discontinuous dimer of the peptide, Chex1-Arg20, undergoes degradation to small fragments at positions Pro6-Arg7 and Val19-Arg20. With the aim of minimizing or abolishing this degradation, a series of Chex1-Arg20 analogs were prepared via Fmoc/tBu solid phase peptide synthesis with D-arginine or, in some cases, peptide backbone N
α -methylated arginine, substitution at these sites. All the peptides were tested for antibacterial activity against the Gram-negative bacterium Klebsiella pneumoniae . The resulting activity of position-7 substitution of Chex1-Arg20 analogs showed that arginine-7 is a crucial residue for maintaining activity against K. pneumoniae . However, arginine-20 substitution had a much less deleterious effect on the antibacterial activity of the peptide. Moreover, none of these peptides displayed any cytotoxicity to HEK and H-4-II-E mammalian cells. These results will aid the development of more effective and stable PrAMPs via judicious amino acid substitutions.- Published
- 2017
- Full Text
- View/download PDF
44. C-Terminal Modification and Multimerization Increase the Efficacy of a Proline-Rich Antimicrobial Peptide.
- Author
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Li W, O'Brien-Simpson NM, Yao S, Tailhades J, Reynolds EC, Dawson RM, Otvos L Jr, Hossain MA, Separovic F, and Wade JD
- Subjects
- Animals, Anti-Bacterial Agents pharmacology, Anti-Bacterial Agents toxicity, Antimicrobial Cationic Peptides pharmacology, Antimicrobial Cationic Peptides toxicity, Cell Line, Cross Infection microbiology, Gram-Negative Bacteria drug effects, Humans, Hydrocarbons, Brominated chemistry, Maleimides chemistry, Proline-Rich Protein Domains, Protein Multimerization, Rats, Anti-Bacterial Agents chemistry, Antimicrobial Cationic Peptides chemistry
- Abstract
Two series of branched tetramers of the proline-rich antimicrobial peptide (PrAMP), Chex1-Arg20, were prepared to improve antibacterial selectivity and potency against a panel of Gram-negative nosocomial pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa. First, tetramerization was achieved by dithiomaleimide (DTM) conjugation of two C-terminal-cysteine bearing dimers that also incorporated C-terminal peptide chemical modification. DTM-linked tetrameric peptides containing a C-terminal hydrazide moiety on each dimer exhibited highly potent activities in the minimum inhibitory concentration (MIC) range of 0.49-2.33 μm. A second series of tetrameric analogues with C-terminal hydrazide modification was prepared by using alternative conjugation linkers including trans-1,4-dibromo-2-butene, α,α'-dibromo-p-xylene, or 6-bismaleimidohexane to determine the effect of length on activity. Each displayed potent and broadened activity against Gram-negative nosocomial pathogens, particularly the butene-linked tetrameric hydrazide. Remarkably, the greatest MIC activity is against P. aeruginosa (0.77 μm/8 μg mL
-1 ) where the monomer is inactive. None of these peptides showed any cytotoxicity to mammalian cells up to 25 times the MIC. A diffusion NMR study of the tetrameric hydrazides showed that the more active antibacterial analogues were those with a more compact structure having smaller hydrodynamic radii. The results show that C-terminal PrAMP hydrazidation together with its rational tetramerization is an effective means for increasing both diversity and potency of PrAMP action., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)- Published
- 2017
- Full Text
- View/download PDF
45. Total Solid-Phase Synthesis of Biologically Active Drosophila Insulin-Like Peptide 2 (DILP2).
- Author
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Lin F, Hossain MA, Post S, Karashchuk G, Tatar M, De Meyts P, and Wade JD
- Abstract
In the fruit fly Drosophila melanogaster , there are eight insulin-like peptides (DILPs) with DILPs 1-7 interacting with a sole insulin-like receptor tyrosine kinase (DInR) while DILP8 interacts with a single G protein-coupled receptor (GPCR), Lgr3. Loss-of-function dilp mutation studies show that the neuropeptide DILP2 has a key role in carbohydrate and lipid metabolism as well as longevity and reproduction. A better understanding of the processes whereby DILP2 mediates its specific actions is required. Consequently we undertook to prepare DILP2 as part of a larger, detailed structure-function relationship study. Use of our well-established insulin-like peptide synthesis protocol that entails separate solid phase assembly of each of the A- and B-chains with selective cysteine S-protection followed by sequential S-deprotection and simultaneous disulfide bond formation produced DILP2 in good overall yield and high purity. The synthetic DILP2 was shown to induce significant DInR phosphorylation and downstream signalling, with it being more potent than human insulin. This peptide will be a valuable tool to provide further insights into its binding to the insulin receptor, the subsequent cell signalling and role in insect metabolism.
- Published
- 2017
- Full Text
- View/download PDF
46. Total Chemical Synthesis of an Intra-A-Chain Cystathionine Human Insulin Analogue with Enhanced Thermal Stability.
- Author
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Karas JA, Patil NA, Tailhades J, Sani MA, Scanlon DB, Forbes BE, Gardiner J, Separovic F, Wade JD, and Hossain MA
- Subjects
- Cystathionine chemistry, Humans, Insulin analogs & derivatives, Molecular Conformation, Cystathionine chemical synthesis, Insulin chemistry, Temperature
- Abstract
Despite recent advances in the treatment of diabetes mellitus, storage of insulin formulations at 4 °C is still necessary to minimize chemical degradation. This is problematic in tropical regions where reliable refrigeration is not ubiquitous. Some degradation byproducts are caused by disulfide shuffling of cystine that leads to covalently bonded oligomers. Consequently we examined the utility of the non-reducible cystine isostere, cystathionine, within the A-chain. Reported herein is an efficient method for forming this mimic using simple monomeric building blocks. The intra-A-chain cystathionine insulin analogue was obtained in good overall yield, chemically characterized and demonstrated to possess native binding affinity for the insulin receptor isoform B. It was also shown to possess significantly enhanced thermal stability indicating potential application to next-generation insulin analogues., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
- Full Text
- View/download PDF
47. A One-Pot Chemically Cleavable Bis-Linker Tether Strategy for the Synthesis of Heterodimeric Peptides.
- Author
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Patil NA, Tailhades J, Karas JA, Separovic F, Wade JD, and Hossain MA
- Subjects
- Disulfides chemistry, Drug Delivery Systems, Molecular Structure, Thionins chemistry, Thionins chemical synthesis
- Abstract
Heterodimeric peptides linked by disulfide bonds are attractive drug targets. However, their chemical assembly can be tedious, time-consuming, and low yielding. Inspired by the cellular synthesis of pro-insulin in which the two constituent peptide chains are expressed as a single-chain precursor separated by a connecting C-peptide, we have developed a novel chemically cleavable bis-linker tether which allows the convenient assembly of two peptide chains as a single "pro"-peptide on the same solid support. Following the peptide cleavage and post-synthetic modifications, this bis-linker tether can be removed in one-step by chemical means. This method was used to synthesize a drug delivery-cargo conjugate, TAT-PKCi peptide, and a two-disulfide bridged heterodimeric peptide, thionin (7-19)-(24-32R), a thionin analogue. To our knowledge, this is the first report of a one-pot chemically cleavable bis-linker strategy for the facile synthesis of cross-bridged two-chain peptides., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
- Full Text
- View/download PDF
48. The N-terminal pro-domain of the kalata B1 cyclotide precursor is intrinsically unstructured.
- Author
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Daly NL, Gunasekera S, Clark RJ, Lin F, Wade JD, Anderson MA, and Craik DJ
- Subjects
- Protein Domains, Cyclotides chemistry, Intrinsically Disordered Proteins chemistry, Oldenlandia chemistry, Plant Proteins chemistry
- Abstract
Cyclotides are plant-derived, gene-encoded, circular peptides with a range of host-defense functions, including insecticidal activity. They also have potential as pharmaceutical scaffolds and understanding their biosynthesis is important to facilitate their large-scale production. Insights into the biosynthesis of cyclotides are emerging but there are still open questions, particularly regarding the influence of the structure of the precursor proteins on processing/biosynthetic pathways. The precursor protein of kalata B1, encoded by the plant Oldenlandia affinis, contains N- and C-terminal propeptides that flank the mature cyclotide domain. The C-terminal region (ctr) is important for the cyclization process, whereas the N-terminal repeat (ntr) has been implicated in vacuolar targeting. In this study we examined the structure and folding of various truncated constructs of the ntr coupled to the mature domain of kalata B1. Despite the ntr having a well-defined helical structure in isolation, once coupled to the natively folded mature domain there is no evidence of an ordered structure. Surprisingly, the ntr appears to be highly disordered and induces self-association of the precursor. This self-association might be associated with the role of the ntr as a vacuolar-targeting signal, as previously shown for unrelated storage proteins., (© 2016 Wiley Periodicals, Inc.)
- Published
- 2016
- Full Text
- View/download PDF
49. Development of a Single-Chain Peptide Agonist of the Relaxin-3 Receptor Using Hydrocarbon Stapling.
- Author
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Hojo K, Hossain MA, Tailhades J, Shabanpoor F, Wong LL, Ong-Pålsson EE, Kastman HE, Ma S, Gundlach AL, Rosengren KJ, Wade JD, and Bathgate RA
- Subjects
- Animals, Cell Line, Cricetulus, Dose-Response Relationship, Drug, Humans, Hydrocarbons chemistry, Male, Models, Molecular, Molecular Structure, Peptides chemical synthesis, Peptides chemistry, Rats, Rats, Sprague-Dawley, Structure-Activity Relationship, Hydrocarbons pharmacology, Peptides pharmacology, Relaxin agonists
- Abstract
Structure-activity studies of the insulin superfamily member, relaxin-3, have shown that its G protein-coupled receptor (RXFP3) binding site is contained within its central B-chain α-helix and this helical structure is essential for receptor activation. We sought to develop a single B-chain mimetic that retained agonist activity. This was achieved by use of solid phase peptide synthesis together with on-resin ruthenium-catalyzed ring closure metathesis of a pair of judiciously placed i,i+4 α-methyl, α-alkenyl amino acids. The resulting hydrocarbon stapled peptide was shown by solution NMR spectroscopy to mimic the native helical conformation of relaxin-3 and to possess potent RXFP3 receptor binding and activation. Alternative stapling procedures were unsuccessful, highlighting the critical need to carefully consider both the peptide sequence and stapling methodology for optimal outcomes. Our result is the first successful minimization of an insulin-like peptide to a single-chain α-helical peptide agonist which will facilitate study of the function of relaxin-3.
- Published
- 2016
- Full Text
- View/download PDF
50. Direct Measurement of Pore Dynamics and Leakage Induced by a Model Antimicrobial Peptide in Single Vesicles and Cells.
- Author
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Burton MG, Huang QM, Hossain MA, Wade JD, Palombo EA, Gee ML, and Clayton AH
- Subjects
- Cell Membrane metabolism, Escherichia coli metabolism, Melitten pharmacology, Cell Membrane chemistry, Escherichia coli chemistry, Melitten chemistry, Unilamellar Liposomes chemistry
- Abstract
Antimicrobial peptides are promising therapeutic alternatives to counter growing antimicrobial resistance. Their precise mechanism of action remains elusive, however, particularly with respect to live bacterial cells. We investigated the interaction of a fluorescent melittin analogue with single giant unilamellar vesicles, giant multilamellar vesicles, and bilamellar Gram-negative Escherichia coli (E. coli) bacteria. Time-lapse fluorescence lifetime imaging microscopy was employed to determine the population distribution of the fluorescent melittin analogue between pore state and membrane surface state, and simultaneously measure the leakage of entrapped fluorescent species from the vesicle (or bacterium) interior. In giant unilamellar vesicles, leakage from vesicle interior was correlated with an increase in level of pore states, consistent with a stable pore formation mechanism. In giant multilamellar vesicles, vesicle leakage occurred more gradually and did not appear to correlate with increased pore states. Instead pore levels remained at a low steady-state level, which is more in line with coupled equilibria. Finally, in single bacterial cells, significant increases in pore levels were observed over time, which were correlated with only partial loss of cytosolic contents. These observations suggested that pore formation, as opposed to complete dissolution of membrane, was responsible for the leakage of contents in these systems, and that the bacterial membrane has an adaptive capacity that resists peptide attack. We interpret the three distinct pore dynamics regimes in the context of the increasing physical and biological complexity of the membranes.
- Published
- 2016
- Full Text
- View/download PDF
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