scholarly journals Mutational and biophysical robustness in a pre-stabilized monobody

2020 ◽  
Author(s):  
Peter G. Chandler ◽  
Li Lynn Tan ◽  
Benjamin T. Porebski ◽  
James S. Green ◽  
Blake T. Riley ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Therapeutic Potential ◽  
Binding Activity ◽  
Protein Domain ◽  
Long Term Stability ◽  
Accelerated Stability ◽  
Fibronectin Type Iii ◽  
The Stability ◽  
Complex Architecture ◽  
Shelf Stable

AbstractThe fibronectin type III (FN3) monobody domain is a promising non-antibody scaffold which features a less complex architecture than an antibody while maintaining analogous binding loops. We previously developed FN3Con, a hyper-stable monobody derivative with diagnostic and therapeutic potential. Pre-stabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain towards biological activity. Here, we aimed to examine if the FN3Con monobody could take on antibody-like binding to therapeutic targets, while retaining its extreme stability. We targeted the first of the Adnectin derivative of monobodies to reach clinical trials, which was engineered by directed evolution for binding to the therapeutic target VEGFR2; however, this function was gained at the expense of large losses in thermostability and increased oligomerisation. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the pre-stabilized FN3Con scaffold to produce a domain that successfully bound with high affinity to the therapeutic target VEGFR2. This FN3Con-anti-VEGFR2 construct also maintains high thermostability, including remarkable long-term stability, retaining binding activity after 2 years of storage at 36 °C. Further investigations into buffer excipients doubled the presence of monomeric monobody in accelerated stability trials. These data suggest that loop grafting onto a pre-stabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics, and is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

The Stability of the WHO Reference Thromboplastin NIBS & C 67/40

1979 ◽  
Vol 42 (04) ◽  
pp. 1135-1140 ◽  
Author(s):  
G I C Ingram
Keyword(s):  
Human Brain ◽  
Collaborative Study ◽  
Calibration Constant ◽  
Long Term Stability ◽  
Freeze Dried ◽  
Show Evidence ◽  
Human Material ◽  
Reference Preparation ◽  
The Stability

SummaryThe International Reference Preparation of human brain thromboplastin coded 67/40 has been thought to show evidence of instability. The evidence is discussed and is not thought to be strong; but it is suggested that it would be wise to replace 67/40 with a new preparation of human brain, both for this reason and because 67/40 is in a form (like Thrombotest) in which few workers seem to use human brain. A �plain� preparation would be more appropriate; and a freeze-dried sample of BCT is recommended as the successor preparation. The opportunity should be taken also to replace the corresponding ox and rabbit preparations. In the collaborative study which would be required it would then be desirable to test in parallel the three old and the three new preparations. The relative sensitivities of the old preparations could be compared with those found in earlier studies to obtain further evidence on the stability of 67/40; if stability were confirmed, the new preparations should be calibrated against it, but if not, the new human material should receive a calibration constant of 1.0 and the new ox and rabbit materials calibrated against that.The types of evidence available for monitoring the long-term stability of a thromboplastin are discussed.

10-step Synthesis of 20-nor-Salvinorin A by Dynamic Strategic Bond Analysis

2017 ◽  
Author(s):  
Jeremy Roach ◽  
Yusuke Sasano ◽  
Cullen Schmid ◽  
Saheem Zaidi ◽  
Vsevolod Katritch ◽  
...  
Keyword(s):  
Total Synthesis ◽  
Opioid Receptors ◽  
Structural Modification ◽  
High Selectivity ◽  
Therapeutic Potential ◽  
Salvinorin A ◽  
High Affinity ◽  
Plant Metabolite ◽  
Complex Architecture ◽  
Property Modification

Salvinorin A (SalA) is a plant metabolite that agonizes the human <i>kappa</i>-opioid receptor (κ-OR) with high affinity and high selectivity over <i>mu- </i>and <i>delta-</i>opioid receptors. Its therapeutic potential has stimulated extensive semi-synthetic studies and total synthesis campaigns. However, structural modification of SalA has been complicated by its instability, and efficient total synthesis has been frustrated by its dense, complex architecture. Treatment of strategic bonds in SalA as dynamic and dependent on structural perturbation enabled the identification of an efficient retrosynthetic pathway. Here we show that deletion of C20 simultaneously stabilizes the SalA skeleton, simplifies its synthesis and retains its high affinity and selectivity for the κ-OR. The resulting 10-step synthesis now opens the SalA scaffold to deep-seated property modification.

scholarly journals Nanoencapsulation of Pomegranate Extract to Increase Stability and Potential Dermatological Protection

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 271
Author(s):  
Lucía Yepes-Molina ◽  
José A. Hernández ◽  
Micaela Carvajal
Keyword(s):  
Oxidative Stress ◽  
Heavy Metals ◽  
Protective Effect ◽  
Uv Radiation ◽  
Entrapment Efficiency ◽  
Hacat Cells ◽  
Biotechnological Applications ◽  
Accelerated Stability ◽  
The Stability ◽  
Brassica Oleracea L

Pomegranate extract (PG-E) has been reported to exert a protective effect on the skin due to its antioxidant activity. Ingredients rich in phenolic compounds are unstable in extract solutions, and, therefore, the use of a suitable nanosystem to encapsulate this type of extract could be necessary in different biotechnological applications. Thus, we investigated the capacity of Brassica oleracea L. (cauliflower) inflorescence vesicles (CI-vesicles) to encapsulate PG-E and determined the stability and the antioxidant capacity of the system over time. In addition, the protective effect against UV radiation and heavy metals in HaCaT cells was also tested. The CI-vesicles had an entrapment efficiency of around 50%, and accelerated stability tests did not show significant changes in the parameters tested. The results for the HaCaT cells showed the non-cytotoxicity of the CI-vesicles containing PG-E and their protection against heavy metals (lead acetate and mercuric chloride) and UV-B radiation through a reduction of oxidative stress. The reduction of the percentage of deleted mtDNA (mtDNA4977, “common deletion”) in UV-treated HaCaT cells due to the presence of CI-vesicles containing PG-E indicated the mechanism of protection. Therefore, the effects of CI-vesicles loaded with PG-E against oxidative stress support their utilization as natural cosmeceuticals to protect skin health against external damage from environmental pollution and UV radiation.

scholarly journals Characteristics and Clinical Application of Extracellular Vesicle-Derived DNA

Cancers ◽  
2021 ◽  
Vol 13 (15) ◽  
pp. 3827
Author(s):  
Jae Young Hur ◽  
Kye Young Lee
Keyword(s):  
Therapeutic Potential ◽  
Extracellular Vesicle ◽  
Clinical Settings ◽  
Biomarker Detection ◽  
Diagnosis And Prognosis ◽  
Double Stranded Dna ◽  
Fundamental Research ◽  
The Stability ◽  
Next Generation Sequencing Ngs ◽  
Generation Sequencing

Extracellular vesicles (EVs) carry RNA, proteins, lipids, and diverse biomolecules for intercellular communication. Recent studies have reported that EVs contain double-stranded DNA (dsDNA) and oncogenic mutant DNA. The advantage of EV-derived DNA (EV DNA) over cell-free DNA (cfDNA) is the stability achieved through the encapsulation in the lipid bilayer of EVs, which protects EV DNA from degradation by external factors. The existence of DNA and its stability make EVs a useful source of biomarkers. However, fundamental research on EV DNA remains limited, and many aspects of EV DNA are poorly understood. This review examines the known characteristics of EV DNA, biogenesis of DNA-containing EVs, methylation, and next-generation sequencing (NGS) analysis using EV DNA for biomarker detection. On the basis of this knowledge, this review explores how EV DNA can be incorporated into diagnosis and prognosis in clinical settings, as well as gene transfer of EV DNA and its therapeutic potential.

scholarly journals Limitations and Challenges in the Stability of Cysteamine Eye Drop Compounded Formulations

2021 ◽  
Vol 15 (1) ◽  
pp. 2
Author(s):  
Cristina Martín-Sabroso ◽  
Mario Alonso-González ◽  
Ana Fernández-Carballido ◽  
Juan Aparicio-Blanco ◽  
Damián Córdoba-Díaz ◽  
...  
Keyword(s):  
Shelf Life ◽  
Stability Study ◽  
Eye Drops ◽  
Microbiological Analysis ◽  
Nitrogen Gas ◽  
Long Term Stability ◽  
Main Degradation Product ◽  
The Stability ◽  
Unstable Molecule

Accumulation of cystine crystals in the cornea of patients suffering from cystinosis is considered pathognomonic and can lead to severe ocular complications. Cysteamine eye drop compounded formulations, commonly prepared by hospital pharmacy services, are meant to diminish the build-up of corneal cystine crystals. The objective of this work was to analyze whether the shelf life proposed for six formulations prepared following different protocols used in hospital pharmacies is adequate to guarantee the quality and efficacy of cysteamine eye drops. The long-term and in-use stabilities of these preparations were studied using different parameters: content of cysteamine and its main degradation product cystamine; appearance, color and odor; pH and viscosity; and microbiological analysis. The results obtained show that degradation of cysteamine was between 20% and 50% after one month of storage in the long-term stability study and between 35% and 60% in the in-use study. These data confirm that cysteamine is a very unstable molecule in aqueous solution, the presence of oxygen being the main degradation factor. Saturation with nitrogen gas of the solutions offers a means of reducing cysteamine degradation. Overall, all the formulae studied presented high instability at the end of their shelf life, suggesting that their clinical efficacy might be dramatically compromised.

scholarly journals The SARS-CoV-2 conserved macrodomain is a mono-ADP-ribosylhydrolase

2020 ◽  
Author(s):  
Yousef M.O. Alhammad ◽  
Maithri M. Kashipathy ◽  
Anuradha Roy ◽  
Jean-Philippe Gagné ◽  
Peter McDonald ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Therapeutic Target ◽  
Viral Infections ◽  
Protein Domain ◽  
Structural Protein ◽  
Potential Therapeutic Target ◽  
Novel Therapeutic Strategies ◽  
Design And Testing ◽  
Translational Process ◽  
Novel Therapeutic

ABSTRACTSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other SARS-like-CoVs encode 3 tandem macrodomains within non-structural protein 3 (nsp3). The first macrodomain, Mac1, is conserved throughout CoVs, and binds to and hydrolyzes mono-ADP-ribose (MAR) from target proteins. Mac1 likely counters host-mediated anti-viral ADP-ribosylation, a posttranslational modification that is part of the host response to viral infections. Mac1 is essential for pathogenesis in multiple animal models of CoV infection, implicating it as a virulence factor and potential therapeutic target. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose. SARS-CoV-2, SARS-CoV and MERS-CoV Mac1 exhibit similar structural folds and all 3 proteins bound to ADP-ribose with low μM affinities. Importantly, using ADP-ribose detecting binding reagents in both a gel-based assay and novel ELISA assays, we demonstrated de-MARylating activity for all 3 CoV Mac1 proteins, with the SARS-CoV-2 Mac1 protein leading to a more rapid loss of substrate compared to the others. In addition, none of these enzymes could hydrolyze poly-ADP-ribose. We conclude that the SARS-CoV-2 and other CoV Mac1 proteins are MAR-hydrolases with similar functions, indicating that compounds targeting CoV Mac1 proteins may have broad anti-CoV activity.IMPORTANCESARS-CoV-2 has recently emerged into the human population and has led to a worldwide pandemic of COVID-19 that has caused greater than 900 thousand deaths worldwide. With, no currently approved treatments, novel therapeutic strategies are desperately needed. All coronaviruses encode for a highly conserved macrodomain (Mac1) that binds to and removes ADP-ribose adducts from proteins in a dynamic post-translational process increasingly recognized as an important factor that regulates viral infection. The macrodomain is essential for CoV pathogenesis and may be a novel therapeutic target. Thus, understanding its biochemistry and enzyme activity are critical first steps for these efforts. Here we report the crystal structure of SARS-CoV-2 Mac1 in complex with ADP-ribose, and describe its ADP-ribose binding and hydrolysis activities in direct comparison to SARS-CoV and MERS-CoV Mac1 proteins. These results are an important first step for the design and testing of potential therapies targeting this unique protein domain.

scholarly journals Correlation between composition and activity of gold nanoparticle conjugates with streptococcal protein G

2020 ◽  
Vol 10 (2) ◽  
pp. 4988-4992
Keyword(s):  
Dissociation Constant ◽  
Equilibrium Constants ◽  
Gold Surface ◽  
Binding Activity ◽  
Protein G ◽  
Binding Ability ◽  
Adsorbed Protein ◽  
Streptococcal Protein G ◽  
The Stability ◽  
Nanoparticle Complex

The composition of the conjugates of gold nanoparticles with streptococcal protein G was studied using fluorescence spectroscopy. The method for determining the composition is based on measuring the intrinsic fluorescence of tryptophan as part of the protein. The equilibrium constants of protein binding by the gold surface were determined using the Sketchard method. An increase in the dissociation constant of the protein–nanoparticle complex for increasing the amount of bound protein was demonstrated, and a relationship was established between the stability of the conjugates, their antigen-binding activity, and the dissociation constant. The effectiveness of the conjugates of different compositions in immunochromatographic assay of specific antibodies against the lipopolysaccharide antigen of Brucella abortus was compared. The binding ability of the conjugates increased along with the amount of protein G to ~200 molecules per nanoparticle. A further increase in the amount of adsorbed protein led to a deterioration in the functional activity of the conjugates.

scholarly journals Insulin-Like Growth Factor-1: A Promising Therapeutic Target for Peripheral Nerve Injury

2021 ◽  
Vol 9 ◽  
Author(s):  
Benjamin R. Slavin ◽  
Karim A. Sarhane ◽  
Nicholas von Guionneau ◽  
Phillip J. Hanwright ◽  
Chenhu Qiu ◽  
...  
Keyword(s):  
Growth Factor ◽  
Peripheral Nerve ◽  
Therapeutic Target ◽  
Surgical Repair ◽  
Therapeutic Potential ◽  
Regenerative Process ◽  
Insulin Like Growth Factor ◽  
Peripheral Nerve Injuries ◽  
Promising Therapeutic Target ◽  
Apoptotic Effects

Patients who sustain peripheral nerve injuries (PNIs) are often left with debilitating sensory and motor loss. Presently, there is a lack of clinically available therapeutics that can be given as an adjunct to surgical repair to enhance the regenerative process. Insulin-like growth factor-1 (IGF-1) represents a promising therapeutic target to meet this need, given its well-described trophic and anti-apoptotic effects on neurons, Schwann cells (SCs), and myocytes. Here, we review the literature regarding the therapeutic potential of IGF-1 in PNI. We appraised the literature for the various approaches of IGF-1 administration with the aim of identifying which are the most promising in offering a pathway toward clinical application. We also sought to determine the optimal reported dosage ranges for the various delivery approaches that have been investigated.

LONG-TERM STABILITY ANALYSIS OF ACOUSTIC ABSORBING BOUNDARY CONDITIONS

2013 ◽  
Vol 23 (11) ◽  
pp. 2129-2154 ◽  
Author(s):  
HÉLÈNE BARUCQ ◽  
JULIEN DIAZ ◽  
VÉRONIQUE DUPRAT
Keyword(s):  
Stability Analysis ◽  
Boundary Conditions ◽  
Absorbing Boundary Conditions ◽  
Computational Domain ◽  
Acoustic Wave Equation ◽  
Absorbing Boundary ◽  
Term Stability ◽  
Long Term Stability ◽  
The Stability

This work deals with the stability analysis of a one-parameter family of Absorbing Boundary Conditions (ABC) that have been derived for the acoustic wave equation. We tackle the problem of long-term stability of the wave field both at the continuous and the numerical levels. We first define a function of energy and show that it is decreasing in time. Its discrete form is also decreasing under a Courant–Friedrichs–Lewy (CFL) condition that does not depend on the ABC. Moreover, the decay rate of the continuous energy can be determined: it is exponential if the computational domain is star-shaped and this property can be illustrated numerically.

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