Dess-Martin periodinane-mediated oxidation of the primary alcohol of cytidine into a carboxylic acid.

Herein the first example of conversion of alcohols into carboxylic acids by use of the Dess-Martin Periodinane (DMP), which is otherwise routinely employed for the conversion to aldehydes, is reported. This methodology will have significant potential utility in the synthesis of cytidine analogues and other related biologically important molecules.

Mutational and biophysical robustness in a prestabilized monobody.

The 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 hyperstable monobody derivative with diagnostic and therapeutic potential. Prestabilization of the scaffold mitigates the stability-function trade-off commonly associated with evolving a protein domain toward 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 oligomerization. In order to mitigate these losses, we grafted the binding loops from Adnectin-anti-VEGFR2 (CT-322) onto the prestabilized 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 prestabilized scaffold is a viable strategy for the development of monobody domains with desirable biophysical characteristics and that FN3Con is therefore well-suited to applications such as the evolution of multiple paratopes or shelf-stable diagnostics and therapeutics.

Chemoselective Solution- and Solid-Phase Synthesis of Disulfide-Linked Glycopeptides.

Glycosylation of peptides and proteins is a widely employed strategy to mimic important post-translational modifications or to modulate the physicochemical properties of peptides to enhance their delivery. Furthermore, glycosylation via a sulfur atom imparts increased chemical and metabolic stability to the resulting glycoconjugates. Herein, we report a simple and chemoselective procedure to prepare disulfide-linked glycopeptides. Acetate-protected glycosylsulfenyl hydrazines are shown to be highly reactive with the thiol group of cysteine residues within peptides, both in solution and as part of conventional solid-phase peptide synthesis protocols. The efficiency of this glycosylation methodology with unprotected carbohydrates is also demonstrated, which avoids the need for deprotection steps and further extends its utility, with disulfide-linked glycopeptides produced in excellent yields. Given the importance of glycosylated peptides in structural glycobiology, pharmacology, and therapeutics, the methodology outlined provides easy access to disulfide-linked glycopeptides as molecules with multiple biological applications.

Design and in-vitro testing of a portable patient isolation chamber for bedside aerosol containment and filtration.

The emergence of the SARS-CoV-2 pandemic has imposed a multitude of complications on healthcare facilities. Healthcare professionals had to develop creative solutions to deal with resource shortages and isolation spaces when caring for COVID positive patients. Among many other solutions, facilities have utilized engineering strategies to mitigate the spread of viral contamination within the hospital environment. One of the standard solutions has been the use of whole room negative pressurization (WRNP) to turn a general patient room into an infection isolation space. However, this has not always been easy due to many limitations, such as direct access to the outdoors and the availability of WRNP units. In operating rooms where a patient is likely to go through aerosol-generating procedures, other solutions must be considered because most operating rooms use positive pressure ventilation to maintain sterility. The research team has designed, built, and tested a Covering for Operations during Viral Emergency Response (COVER), a low-cost, portable isolation chamber that fits over a patient's torso on a hospital bed to contain and remove the pathogenic agents at the source (i.e., patient's mouth and nose). This study tests the performance of the COVER system under various design and performance scenarios using particle tracing techniques and compares its efficiency with WRNP units. The results show that COVER can dramatically reduce the concentration of particles within the room, while WRNP is only effective in preventing the room-induced particles from migrating to adjacent spaces.

Applications of isothermal titration calorimetry in pure and applied research from 2016 to 2020.

The last 5 years have seen a series of advances in the application of isothermal titration microcalorimetry (ITC) and interpretation of ITC data. ITC has played an invaluable role in understanding multiprotein complex formation including proteolysis-targeting chimeras (PROTACS), and mitochondrial autophagy receptor Nix interaction with LC3 and GABARAP. It has also helped elucidate complex allosteric communication in protein complexes like trp RNA-binding attenuation protein (TRAP) complex. Advances in kinetics analysis have enabled the calculation of kinetic rate constants from pre-existing ITC data sets. Diverse strategies have also been developed to study enzyme kinetics and enzyme-inhibitor interactions. ITC has also been applied to study small molecule solvent and solute interactions involved in extraction, separation, and purification applications including liquid-liquid separation and extractive distillation. Diverse applications of ITC have been developed from the analysis of protein instability at different temperatures, determination of enzyme kinetics in suspensions of living cells to the adsorption of uremic toxins from aqueous streams.

Glycosyl disulfides: importance, synthesis and application to chemical and biological systems.

The disulfide bond plays an important role in the formation and stabilisation of higher order structures of peptides and proteins, while in recent years interest in this functional group has been extended to carbohydrate chemistry. Rarely found in nature, glycosyl disulfides have attracted significant attention as glycomimetics, with wide biological applications including lectin binding, as key components of dynamic libraries to study carbohydrate structures, the study of metabolic and enzymatic studies, and even as potential drug molecules. This interest has been accompanied and fuelled by the continuous development of new methods to construct the disulfide bond at the anomeric centre. Glycosyl disulfides have also been exploited as versatile intermediates in carbohydrate synthesis, particularly as glycosyl donors. This review focuses on the importance of the disulfide bond in glycobiology and in chemistry, evaluating the different methods available to synthesise glycosyl disulfides. Furthermore, we review the role of glycosyl disulfides as intermediates and/or glycosyl donors for the synthesis of neoglycoproteins and oligosaccharides, before finally considering examples of how this important class of carbohydrates have made an impact in biological and therapeutic contexts.

Phytase catalysis of dephosphorylation studied using isothermal titration calorimetry and electrospray ionization time-of-flight mass spectroscopy.

Phytases are important commercial enzymes that catalyze the dephosphorylation of myo-inositol hexakisphosphate (phytate) to its lower inositol phosphate (IP) esters, IP6 to IP1. Digestion of phytate by Citrobacter braakii 6-phytase deviates significantly from monophasic Michaelis-Menten kinetics. Analysis of phytate digestion using isothermal titration calorimetry (ITC) using the single injection method produced a thermogram with two peaks consistent with two periods of high enzyme activity. Continuous-flow electrospray ionization time-of-flight mass spectroscopy (ESI-ToF-MS) provided real-time analysis of phytase catalysis. It was able to show that the first two cleavage steps were rapid and concurrent but the third cleavage step from IP4 to IP3 was slow. The third (IP4 to IP3), fourth (IP3 to IP2) and fifth (IP2 to IP1) cleavages were effectively sequential due to the preferred association of the more phosphorylated species with the phytase catalytic site. This created a bottleneck during the cleavage of IP4 to IP3 until the point at which IP4 was exhausted and was followed by the rapid cleavage of IP3 to IP2, which was observed as the second peak in the ITC thermogram. This work illustrates the importance of an orthogonal approach when studying non-specific or complex enzyme catalyzed reactions.

An efficient assay for identification and quantitative evaluation of potential polysialyltransferase inhibitors.

The polysialyltransferases (polySTs) catalyse the polymerisation of polysialic acid, which plays an important role in tumour metastasis. While assays are available to assess polyST enzyme activity, there is no methodology available specifically optimised for identification and quantitative evaluation of potential polyST inhibitors. The development of an HPLC-fluorescence-based enzyme assay described within includes a comprehensive investigation of assay conditions, including evaluation of metal ion composition, enzyme, substrate and acceptor concentrations, temperature, pH, and tolerance to DMSO, followed by validation using known polyST inhibitors. Thorough analysis of each of the assay components provided a set of optimised conditions. Under these optimised conditions, the experimentally observed Ki value for CMP, a competitive polyST inhibitor, was strongly correlated with the predicted Ki value, based on the classical Cheng-Prusoff equation [average fold error (AFE) = 1.043]. These results indicate that this assay can provide medium-throughput analysis for enzyme inhibitors with high accuracy, through determining the corresponding IC50 values with substrate concentration at the KM, without the need to perform extensive kinetic studies for each compound. In conclusion, an in vitro cell-free assay for accurate assessment of polyST inhibition is described. The utility of the assay for routine identification of potential polyST inhibitors is demonstrated, allowing quantitative measurement of inhibition to be achieved, and exemplified through assessment of full competitive inhibition. Given the considerable and growing interest in the polySTs as important anti-metastatic targets in cancer drug discovery, this is a vital tool to enable preclinical identification and evaluation of novel polyST inhibitors.

Exploring and Exploiting Acceptor Preferences of the Human Polysialyltransferases as a Basis for an Inhibitor Screen.

α2,8-Linked polysialic acid (polySia) is an oncofoetal antigen with high abundance during embryonic development. It reappears in malignant tumours of neuroendocrine origin. Two polysialyltransferases (polySTs) ST8SiaII and IV are responsible for polySia biosynthesis. During development, both enzymes are essential to control polySia expression. However, in tumours ST8SiaII is the prevalent enzyme. Consequently, ST8SiaII is an attractive target for novel cancer therapeutics. A major challenge is the high structural and functional conservation of ST8SiaII and -IV. An assay system that enables differential testing of ST8SiaII and -IV would be of high value to search for specific inhibitors. Here we exploited the different modes of acceptor recognition and elongation for this purpose. With DMB-DP3 and DMB-DP12 (fluorescently labelled sialic acid oligomers with a degree of polymerisation of 3 and 12, respectively) we identified stark differences between the two enzymes. The new acceptors enabled the simple comparative testing of the polyST initial transfer rate for a series of CMP-activated and N-substituted sialic acid derivatives. Of these derivatives, the non-transferable CMP-Neu5Cyclo was found to be a new, competitive ST8SiaII inhibitor.

Applications of isothermal titration calorimetry - the research and technical developments from 2011 to 2015.

Isothermal titration calorimetry is a widely used biophysical technique for studying the formation or dissociation of molecular complexes. Over the last 5 years, much work has been published on the interpretation of isothermal titration calorimetry (ITC) data for single binding and multiple binding sites. As over 80% of ITC papers are on macromolecules of biological origin, this interpretation is challenging. Some researchers have attempted to link the thermodynamics constants to events at the molecular level. This review highlights work carried out using binding sites characterized using x-ray crystallography techniques that allow speculation about individual bond formation and the displacement of individual water molecules during ligand binding and link these events to the thermodynamic constants for binding. The review also considers research conducted with synthetic binding partners where specific binding events like anion-π and π-π interactions were studied. The revival of assays that enable both thermodynamic and kinetic information to be collected from ITC data is highlighted. Lastly, published criticism of ITC research from a physical chemistry perspective is appraised and practical advice provided for researchers unfamiliar with thermodynamics and its interpretation. Copyright © 2016 John Wiley & Sons, Ltd.

An optimised assay for quantitative, high-throughput analysis of polysialyltransferase activity.

The polysialyltransferases are biologically important glycosyltransferase enzymes responsible for the biosynthesis of polysialic acid, a carbohydrate polymer that plays a critical role in the progression of several diseases, notably cancer. Having improved the chemical synthesis and purification of the fluorescently-labelled DMB-DP3 acceptor, we report optimisation and validation of a highly sensitive cell-free high-throughput HPLC-based assay for assessment of human polysialyltransferase activity.

Novel strategies for the synthesis of unsymmetrical glycosyl disulfides.

Novel strategies for the efficient synthesis of unsymmetrical glycosyl disulfides are reported. Glycosyl disulfides are increasingly important as glycomimetics and molecular probes in glycobiology. Sialosyl disulfides are synthesised directly from the chlorosialoside Neu5Ac2Cl, proceeding via a thiol-disulfide exchange reaction between the sialosyl thiolate and symmetrical disulfides. This methodology was adapted and found to be successfully applicable to the synthesis of unsymmetrical glucosyl disulfides under mild conditions.

A study of the relationship between water and anions of the Hofmeister series using pressure perturbation calorimetry.

Pressure perturbation calorimetry (PPC) was used to study the relationship between water and sodium salts with a range of different anions. At temperatures around 25 °C the heat on pressurisation (ΔQ) from 1 to 5 bar was negative for all solutions relative to pure water. The raw data showed that as the temperature rose, the gradient was positive relative to pure water and the transition temperature where ΔQ was zero was related to anion surface charge density and was more pronounced for the low-charge density anions. A three component model was developed comprising bulk water, the hydration layer and the solute to calculate the molar expansivity of the hydration layer around the ions in solution. The calculated molar expansivities of water in the hydration layer around the ions were consistently less than pure water. ΔQ at different disodium hydrogen phosphate concentrations showed that the change in molar enthalpy relative to pure water was not linear even as it approached infinite dilution suggesting that while hydration layers can be allocated to the water around ions this does not rule out interactions between water and ions extending beyond the immediate hydration layer.

Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy.

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.

Tumor-targeted prodrug ICT2588 demonstrates therapeutic activity against solid tumors and reduced potential for cardiovascular toxicity.

Development of therapeutic strategies for tumor-selective delivery of therapeutics through exploitation of the proteolytic tumor phenotype has significant scope for improvement of cancer treatment. ICT2588 is a peptide-conjugated prodrug of the vascular disrupting agent (VDA) azademethylcolchicine developed to be selectively hydrolyzed by matrix metalloproteinase-14 (MMP-14) within the tumor. In this report, we extend our previous proof-of-concept studies and demonstrate the therapeutic potential of this agent against models of human colorectal, lung, breast, and prostate cancer. In all tumor types, ICT2588 was superior to azademethylcolchicine and was greater or comparable to standard clinically used agents for the respective tumor type. Prodrug activation in clinical human lung tumor homogenates relative to stability in human plasma and liver was observed, supporting clinical translation potential. A major limiting factor to the clinical value of VDAs is their inherent cardiovascular toxicity. No increase in plasma von Willebrand factor (vWF) levels, an indicator of systemic vascular dysfunction and acute cardiovascular toxicity, was detected with ICT2588, thereby supporting the tumor-selective activation and reduced potential of ICT2588 to cause cardiovascular toxicity. Our findings reinforce the improved therapeutic index and tumor-selective approach offered by ICT2588 and this nanotherapeutic approach.

Analysis of the hydration water around bovine serum albumin using terahertz coherent synchrotron radiation.

Terahertz spectroscopy was used to study the absorption of bovine serum albumin (BSA) in water. The Diamond Light Source operating in a low alpha mode generated coherent synchrotron radiation that covered a useable spectral bandwidth of 0.3-3.3 THz (10-110 cm(-1)). As the BSA concentration was raised, there was a nonlinear change in absorption inconsistent with Beer's law. At low BSA concentrations (0-1 mM), the absorption remained constant or rose slightly. Above a concentration of 1 mM BSA, a steady decrease in absorption was observed, which was followed by a plateau that started at 2.5 mM. Using a overlapping hydration layer model, the hydration layer was estimated to extend 15 Å from the protein. Calculation of the corrected absorption coefficient (αcorr) for the water around BSA by subtracting the excluded volume of the protein provides an alternative approach to studying the hydration layer that provides evidence for complexity in the population of water around BSA.

Biopharmaceutical liquid formulation: a review of the science of protein stability and solubility in aqueous environments.

Formulation scientists employed in the biopharmaceutical industry face the challenge of creating liquid aqueous formulations for proteins that never had evolutionary pressure to be exceptionally stable or soluble. Yet commercial products usually need a shelf life of 2 years to be economically viable. The research done in this field is dominated by physical chemists who have developed theories like preferential interaction, preferential hydration and excluded volume to explain the mechanisms for the interaction between salt, small organic molecules and proteins. This review aims to translate the research findings on protein stability and solubility produced by the physical chemists and make it accessible to formulation scientists working within the biopharmaceutical industry.

Hypoxia-Responsive Prodrug of ATR Inhibitor, AZD6738, Selectively Eradicates Treatment-Resistant Cancer Cells.

Targeted therapy remains the future of anti-cancer drug development, owing to the lack of specificity of current treatments which lead to damage in healthy normal tissues. ATR inhibitors have in recent times demonstrated promising clinical potential, and are currently being evaluated in the clinic. However, despite the considerable optimism for clinical success of these inhibitors, reports of associated normal tissues toxicities remain a concern and can compromise their utility. Here, ICT10336 is reported, a newly developed hypoxia-responsive prodrug of ATR inhibitor, AZD6738, which is hypoxia-activated and specifically releases AZD6738 only in hypoxic conditions, in vitro. This hypoxia-selective release of AZD6738 inhibited ATR activation (T1989 and S428 phosphorylation) and subsequently abrogated HIF1a-mediated adaptation of hypoxic cancers cells, thus selectively inducing cell death in 2D and 3D cancer models. Importantly, in normal tissues, ICT10336 is demonstrated to be metabolically stable and less toxic to normal cells than its active parent agent, AZD6738. In addition, ICT10336 exhibited a superior and efficient multicellular penetration ability in 3D tumor models, and selectively eradicated cells at the hypoxic core compared to AZD6738. In summary, the preclinical data demonstrate a new strategy of tumor-targeted delivery of ATR inhibitors with significant potential of enhancing the therapeutic index.

The DNA repair kinase ATM regulates CD13 expression and cell migration.

Classically, ATM is known for its role in sensing double-strand DNA breaks, and subsequently signaling for their repair. Non-canonical roles of ATM include transcriptional silencing, ferroptosis, autophagy and angiogenesis. Angiogenesis mediated by ATM signaling has been shown to be VEGF-independent via p38 signaling. Independently, p38 signaling has been shown to upregulate metalloproteinase expression, including MMP-2 and MMP-9, though it is unclear if this is linked to ATM. Here, we demonstrate ATM regulates aminopeptidase-N (CD13/APN/ANPEP) at the protein level. Positive correlation was seen between ATM activity and CD13 protein expression using both "wildtype" (WT) and knockout (KO) ataxia telangiectasia (AT) cells through western blotting; with the same effect shown when treating neuroblastoma cancer cell line SH-SY5Y, as well as AT-WT cells, with ATM inhibitor (ATMi; KU55933). However, qPCR along with publically available RNAseq data from Hu et al. (J. Clin. Invest., 2021, 131, e139333), demonstrated no change in mRNA levels of CD13, suggesting that ATM regulates CD13 levels via controlling protein degradation. This is further supported by the observation that incubation with proteasome inhibitors led to restoration of CD13 protein levels in cells treated with ATMi. Migration assays showed ATM and CD13 inhibition impairs migration, with no additional effect observed when combined. This suggests an epistatic effect, and that both proteins may be acting in the same signaling pathway that influences cell migration. This work indicates a novel functional interaction between ATM and CD13, suggesting ATM may negatively regulate the degradation of CD13, and subsequently cell migration.

2023 International Consensus Meeting on musculoskeletal infection: Summary from the treatment workgroup and consensus on treatment in preclinical models.

In vitro and in vivo studies are critical for the preclinical efficacy assessment of novel therapies targeting musculoskeletal infections (MSKI). Many preclinical models have been developed and applied as a prelude to evaluating safety and efficacy in human clinical trials. In performing these studies, there is both a requirement for a robust assessment of efficacy, as well as a parallel responsibility to consider the burden on experimental animals used in such studies. Since MSKI is a broad term encompassing infections varying in pathogen, anatomical location, and implants used, there are also a wide range of animal models described modeling these disparate infections. Although some of these variations are required to adequately evaluate specific interventions, there would be enormous value in creating a unified and standardized criteria to animal testing in the treatment of MSKI. The Treatment Workgroup of the 2023 International Consensus Meeting on Musculoskeletal Infection was responsible for questions related to preclinical models for treatment of MSKI. The main objective was to review the literature related to priority questions and estimate consensus opinion after voting. This document presents that process and results for preclinical models related to (1) animal model considerations, (2) outcome measurements, and (3) imaging.