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.

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.

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.

Cancer-specific glycosylation of CD13 impacts its detection and activity in preclinical cancer tissues.

Harnessing the differences between cancer and non-cancer tissues presents new opportunities for selective targeting by anti-cancer drugs. CD13, a heavily glycosylated protein, is one example with significant unmet clinical potential in cancer drug discovery. Despite its high expression and activity in cancers, CD13 is also expressed in many normal tissues. Here, we report differential tissue glycosylation of CD13 across tissues and demonstrate for the first time that the nature and pattern of glycosylation of CD13 in preclinical cancer tissues are distinct compared to normal tissues. We identify cancer-specific O-glycosylation of CD13, which selectively blocks its detection in cancer models but not in normal tissues. In addition, the metabolism activity of cancer-expressed CD13 was observed to be critically dependent on its unique glycosylation. Thus, our data demonstrate the existence of discrete cancer-specific CD13 glycoforms and propose cancer-specific CD13 glycoforms as a clinically useful target for effective cancer-targeted therapy.

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.

Targeted delivery of a colchicine analogue provides synergy with ATR inhibition in cancer cells.

Despite significant preclinical promise as anticancer agents, vascular-disrupting agents have yet to fulfil their clinical potential due to systemic toxicities. ICT2588 is a tumour-selective MT1-MMP-targeted prodrug of azademethylcolchicine, ICT2552. We investigate activation of ICT2588 and subsequent release of ICT2552 in tumour cells, and examine its ability to induce G2/M cell cycle arrest. We also explore synergism between ICT2588 and ATR inhibition, since colchicine, in addition to its vascular-disrupting properties, is known to induce G2/M arrest, DNA damage, and trigger apoptosis. Several ATR inhibitors are currently undergoing clinical evaluation. The cellular activation of ICT2588 was observed to correlate with MT1-MMP expression, with selective release of ICT2552 not compromised by cellular uptake and prodrug activation mechanisms. ICT2588 induced G2/M arrest, and triggered apoptosis in MT1-MMP-expressing cells, but not in cells lacking MT1-MMP expression, while ICT2552 itself induced G2/M arrest and triggered apoptosis in both cell lines. Interestingly, we uncovered that the intracellular release and accumulation dynamics of ICT2552 subsequent to prodrug activation provided synergism with an ATR inhibitor in a way not observed with direct administration of ICT2552. These findings have important potential implications for clinical combinations of ICT2588 and DNA repair inhibitors.

Development and efficacy testing of a portable negative pressure enclosure for airborne infection containment.

OBJECTIVES: To overcome the shortage of personal protective equipment and airborne infection isolation rooms (AIIRs) in the COVID-19 pandemic, a collaborative team of research engineers and clinical physicians worked to build a novel negative pressure environment in the hopes of improving healthcare worker and patient safety. The team then sought to test the device's efficacy in generating and maintaining negative pressure. The goal proved prescient as the US Food and Drug Administration (FDA) later recommended that all barrier devices use negative pressure. METHODS: Initially, engineers observed simulations of various aerosol- and droplet-generating procedures using hospital beds and stretchers to determine the optimal working dimensions of the containment device. Several prototypes were made based on these dimensions which were combined with filters and various flow-generating devices. Then, the airflow generated and the pressure differential within the device during simulated patient care were measured, specifically assessing its ability to create a negative pressure environment consistent with standards published by the Centers for Disease Control and Prevention (CDC). RESULTS: The portable fans were unable to generate any airflow and were dropped from further testing. The vacuums tested were all able to generate a negative pressure environment with the magnitude of pressure differential increasing with the vacuum horsepower. Only the 3.5-horsepower Shop-Vac, however, generated a -3.0 pascal (Pa) pressure gradient, exceeding the CDC-recommended minimum of -2.5 Pa for AIIRs. CONCLUSION: A collaborative team of physicians and engineers demonstrated the efficacy of a prototype portable negative pressure environment, surpassing the negative pressure differential recommended by the CDC.

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.

Progress towards a clinically-successful ATR inhibitor for cancer therapy.

The DNA damage response (DDR) is now known to play an important role in both cancer development and its treatment. Targeting proteins such as ATR (Ataxia telangiectasia mutated and Rad3-related) kinase, a major regulator of DDR, has demonstrated significant therapeutic potential in cancer treatment, with ATR inhibitors having shown anti-tumour activity not just as monotherapies, but also in potentiating the effects of conventional chemotherapy, radiotherapy, and immunotherapy. This review focuses on the biology of ATR, its functional role in cancer development and treatment, and the rationale behind inhibition of this target as a therapeutic approach, including evaluation of the progress and current status of development of potent and specific ATR inhibitors that have emerged in recent decades. The current applications of these inhibitors both in preclinical and clinical studies either as single agents or in combinations with chemotherapy, radiotherapy and immunotherapy are also extensively discussed. This review concludes with some insights into the various concerns raised or observed with ATR inhibition in both the preclinical and clinical settings, with some suggested solutions.

Is tumour-expressed aminopeptidase N (APN/CD13) structurally and functionally unique?

Aminopeptidase N (APN/CD13) is a multifunctional glycoprotein that acts as a peptidase, receptor, and signalling molecule in a tissue-dependent manner. The activities of APN have been implicated in the progression of many cancers, pointing toward significant therapeutic potential for cancer treatment. However, despite the tumour-specific functions of this protein that have been uncovered, the ubiquitous nature of its expression in normal tissues as generally reported remains a limitation to the potential utility of APN as a target for cancer therapeutics and drug discovery. With this in mind, we have extensively explored the literature, and present a comprehensive review that for the first-time provides evidence to support the suggestion that tumour-expressed APN may in fact be unique in structure, function, substrate specificity and activity, contrary to its nature in normal tissues. The review also focuses on the biology of APN, and its "moonlighting" functional roles in both normal physiology and cancer development. Several APN-targeting approaches that have been explored over recent decades as therapeutic strategies in cancer treatment, including APN-targeting agents reported both in preclinical and clinical studies, are also extensively discussed. This review concludes by posing critical questions about APN that remain unanswered and unexplored, hence providing opportunities for further research.

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.

Latent Anti-nutrients and Unintentional Breeding Consequences in Australian Sorghum bicolor Varieties.

Modern feed quality sorghum grain has been bred to reduce anti-nutrients, most conspicuously condensed tannins, but its inclusion in the diets of monogastric animals can still result in variable performance that is only partially understood. Sorghum grain contains several negative intrinsic factors, including non-tannin phenolics and polyphenols, phytate, and kafirin protein, which may be responsible for these muted feed performances. To better understand the non-tannin phenolic and polyphenolic metabolites that may have negative effects on nutritional parameters, the chemical composition of sorghum grain polyphenol extracts from three commercial varieties (MR-Buster, Cracka, and Liberty) was determined through the use of an under-studied, alternative analytical approach involving Fourier-transform infrared (FT-IR) spectroscopy and direct ionization mass spectrometry. Supervised analyses and interrogation of the data contributing to variation resulted in the identification of a variety of metabolites, including established polyphenols, lignin-like anti-nutrients, and complex sugars, as well as high levels of fatty acids which could contribute to nutritional variation and underperformance in monogastrics. FT-IR and mass spectrometry could both discriminate among the different sorghum varieties indicating that FT-IR, rather than more sophisticated chromatographic and mass spectrometric methods, could be incorporated into quality control applications.

An assay for quantitative analysis of polysialic acid expression in cancer cells.

Polysialic acid (polySia) is a linear polysaccharide comprised of N-acetylneuraminic acid residues and its over-expression in cancer cells has been correlated with poor clinical prognosis. An assay has been developed for quantitative analysis of cellular polySia expression. This was achieved by extracting and purifying released polySia from glycoproteins by mild acid hydrolysis and optimised organic extraction. The polySia was further hydrolysed into Sia monomers, followed by fluorescent labelling and quantitative analysis. The assay was qualified utilising endoneuraminidase-NF to remove polySia from the surface of C6-ST8SiaII cancer cells (EC50 = 2.13 ng/mL). The result was comparable to that obtained in a polySia-specific cellular ELISA assay. Furthermore, the assay proved suitable for evaluation of changes in polySia expression following treatment with a small molecule inhibitor of polysialylation. Given the importance of polySia in multiple disease states, notably cancer, this is a potentially vital tool with applications in the fields of drug discovery and glycobiology.

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.

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.

The use of catechins in Chinese hamster ovary cell media for the improvement of monoclonal antibody yields and a reduction of acidic species.

Catechin compounds have potential benefits for recombinant monoclonal antibody (Mab) production as chemical additives in cell culture media. In this study, four catechin compounds catechin (Cat), epicatechin (EC), gallocatechin-gallate (GCG), and epigallocatechin-gallate (EGCG) were added to cell culture media (at 50 µM) and their effects on the recombinant Chinese hamster ovary (CHO) cell culture, specific productivity, and Mab quality were assessed. The results indicate that the improvement of specific productivity was linked to cell growth inhibition. All catechins caused cell phase growth arrest by lowering the number of cells in the G1/G0 phase and increasing the cells in the S and G2/M phases. Late addition of the catechin resulted in a significantly higher final IgG concentration. Cat and EC caused an improvement in the final antibody titer of 1.5 ± 0.1 and 1.3 ± 0.1 fold, respectively. Catechins with a galloyl group (GCG and EGCG) arrested cell growth and reduced cell specific productivity at the concentrations tested. The Cat-treated IgG was found to have reduced acidic species with a corresponding increase in the main peak.

Resveratrol addition to Chinese hamster ovary cell culture media: The effect on cell growth, monoclonal antibody synthesis, and its chemical modification.

The effect of the addition of resveratrol to cell culture media during the production of monoclonal antibodies was investigated. Treatments of Chinese hamster ovary (CHO) cells expressing immunoglobulin G (IgG) with 25 and 50 µM resveratrol showed that resveratrol was capable of slowing cell growth while almost doubling cell-specific productivity to 4.7 ± 0.6 pg IgG/cell·day, resulting in up to a 1.37-fold increase of the final IgG titer. A resveratrol concentration of 50 µM slowed the progression through the cell cycle temporarily by trapping cells in the S-phase. Cation exchange chromatography showed no significant difference in the composition of acidic or basic IgG species and size exclusion chromatography indicated no change in fragmentation or aggregation of the recombinant IgG in the treatment groups. Resveratrol could be used as a chemical additive to CHO media where it would enhance IgG productivity and provide a degree of protection against hydroxyl and superoxide free radicals, expanding the range of options for process improvement available to monoclonal antibody manufacturers.