Erratum: iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

[This corrects the article DOI: 10.1016/j.isci.2023.107059.].

iTAG an optimized IMiD-induced degron for targeted protein degradation in human and murine cells.

To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented "hook effect" of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome.

C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays.

Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 µM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells.

Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species.

CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies.

Patient expectations, eating disorder severity and personality features: Impact on eating pathology in psychological therapy for eating disorders.

Many factors have been shown to impact treatment outcome in eating disorders, including illness related variables (e.g. symptom severity), and broader clinical features (e.g. personality pathology). Less is known about the potential impact of patient related variables, such as patients' views regarding treatment suitability and success. This study explored the impact of eating disorder severity, personality features and patient expectations regarding treatment suitability and success, on eating pathology at treatment end. Participants were 128 adults with diagnosed eating disorders who completed a course of evidence-based psychological therapy in a community eating disorders service in the UK. Patients completed measures of eating disorder psychopathology and personality features at treatment commencement, and a measure of patient expectations regarding change after session six. They repeated the measure of eating disorder psychopathology at treatment end. All patients had some level of positive expectations regarding the suitability and potential success of treatment after session six. Eating disorder severity significantly predicted eating pathology at treatment end. However, strength of positive expectations regarding treatment success significantly predicted eating pathology over and above baseline level of eating disorder severity. Personality features at baseline were not significantly related to treatment outcome. Findings suggest that patients' expectations regarding the likely success of therapy have a significant impact on treatment outcome. Patients who develop a strong belief that treatment will work may be more likely to actively engage in the process of therapy to achieve their recovery goals. These findings have important implications for the early phase of therapy and how patients are oriented towards treatment.

Importance of the Unstirred Water Layer and Hepatocyte Membrane Integrity In Vitro for Quantification of Intrinsic Metabolic Clearance.

Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using saponin showed either full or minimal P450 activity, depending on the presence or absence of 1 mM NADPH, respectively. The shaking of incubations facilitated P450 metabolic rates up to 5-fold greater than static incubation, depending on intrinsic clearance, indicating the critical influence of the unstirred water layer (UWL). Permeabilization allowed static incubation metabolic rates to approach those of shaking for intact cells, indicating an artificially induced breakdown of the UWL. Permeabilization combined with shaking allowed an increased metabolic rate for saquinavir, resolving the membrane permeability limitation for this drug. These findings advance the interpretation of the rate-limiting processes involved in intrinsic clearance measurements and could be critical for successful in vitro prediction.

CBT for eating disorders: The impact of early changes in eating pathology on later changes in personality pathology, anxiety and depression.

Whilst studies have consistently identified early symptom reduction as an important predictor of treatment outcome, the impact of early change on common comorbid features has not been investigated. This study of CBT for eating disorders explored patterns of early change in eating pathology and longer-term change in personality pathology, anxiety and depression. It also explored the impact of early change in eating pathology on overall change in personality pathology, anxiety and depression. Participants were 179 adults diagnosed with eating disorders who were offered a course of CBT in an out-patient community eating disorders service in the UK. Patients completed a measure of eating disorder psychopathology at the start of treatment and following the 6th session. They also completed measures of personality disorder cognitions, anxiety and depression at the start and end of treatment. There were significant changes in eating pathology over the first six sessions of treatment. Significant improvements were also seen in personality disorder pathology, anxiety and depression by the end of therapy. Effect sizes were medium to large for both completer and intention to treat analyses. Early changes in eating pathology were associated with later changes in common comorbid features, with early reduction in restraint being a key predictor. These findings demonstrate that early symptom change can be achieved in CBT for eating disorders when delivered in routine clinical practice. Such change has long-term benefits that go beyond the domain of eating pathology, enhancing change in personality pathology, anxiety and depression.