A covalent BTK ternary complex compatible with targeted protein degradation.

Targeted protein degradation using heterobifunctional chimeras holds the potential to expand target space and grow the druggable proteome. Most acutely, this provides an opportunity to target proteins that lack enzymatic activity or have otherwise proven intractable to small molecule inhibition. Limiting this potential, however, is the remaining need to develop a ligand for the target of interest. While a number of challenging proteins have been successfully targeted by covalent ligands, unless this modification affects form or function, it may lack the ability to drive a biological response. Bridging covalent ligand discovery with chimeric degrader design has emerged as a potential mechanism to advance both fields. In this work, we employ a set of biochemical and cellular tools to deconvolute the role of covalent modification in targeted protein degradation using Bruton's tyrosine kinase. Our results reveal that covalent target modification is fundamentally compatible with the protein degrader mechanism of action.

Delivering on the promise of protein degraders.

Over the past 3 years, the first bivalent protein degraders intentionally designed for targeted protein degradation (TPD) have advanced to clinical trials, with an initial focus on established targets. Most of these clinical candidates are designed for oral administration, and many discovery efforts appear to be similarly focused. As we look towards the future, we propose that an oral-centric discovery paradigm will overly constrain the chemical designs that are considered and limit the potential to drug novel targets. In this Perspective, we summarize the current state of the bivalent degrader modality and propose three categories of degrader designs, based on their likely route of administration and requirement for drug delivery technologies. We then describe a vision for how parenteral drug delivery, implemented early in research and supported by pharmacokinetic-pharmacodynamic modelling, can enable exploration of a broader drug design space, expand the scope of accessible targets and deliver on the promise of protein degraders as a therapeutic modality.

Intramolecular Ring-Opening Decomposition of Aryl Azetidines.

The ring strain present in azetidines can lead to undesired stability issues. Herein, we described a series of N-substituted azetidines which undergo an acid-mediated intramolecular ring-opening decomposition via nucleophilic attack of a pendant amide group. Studies were conducted to understand the decomposition mechanism enabling the design of stable analogues.

Informing the classification and assessment of positive emotional experiences: A multisample examination of hierarchical positive emotionality models.

Despite being multifaceted in nature, positive emotional (PE) experiences often are studied using only global PE ratings, and measures assessing more specific PE facets do not converge in their assessment approaches. To address these issues, we examined hierarchical factor structures of ratings of positive emotionality, which reflect propensities toward experiencing PE, in both online community adult (N = 375) and undergraduate (N = 447) samples. Preregistered analyses indicated (a) a broad distinction between tendencies to experience social affection and other PE types, and that (b) PE ratings can be differentiated by as many as four replicable factors of Joviality, Social Affection, Serenity, and Attentiveness. These PE dimensions were associated with distinct personality and psychopathology profiles. Examples of these distinctive associations included Joviality displaying robust positive associations with grandiosity and exhibitionism; conversely, although Social Affection and Joviality were strongly correlated, Social Affection showed associations in the opposite direction with grandiosity and exhibitionism. Other notable results include Serenity (e.g., feeling relaxed) showing negative associations with negative emotionality at a magnitude indicating that Serenity may reflect low levels of negative emotionality to a considerable degree. Collectively, these results highlight the need to consider distinct PE facets in addition to global PE ratings when assessing PE, as important nuance may be lost otherwise. Furthermore, our results indicate the need for additional research clarifying PE structure at different levels of abstraction to inform future measure development efforts and assessment approaches. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Examining the Item-Level Structure of the Triarchic Psychopathy Measure: Sharpening Assessment of Psychopathy Constructs.

The Triarchic Psychopathy Measure (TriPM) has emerged as a widely used measure for assessing a three-trait model of disinhibition, meanness, and boldness. Building on recent psychometric work, we examined the TriPM's item-level factor structure and correlates in both a clinically oriented community sample (n = 700) and in undergraduates (n = 527). Our results indicated a replicable three-factor structure generally corresponding with disinhibition, meanness, and boldness, although many items were not clear indicators of their assigned TriPM domain scales. Consequently, these dimensions may be better represented by Alternate Disinhibition (14 items), Boldness (13 items), and Meanness (8 items) domain scales. Additionally, we identified sets of items defining distinct Self-Assurance and Fearlessness dimensions within Boldness and Irresponsibility and Impulsivity dimensions within Disinhibition. We discuss these findings in the context of other recent studies examining the TriPM's item-level structure, highlighting key future directions for sharpening measurement of the externalizing spectrum.

Snapshots and ensembles of BTK and cIAP1 protein degrader ternary complexes.

Heterobifunctional chimeric degraders are a class of ligands that recruit target proteins to E3 ubiquitin ligases to drive compound-dependent protein degradation. Advancing from initial chemical tools, protein degraders represent a mechanism of growing interest in drug discovery. Critical to the mechanism of action is the formation of a ternary complex between the target, degrader and E3 ligase to promote ubiquitination and subsequent degradation. However, limited insights into ternary complex structures exist, including a near absence of studies on one of the most widely co-opted E3s, cellular inhibitor of apoptosis 1 (cIAP1). In this work, we use a combination of biochemical, biophysical and structural studies to characterize degrader-mediated ternary complexes of Bruton's tyrosine kinase and cIAP1. Our results reveal new insights from unique ternary complex structures and show that increased ternary complex stability or rigidity need not always correlate with increased degradation efficiency.

Advancing understanding of the classification of broad autism phenotype and attention-deficit/hyperactivity disorder symptom dimensions within the Hierarchical Taxonomy of Psychopathology.

Research on personality and psychopathology associations has informed the classification of many symptom dimensions within the Hierarchical Taxonomy of Psychopathology (HiTOP). However, classification of symptom dimensions defining autism and attention-deficit/hyperactivity disorder (ADHD) within the HiTOP framework remains unclear in many ways. To address this issue, we examined the joint factor structure of (a) measures assessing characteristics relevant to ADHD and autism and (b) normal range personality traits in a sample of 547 adults recruited from Amazon Mechanical Turk, many of whom reported elevated autism-relevant and ADHD-relevant characteristics. We also examined how factors identified in these analyses correlated with measures of internalizing symptoms and select externalizing traits. Our results indicated that some measures assessing autism-relevant and ADHD-relevant characteristics (e.g. communication issues, hyperactivity/impulsivity) defined a distinct Attention and Communication Difficulties factor, with scores on this factor correlating strongly with internalizing symptom ratings. However, other relevant characteristics such as aloofness may be indicators of existing HiTOP spectra such as detachment. We discuss how these findings inform classification of autism-relevant and ADHD-relevant characteristics within the HiTOP, as well as key future directions for extending the limited research in this area.

Discovery of Tyrosine Kinase 2 (TYK2) Inhibitor (PF-06826647) for the Treatment of Autoimmune Diseases.

Tyrosine kinase 2 (TYK2) is a member of the JAK kinase family that regulates signal transduction downstream of receptors for the IL-23/IL-12 pathways and type I interferon family, where it pairs with JAK2 or JAK1, respectively. On the basis of human genetic and emerging clinical data, a selective TYK2 inhibitor provides an opportunity to treat autoimmune diseases delivering a potentially differentiated clinical profile compared to currently approved JAK inhibitors. The discovery of an ATP-competitive pyrazolopyrazinyl series of TYK2 inhibitors was accomplished through computational and structurally enabled design starting from a known kinase hinge binding motif. With understanding of PK/PD relationships, a target profile balancing TYK2 potency and selectivity over off-target JAK2 was established. Lead optimization involved modulating potency, selectivity, and ADME properties which led to the identification of the clinical candidate PF-06826647 (22).

Assessing Post-Traumatic Tonic Immobility Responses: The Scale for Tonic Immobility Occurring Post-Trauma.

BACKGROUND: Peri-traumatic tonic immobility has been associated with the development and course of post-traumatic stress disorder. Despite serving as an adaptive late-stage defense response, tonic immobility that continues in response to post-traumatic reminders may lead to reduced functioning and a diminished sense of well-being. At present, no validated self-report measures assess post-traumatic tonic immobility responses specifically. METHODS: The primary objective of the present study was to evaluate the Scale for Tonic immobility Occurring Post-trauma (STOP), the first self-report measure developed to assess for the presence and severity of tonic immobility responses that persist following trauma exposure as part of post-traumatic symptomatology. Trauma-exposed clinical and non-clinical participants (N = 462) with a history of tonic immobility completed a demographic questionnaire, the STOP, and measures of post-traumatic symptoms, dissociation, anxiety, and depression. RESULTS: STOP assessed four latent constructs, which were interpreted following the human defense cascade model. Together, these factors capture the sensorimotor and perceptual alterations, and dissociative experiences, associated with post-traumatic tonic immobility as a trauma-related altered state. Residual symptoms and the experience of negative affect following this response (including guilt and shame) are also represented. STOP scores demonstrated excellent reliability, as well as good construct and convergent validity, with other measures of dissociation and post-traumatic stress disorder. Results from the present study suggest tonic immobility is most consistent with other dissociative post-traumatic symptomatology. CONCLUSIONS: STOP demonstrates excellent preliminary psychometric properties and may be useful for researchers and clinicians wishing to assess chronic forms of tonic immobility across trauma-exposed, clinical and community samples.

Dual Inhibition of TYK2 and JAK1 for the Treatment of Autoimmune Diseases: Discovery of (( S)-2,2-Difluorocyclopropyl)((1 R,5 S)-3-(2-((1-methyl-1 H-pyrazol-4-yl)amino)pyrimidin-4-yl)-3,8-diazabicyclo[3.2.1]octan-8-yl)methanone (PF-06700841).

Cytokine signaling is an important characteristic of autoimmune diseases. Many pro-inflammatory cytokines signal through the Janus kinase (JAK)/Signal transducer and activator of transcription (STAT) pathway. JAK1 is important for the γ-common chain cytokines, interleukin (IL)-6, and type-I interferon (IFN) family, while TYK2 in addition to type-I IFN signaling also plays a role in IL-23 and IL-12 signaling. Intervention with monoclonal antibodies (mAbs) or JAK1 inhibitors has demonstrated efficacy in Phase III psoriasis, psoriatic arthritis, inflammatory bowel disease, and rheumatoid arthritis studies, leading to multiple drug approvals. We hypothesized that a dual JAK1/TYK2 inhibitor will provide additional efficacy, while managing risk by optimizing selectivity against JAK2 driven hematopoietic changes. Our program began with a conformationally constrained piperazinyl-pyrimidine Type 1 ATP site inhibitor, subsequent work led to the discovery of PF-06700841 (compound 23), which is in Phase II clinical development (NCT02969018, NCT02958865, NCT03395184, and NCT02974868).

Delineating the role of cooperativity in the design of potent PROTACs for BTK.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional small molecules that simultaneously bind to a target protein and an E3 ligase, thereby leading to ubiquitination and subsequent degradation of the target. They present an exciting opportunity to modulate proteins in a manner independent of enzymatic or signaling activity. As such, they have recently emerged as an attractive mechanism to explore previously "undruggable" targets. Despite this interest, fundamental questions remain regarding the parameters most critical for achieving potency and selectivity. Here we employ a series of biochemical and cellular techniques to investigate requirements for efficient knockdown of Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase essential for B cell maturation. Members of an 11-compound PROTAC library were investigated for their ability to form binary and ternary complexes with BTK and cereblon (CRBN, an E3 ligase component). Results were extended to measure effects on BTK-CRBN cooperative interactions as well as in vitro and in vivo BTK degradation. Our data show that alleviation of steric clashes between BTK and CRBN by modulating PROTAC linker length within this chemical series allows potent BTK degradation in the absence of thermodynamic cooperativity.

Identification of N-{cis-3-[Methyl(7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino]cyclobutyl}propane-1-sulfonamide (PF-04965842): A Selective JAK1 Clinical Candidate for the Treatment of Autoimmune Diseases.

Janus kinases (JAKs) are intracellular tyrosine kinases that mediate the signaling of numerous cytokines and growth factors involved in the regulation of immunity, inflammation, and hematopoiesis. As JAK1 pairs with JAK2, JAK3, and TYK2, a JAK1-selective inhibitor would be expected to inhibit many cytokines involved in inflammation and immune function while avoiding inhibition of the JAK2 homodimer regulating erythropoietin and thrombopoietin signaling. Our efforts began with tofacitinib, an oral JAK inhibitor approved for the treatment of rheumatoid arthritis. Through modification of the 3-aminopiperidine linker in tofacitinib, we discovered highly selective JAK1 inhibitors with nanomolar potency in a human whole blood assay. Improvements in JAK1 potency and selectivity were achieved via structural modifications suggested by X-ray crystallographic analysis. After demonstrating efficacy in a rat adjuvant-induced arthritis (rAIA) model, PF-04965842 (25) was nominated as a clinical candidate for the treatment of JAK1-mediated autoimmune diseases.

Exploiting Radiation-Induced Signaling to Increase the Susceptibility of Resistant Cancer Cells to Targeted Drugs: AKT and mTOR Inhibitors as an Example.

Implementing targeted drug therapy in radio-oncologic treatment regimens has greatly improved the outcome of cancer patients. However, the efficacy of molecular targeted drugs such as inhibitory antibodies or small molecule inhibitors essentially depends on target expression and activity, which both can change during the course of treatment. Radiotherapy has previously been shown to activate prosurvival pathways, which can help tumor cells to adapt and thereby survive treatment. Therefore, we aimed to identify changes in signaling induced by radiation and evaluate the potential of targeting these changes with small molecules to increase the therapeutic efficacy on cancer cell survival. Analysis of "The Cancer Genome Atlas" database disclosed a significant overexpression of AKT1, AKT2, and MTOR genes in human prostate cancer samples compared with normal prostate gland tissue. Multifractionated radiation of three-dimensional-cultured prostate cancer cell lines with a dose of 2 Gy/day as a clinically relevant schedule resulted in an increased protein phosphorylation and enhanced protein-protein interaction between AKT and mTOR, whereas gene expression of AKT, MTOR, and related kinases was not altered by radiation. Similar results were found in a xenograft model of prostate cancer. Pharmacologic inhibition of mTOR/AKT signaling after activation by multifractionated radiation was more effective than treatment prior to radiotherapy. Taken together, our findings provide a proof-of-concept that targeting signaling molecules after activation by radiotherapy may be a novel and promising treatment strategy for cancers treated with multifractionated radiation regimens such as prostate cancer to increase the sensitivity of tumor cells to molecular targeted drugs. Mol Cancer Ther; 17(2); 355-67. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans.

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Clearance Prediction of Targeted Covalent Inhibitors by In Vitro-In Vivo Extrapolation of Hepatic and Extrahepatic Clearance Mechanisms.

The concept of target-specific covalent enzyme inhibitors appears attractive from both an efficacy and a selectivity viewpoint considering the potential for enhanced biochemical efficiency associated with an irreversible mechanism. Aside from potential safety concerns, clearance prediction of covalent inhibitors represents a unique challenge due to the inclusion of nontraditional metabolic pathways of direct conjugation with glutathione (GSH) or via GSH S-transferase-mediated processes. In this article, a novel pharmacokinetic algorithm was developed using a series of Pfizer kinase selective acrylamide covalent inhibitors based on their in vitro-in vivo extrapolation of systemic clearance in rats. The algorithm encompasses the use of hepatocytes as an in vitro model for hepatic clearance due to oxidative metabolism and GSH conjugation, and the use of whole blood as an in vitro surrogate for GSH conjugation in extrahepatic tissues. Initial evaluations with clinical covalent inhibitors suggested that the scaling algorithm developed from rats may also be useful for human clearance prediction when species-specific parameters, such as hepatocyte and blood stability and blood binding, were considered. With careful consideration of clearance mechanisms, the described in vitro-in vivo extrapolation approach may be useful to facilitate candidate optimization, selection, and prediction of human pharmacokinetic clearance during the discovery and development of targeted covalent inhibitors.

Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate.

Most gastrointestinal stromal tumors (GISTs) are caused by activating mutations of the KIT receptor tyrosine kinase. The small molecule inhibitor imatinib mesylate was initially developed to target the ABL1 kinase, which is constitutively activated through chromosomal translocation in BCR-ABL1-positive chronic myeloid leukemia. Because of cross-reactivity of imatinib against the KIT kinase, the drug is also successfully used for the treatment of GIST. Although inhibition of KIT clearly has a major role in the therapeutic response of GIST to imatinib, the contribution of concomitant inhibition of ABL in this context has never been explored. We show here that ABL1 is expressed in the majority of GISTs, including human GIST cell lines. Using siRNA-mediated knockdown, we demonstrate that depletion of KIT in conjunction with ABL1 - hence mimicking imatinib treatment - leads to reduced apoptosis induction and attenuated inhibition of cellular proliferation when compared to depletion of KIT alone. These results are explained by an increased activity of the AKT survival kinase, which is mediated by the cyclin-dependent kinase CDK2, likely through direct phosphorylation. Our results highlight that distinct inhibitory properties of targeted agents can impede antitumor effects and hence provide insights for rational drug development. Novel KIT-targeted agents to treat GIST should therefore comprise an increased specificity for KIT while at the same time displaying a reduced ability to inhibit ABL1.

Discovery of a JAK3-Selective Inhibitor: Functional Differentiation of JAK3-Selective Inhibition over pan-JAK or JAK1-Selective Inhibition.

PF-06651600, a newly discovered potent JAK3-selective inhibitor, is highly efficacious at inhibiting γc cytokine signaling, which is dependent on both JAK1 and JAK3. PF-06651600 allowed the comparison of JAK3-selective inhibition to pan-JAK or JAK1-selective inhibition, in relevant immune cells to a level that could not be achieved previously without such potency and selectivity. In vitro, PF-06651600 inhibits Th1 and Th17 cell differentiation and function, and in vivo it reduces disease pathology in rat adjuvant-induced arthritis as well as in mouse experimental autoimmune encephalomyelitis models. Importantly, by sparing JAK1 function, PF-06651600 selectively targets γc cytokine pathways while preserving JAK1-dependent anti-inflammatory signaling such as the IL-10 suppressive functions following LPS treatment in macrophages and the suppression of TNFα and IL-1ß production in IL-27-primed macrophages. Thus, JAK3-selective inhibition differentiates from pan-JAK or JAK1 inhibition in various immune cellular responses, which could potentially translate to advantageous clinical outcomes in inflammatory and autoimmune diseases.

Discovery of a Selective Covalent Inhibitor of Lysophospholipase-like 1 (LYPLAL1) as a Tool to Evaluate the Role of this Serine Hydrolase in Metabolism.

Lysophospholipase-like 1 (LYPLAL1) is an uncharacterized metabolic serine hydrolase. Human genome-wide association studies link variants of the gene encoding this enzyme to fat distribution, waist-to-hip ratio, and nonalcoholic fatty liver disease. We describe the discovery of potent and selective covalent small-molecule inhibitors of LYPLAL1 and their use to investigate its role in hepatic metabolism. In hepatocytes, selective inhibition of LYPLAL1 increased glucose production supporting the inference that LYPLAL1 is a significant actor in hepatic metabolism. The results provide an example of how a selective chemical tool can contribute to evaluating a hypothetical target for therapeutic intervention, even in the absence of complete biochemical characterization.

Latent profile analysis and principal axis factoring of the DSM-5 dissociative subtype.

OBJECTIVE: A dissociative subtype has been recognized based on the presence of experiences of depersonalization and derealization in relation to DSM-IV posttraumatic stress disorder (PTSD). However, the dissociative subtype has not been assessed in a community sample in relation to the revised DSM-5 PTSD criteria. Moreover, the 20-item PTSD Checklist for DSM-5 (PCL-5) currently does not assess depersonalization and derealization. METHOD: We therefore evaluated two items for assessing depersonalization and derealization in 557 participants recruited online who endorsed PTSD symptoms of at least moderate severity on the PCL-5. RESULTS: A five-class solution identified two PTSD classes who endorsed dissociative experiences associated with either 1) severe or 2) moderate PTSD symptom severity (D-PTSD classes). Those in the severe dissociative class were particularly likely to endorse histories of childhood physical and sexual abuse. A principal axis factor analysis of the symptom list identified six latent variables: 1) Reexperiencing, 2) Emotional Numbing/Anhedonia, 3) Dissociation, 4) Negative Alterations in Cognition & Mood, 5) Avoidance, and 6) Hyperarousal. CONCLUSIONS: The present results further support the presence of a dissociative subtype within the DSM-5 criteria for PTSD.

Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors.

Interest in drugs that covalently modify their target is driven by the desire for enhanced efficacy that can result from the silencing of enzymatic activity until protein resynthesis can occur, along with the potential for increased selectivity by targeting uniquely positioned nucleophilic residues in the protein. However, covalent approaches carry additional risk for toxicities or hypersensitivity reactions that can result from covalent modification of unintended targets. Here we describe methods for measuring the reactivity of covalent reactive groups (CRGs) with a biologically relevant nucleophile, glutathione (GSH), along with kinetic data for a broad array of electrophiles. We also describe a computational method for predicting electrophilic reactivity, which taken together can be applied to the prospective design of thiol-reactive covalent inhibitors.