A chemical probe to modulate human GID4 Pro/N-degron interactions.

The C-terminal to LisH (CTLH) complex is a ubiquitin ligase complex that recognizes substrates with Pro/N-degrons via its substrate receptor Glucose-Induced Degradation 4 (GID4), but its function and substrates in humans remain unclear. Here, we report PFI-7, a potent, selective and cell-active chemical probe that antagonizes Pro/N-degron binding to human GID4. Use of PFI-7 in proximity-dependent biotinylation and quantitative proteomics enabled the identification of GID4 interactors and GID4-regulated proteins. GID4 interactors are enriched for nucleolar proteins, including the Pro/N-degron-containing RNA helicases DDX21 and DDX50. We also identified a distinct subset of proteins whose cellular levels are regulated by GID4 including HMGCS1, a Pro/N-degron-containing metabolic enzyme. These data reveal human GID4 Pro/N-degron targets regulated through a combination of degradative and nondegradative functions. Going forward, PFI-7 will be a valuable research tool for investigating CTLH complex biology and facilitating development of targeted protein degradation strategies that highjack CTLH E3 ligase activity.

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.

Understanding Data Noise and Uncertainty through Analysis of Replicate Samples in DNA-Encoded Library Selection.

By analyzing data sets of replicate DNA-Encoded Library (DEL) selections, an approach for estimating the noise level of the experiment has been developed. Using a logarithm transformation of the number of counts associated with each compound and a subset of compounds with the highest number of counts, it is possible to assess the quality of the data through normalizing the replicates and use this same data to estimate the noise in the experiment. The noise level is seen to be dependent on sequencing depth as well as specific selection conditions. The noise estimation is independent of any cutoff used to remove low frequency compounds from the data analysis. The removal of compounds with only 1-5 read counts greatly reduces some of the challenges encountered in DEL data analysis as it can reduce the data set by greater than 100-fold without impacting the interpretation of the results.

Toward the assembly and characterization of an encoded library hit confirmation platform: Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS).

The ability to predict chemical structure from DNA sequence has to date been a necessary cornerstone of DNA-encoded library technology. DNA-encoded libraries (DELs) are typically screened by immobilized affinity selection and enriched library members are identified by counting the number of times an individual compound's sequence is observed in the resultant dataset. Those with high signal reads (DEL hits) are subsequently followed up through off-DNA synthesis of the predicted small molecule structures. However, hits followed-up in this manner often fail to translate to confirmed ligands. To address this low conversion rate of DEL hits to off-DNA ligands, we have developed an approach that eliminates the reliance on chemical structure prediction from DNA sequence. Here we describe our method of combining non-combinatorial resynthesis on-DNA following library procedures as a rapid means to assess the probable molecules attached to the DNA barcode. Furthermore, we apply our Bead-Assisted Ligand Isolation Mass Spectrometry (BALI-MS) technique to identify the true binders found within the mixtures of on-DNA synthesis products. Finally, we describe a Normalized Enrichment (NE) metric that allows for the quantitative assessment of affinity selection in these studies. We exemplify how this combined approach enables the identification of putative hit matter against a clinically relevant therapeutic target bisphosphoglycerate mutase, BPGM.

Which Risk Factors Are Associated with Pain and Patient-reported Function in Patients with a Rotator Cuff Tear?

BACKGROUND: Patient-reported measures guide physicians in clinical decision making and therefore it is critical to determine what clinical factors are associated with these scores. Psychological and physical factors are commonly studied separately in patients with rotator cuff tears to determine their influence on outcomes. It is well established that psychological distress and scapular motion change in the presence of a symptomatic rotator cuff tear. However, these factors have not been studied simultaneously in a clinical setting to determine their association with shoulder outcome scores. QUESTION/PURPOSE: After controlling for relevant confounding variables, what physical and psychological factors are associated with better (1) American Shoulder and Elbow Surgeons (ASES) scores for function, (2) ASES pain scores, and (3) total ASES scores? METHODS: Fifty-nine patients with a potential symptomatic rotator cuff tear were recruited and agreed to participate in this cross-sectional study. Of those, 85% (50 of 59) met eligibility criteria for a primary diagnosis of an MRI-confirmed symptomatic partial-thickness or full-thickness rotator cuff tear without a history of shoulder surgery. Demographics, rotator cuff tear size, arm flexion, and clinical scapular motion during active arm flexion were evaluated by experienced examiners using standardized procedures. Patients completed the ASES questionnaire and the Optimal Screening for Prediction of Referral and Outcomes-Yellow Flag assessment form, which measures 11 different pain-related psychological distress symptoms. Three separate stepwise multiple linear regression analyses were performed for ASES pain, function, and total scores, with significance set at p < 0.05. RESULTS: This model found that ASES function scores were associated with four factors: older age, increased arm flexion, increased percentage of scapular external rotation during arm flexion, and increased scores for acceptance of chronic pain (adjusted r2 = 0.67; p = 0.01). Those four factors appear to explain 67% of the observed variance in ASES function scores in patients with rotator cuff tears. Furthermore, increased percentage of scapular external rotation during arm flexion and decreased fear-avoidance beliefs related to physical activity scores (adjusted r2 = 0.36; p < 0.01) were associated with better ASES pain scores. And finally, better ASES total scores were associated with four factors: increased arm flexion, increased percentage of scapular upward rotation, increased scapular external rotation during arm flexion, and decreased fear-avoidance beliefs related to physical activity scores (adjusted r2 = 0.65; p < 0.001). CONCLUSION: Our results favor adopting a comprehensive biopsychological clinical assessment for patients with rotator cuff tears that specifically includes humeral and scapular motion, fear-avoidance behaviors, and pain coping behaviors along with demographics. These particular physical and psychological variables were found to be associated with the ASES and, therefore, should be clinically examined simultaneously and targeted as part of a tailored treatment plan. LEVEL OF EVIDENCE: Level II, prognostic study.

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.

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.

Chemical tools for the Gid4 subunit of the human E3 ligase C-terminal to LisH (CTLH) degradation complex.

We have developed a novel chemical handle (PFI-E3H1) and a chemical probe (PFI-7) as ligands for the Gid4 subunit of the human E3 ligase CTLH degradation complex. Through an efficient initial hit-ID campaign, structure-based drug design (SBDD) and leveraging the sizeable Pfizer compound library, we identified a 500 nM ligand for this E3 ligase through file screening alone. Further exploration identified a vector that is tolerant to addition of a linker for future chimeric molecule design. The chemotype was subsequently optimized to sub-100 nM Gid4 binding affinity for a chemical probe. These novel tools, alongside the suitable negative control also identified, should enable the interrogation of this complex human E3 ligase macromolecular assembly.

Rectus abdominus tear in a collegiate cheerleader.

Acute muscle injuries can occur at all skill levels across multiple different sports. Imaging is often used for diagnosis and is necessary for recognizing the extent of injury, complications, and overall prognosis. Ultrasound can be used in diagnosing muscle tears. However, there are less than a dozen documented cases that use ultrasound in the evaluation and diagnosis of a rectus abdominus tear, though the actual number of cases are most likely underreported in the literature. This case discusses an 18-year-old college athlete that suffered a distal rectus abdominus tear after performing a stunt as a cheerleader and her subsequent treatment.

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents.

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.

Imaging findings of a biopsy-proven desmoid tumor of the axilla in a young female.

Desmoid tumors (fibromatoses) are rare but locally aggressive tumors that do not metastasize. They are non-encapsulated, well-differentiated lesions made of fibroblasts and collagen, which mainly appear in the mesentery and abdominal wall. Rarely, these tumors can also occur in breasts, making up approximately 0.2% of all breast neoplasms. Treatment typically includes surgical excision and/or medical management. We describe a case of a 31-year-old female presenting with a mass in her left axilla that was biopsy proven to be a desmoid tumor. In this case report, we discuss the various imaging findings present on ultrasound, mammography, computed tomography, and magnetic resonance imaging.

Isolated distal semitendinosus tendon tear in a collegiate athlete.

Complete distal semitendinosus tendon tears are rare hamstring injuries. They often present with a "pop" followed by delayed pain and swelling. We describe the case of a collegiate athlete with an isolated distal semitendinosus tendon tear diagnosed by MRI and ultrasound after being treated for a stress fracture of the anterior tibial shaft with an intramedullary nail. The tear progressed from partial to complete after 2 weeks of rehabilitation and return to practice. The patient was ultimately successfully treated with semitendinosus tenodesis.

Bilateral popliteal entrapment syndrome in a young athlete diagnosed with ultrasound.

Popliteal artery entrapment syndrome (PAES) occurs when the popliteal artery is compressed by abnormally developed or hypertrophied muscles adjacent to the popliteal fossa. When symptomatic, it most frequently presents with leg cramping while walking or running. We describe the case of an 18-year-old female runner presenting with claudication and exercise intolerance. After MRI was non-diagnostic, diagnostic ultrasound demonstrated that she had functional (Type VI) PAES. She subsequently underwent popliteal artery release surgery. Type VI PAES should be considered in young, healthy patients who present with claudication, particularly athletes.

Mommy's thumb: De Quervain's tenosynovitis in a new mother with cardiomyopathy.

Mommy's thumb is a lay term for de Quervain's tenosynovitis in mothers of newborn to young children. It is most commonly the result of carrying the child, leading to overuse of the wrist. Less commonly, it can also result from fluid retention resulting from lactational changes. We present a case report of a first-time mother presenting with bilateral de Quirvain's tenosynovitis which may be attributed to a previously undiagnosed cardiomyopathy leading to fluid retention.

Slipping rib syndrome presentation in a young woman.

Slipping rib syndrome (SRS) disorder is the hypermobility of the costal cartilages attached to the floating ribs. Causes include weakness of the interchondral ligaments, and less commonly congenital rib deformities or direct trauma. Due to its location (right upper quadrant) and symptomatic presentation, the differential for this syndrome is especially broad, and as a result, even though this syndrome may make up to 5% of visits for lower chest/upper abdominal pain, it is frequently underdiagnosed, and patients often undergo excessive workup. Treatment includes conservative management, physical therapy, intercostal nerve blocks and for refractory cases, surgical intervention. We describe a case of a 43-year-old female presenting with Slipping Rib Syndrome (SRS) that was confirmed with an ultrasound. In this case report, we discuss presentation of SRS, diagnostic maneuvers and image finding of SRS, and the treatment of SRS.

Selecting Approaches for Hit Identification and Increasing Options by Building the Efficient Discovery of Actionable Chemical Matter from DNA-Encoded Libraries.

Over the past 20 years, the toolbox for discovering small-molecule therapeutic starting points has expanded considerably. Pharmaceutical researchers can now choose from technologies that, in addition to traditional high-throughput knowledge-based and diversity screening, now include the screening of fragment and fragment-like libraries, affinity selection mass spectrometry, and selection against DNA-encoded libraries (DELs). Each of these techniques has its own unique combination of advantages and limitations that makes them more, or less, suitable for different target classes or discovery objectives, such as desired mechanism of action. Layered on top of this are the constraints of the drug-hunters themselves, including budgets, timelines, and available platform capacity; each of these can play a part in dictating the hit identification strategy for a discovery program. In this article, we discuss some of the factors that we use to govern our building of a hit identification roadmap for a program and describe the increasing role that DELs are playing in our discovery strategy. Furthermore, we share our learning during our initial exploration of DEL and highlight the approaches we have evolved to maximize the value returned from DEL selections. Topics addressed include the optimization of library design and production, reagent validation, data analysis, and hit confirmation. We describe how our thinking in these areas has led us to build a DEL platform that has begun to deliver tractable matter to our global discovery portfolio.

Discovery and SAR of benzyl phenyl ethers as inhibitors of bacterial phenylalanyl-tRNA synthetase.

A series of benzyl phenyl ethers (BPEs) is described that displays potent inhibition of bacterial phenylalanyl-tRNA synthetase. The synthesis, SAR, and select ADMET data are provided.

A Solution Phase Platform to Characterize Chemical Reaction Compatibility with DNA-Encoded Chemical Library Synthesis.

DNA-encoded chemical library (DECL) synthesis must occur in aqueous media under conditions that preserve the integrity of the DNA encoding tag. While the identification of "DNA-compatible" reaction conditions is critical for the development of DECL designs that explore previously inaccessible chemical space, reports measuring such compatibility have been largely restricted to methods that do not faithfully capture the impact of reaction conditions on DNA fidelity in solution phase. Here we report a comprehensive methodology that uses soluble DNA substrates that exactly recapitulate DNA's exposure to the chemically reactive species of DECL synthesis. This approach includes the assessment of chemical fidelity (reaction yield and purity), encoding fidelity (ligation efficiency), and readability (DNA compatibility), revealing the fate of the DNA tag during DECL chemistry from a single platform.

Determining molecular properties with differential mobility spectrometry and machine learning.

The fast and accurate determination of molecular properties is highly desirable for many facets of chemical research, particularly in drug discovery where pre-clinical assays play an important role in paring down large sets of drug candidates. Here, we present the use of supervised machine learning to treat differential mobility spectrometry - mass spectrometry data for ten topological classes of drug candidates. We demonstrate that the gas-phase clustering behavior probed in our experiments can be used to predict the candidates' condensed phase molecular properties, such as cell permeability, solubility, polar surface area, and water/octanol distribution coefficient. All of these measurements are performed in minutes and require mere nanograms of each drug examined. Moreover, by tuning gas temperature within the differential mobility spectrometer, one can fine tune the extent of ion-solvent clustering to separate subtly different molecular geometries and to discriminate molecules of very similar physicochemical properties.

Discovery of Trifluoromethyl Glycol Carbamates as Potent and Selective Covalent Monoacylglycerol Lipase (MAGL) Inhibitors for Treatment of Neuroinflammation.

Monoacylglycerol lipase (MAGL) inhibition provides a potential treatment approach to neuroinflammation through modulation of both the endocannabinoid pathway and arachidonoyl signaling in the central nervous system (CNS). Herein we report the discovery of compound 15 (PF-06795071), a potent and selective covalent MAGL inhibitor, featuring a novel trifluoromethyl glycol leaving group that confers significant physicochemical property improvements as compared with earlier inhibitor series with more lipophilic leaving groups. The design strategy focused on identifying an optimized leaving group that delivers MAGL potency, serine hydrolase selectivity, and CNS exposure while simultaneously reducing log  D, improving solubility, and minimizing chemical lability. Compound 15 achieves excellent CNS exposure, extended 2-AG elevation effect in vivo, and decreased brain inflammatory markers in response to an inflammatory challenge.