Characterization of Reference Materials for DPYD: A GeT-RM Collaborative Project.

The DPYD gene encodes dihydropyrimidine dehydrogenase (DPD), which is involved in the catalysis of uracil and thymine, as well as 5-fluorouracil (5-FU), which is used to treat solid tumors. Patients with decreased DPD activity are at risk of serious, sometimes fatal, adverse drug reactions to this important cancer drug. Pharmacogenetic testing for DPYD is increasingly provided by clinical and research laboratories; however, only a limited number of quality control and reference materials are currently available for clinical DPYD testing. To address this need, the Division of Laboratory Systems, Centers for Disease Control and Prevention-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Institute for Medical Research, has characterized 33 DNA samples derived from Coriell cell lines for DPYD. Samples were distributed to four volunteer laboratories for genetic testing using a variety of commercially available and laboratory-developed tests. Sanger sequencing was used by one laboratory and publicly available whole-genome sequence data from the 1000 Genomes Project were used by another to inform genotype. Thirty-three distinct DPYD variants were identified among the 33 samples characterized. These publicly available and well-characterized materials can be used to support the quality assurance and quality control programs of clinical laboratories performing clinical pharmacogenetic testing.

Assessing Patient Values and Preferences to Inform the 2023 American College of Rheumatology/American College of Chest Physicians Interstitial Lung Disease Guidelines.

OBJECTIVE: Patient engagement is critical to clinical practice guideline (CPG) development. This work presents our approach to ascertaining patients' values and preferences to inform the American College of Rheumatology guidelines for screening, monitoring, and treatment of interstitial lung disease (ILD) in people with systemic autoimmune rheumatic diseases (SARDs). METHODS: We conducted a cross-sectional qualitative study of a purposefully sampled Patient Panel using a modified content analytic approach. The study team reviewed text transcripts from the Patient Panel discussion to identify themes and develop a clustered thematic schema. RESULTS: Twenty-one patients (75% women) participated, with a mean age of 53 years (range 33-73). Patients had one or more SARDs: systemic sclerosis (38%), Sjögren disease (38%), idiopathic inflammatory myopathy (33%), rheumatoid arthritis (24%), and mixed connective tissue disease (10%). We identified 10 themes in 4 thematic clusters: communication, screening and monitoring, treatment goals, and treatment adverse effects. Patients prioritized recognizing ILD symptoms, importance of ILD screening and close monitoring, goals of survival and improving quality of life, and willingness to accept treatment risks provided that there is close communication with providers. Patient representatives shared patients' priorities and insight at the Voting Panel meeting, influencing multiple guideline recommendations. CONCLUSION: Patient engagement fosters a holistic approach to CPG development, leading to recommendations aiming for the best clinical outcomes while prioritizing outcomes important for patients. The patient-identified themes played a critical role in ILD guideline development and provide core elements for shared decision-making as clinicians make management and therapeutic decisions with patients with SARD-associated ILD.

DPYD Genotyping Recommendations: A Joint Consensus Recommendation of the Association for Molecular Pathology, American College of Medical Genetics and Genomics, Clinical Pharmacogenetics Implementation Consortium, College of American Pathologists, Dutch Pharmacogenetics Working Group of the Royal Dutch Pharmacists Association, European Society for Pharmacogenomics and Personalized Therapy, Pharmacogenomics Knowledgebase, and Pharmacogene Variation Consortium.

The goals of the Association for Molecular Pathology Clinical Practice Committee's Pharmacogenomics (PGx) Working Group are to define the key attributes of pharmacogenetic alleles recommended for clinical testing and a minimum set of variants that should be included in clinical PGx genotyping assays. This document series provides recommendations for a minimum set of variant alleles (tier 1) and an extended list of variant alleles (tier 2) that will aid clinical laboratories when designing assays for PGx testing. The Association for Molecular Pathology PGx Working Group considered the functional impact of the variant alleles, allele frequencies in multiethnic populations, the availability of reference materials, and other technical considerations for PGx testing when developing these recommendations. The goal of this Working Group is to promote standardization of PGx testing across clinical laboratories. This document will focus on clinical DPYD PGx testing that may be applied to all dihydropyrimidine dehydrogenase-related medications. These recommendations are not to be interpreted as prescriptive but to provide a reference guide.

Neutralizing Antibody Sample Testing and Report Harmonization.

Immunogenicity testing and characterization is an important part of understanding the immune response to administration of a protein therapeutic. Neutralizing antibody (NAb) assays are used to characterize a positive anti-drug antibody (ADA) response. Harmonization of reporting of NAb assay performance and results enables efficient communication and expedient review by industry and health authorities. Herein, a cross-industry group of NAb assay experts have harmonized NAb assay reporting recommendations and provided a bioanalytical report (BAR) submission editable template developed to facilitate agency filings. This document addresses key bioanalytical reporting gaps and provides a report structure for documenting clinical NAb assay performance and results. This publication focuses on the content and presentation of the NAb sample analysis report including essential elements such as the method, critical reagents and equipment, data analysis, study samples, and results. The interpretation of immunogenicity data, including the evaluation of the impact of NAb on safety, exposure, and efficacy, is out of scope of this publication.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ~5% of all human cancers worldwide, including most cervical cancer cases and a growing number of anogenital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited the oncogenic phenotype in vitro, whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we demonstrate that the LASP1 SH3 domain is essential for LASP1-mediated oncogenicity in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulates LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrate that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival in cervical cancer, thus identifying a potential therapeutic target in these cancers.

Transcriptional Variabilities in Human hiPSC-derived Cardiomyocytes: All Genes Are Not Equal and Their Robustness May Foretell Donor's Disease Susceptibility.

Human induced pluripotent stem cells (hiPSCs) are frequently used to study disease-associated variations. We characterized transcriptional variability from a hiPSC-derived cardiomyocyte (hiPSC-CM) study of left ventricular hypertrophy (LVH) using donor samples from the HyperGEN study. Multiple hiPSC-CM differentiations over reprogramming events (iPSC generation) across 7 donors were used to assess variabilities from reprogramming, differentiation, and donor LVH status. Variability arising from pathological alterations was assessed using a cardiac stimulant applied to the hiPSC-CMs to trigger hypertrophic responses. We found that for most genes (73.3%~85.5%), technical variability was smaller than biological variability. Further, we identified and characterized lists of "noise" genes showing greater technical variability and "signal" genes showing greater biological variability. Together, they support a "genetic robustness" hypothesis of disease-modeling whereby cellular response to relevant stimuli in hiPSC-derived somatic cells from diseased donors tends to show more transcriptional variability. Our findings suggest that hiPSC-CMs can provide a valid model for cardiac hypertrophy and distinguish between technical and disease-relevant transcriptional changes.

Lack of standardization and faculty development in pediatric colonoscopy: A qualitative study.

A standard curriculum for pediatric colonoscopy training has neither been required nor universally implemented in North American fellowship programs. This qualitative study assessed the needs of colonoscopy training in pediatric gastroenterology to determine the standardized components of procedural teaching. Focus groups with pediatric gastroenterology attendings, fellows, procedural nurses, and interviews with advanced endoscopists, all practicing at a single institution, were conducted between March and June 2018. Data were analyzed using thematic analysis principles. Four themes emerged: (1) lack of standardization of colonoscopy performance, (2) lack of professional development of procedure teaching skills, (3) need for teaching behaviors that promote learner's performance, and (4) barriers to effective teaching and learning. A conceptual framework was created for developing a standardized "train-the-trainer" curriculum. Our needs assessment supports expansion of efforts to make this comprehensive training available to all pediatric gastroenterologists involved in procedure teaching.

Predicting oncology drug-induced cardiotoxicity with donor-specific iPSC-CMs-a proof-of-concept study with doxorubicin.

Many oncology drugs have been found to induce cardiotoxicity in a subset of patients, which significantly limits their clinical use and impedes the benefit of lifesaving anticancer treatments. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carry donor-specific genetic information and have been proposed for exploring the interindividual difference in oncology drug-induced cardiotoxicity. Herein, we evaluated the inter- and intraindividual variability of iPSC-CM-related assays and presented a proof of concept to prospectively predict doxorubicin (DOX)-induced cardiotoxicity (DIC) using donor-specific iPSC-CMs. Our findings demonstrated that donor-specific iPSC-CMs exhibited greater line-to-line variability than the intraindividual variability in impedance cytotoxicity and transcriptome assays. The variable and dose-dependent cytotoxic responses of iPSC-CMs resembled those observed in clinical practice and largely replicated the reported mechanisms. By categorizing iPSC-CMs into resistant and sensitive cell lines based on their time- and concentration-related phenotypic responses to DOX, we found that the sensitivity of donor-specific iPSC-CMs to DOX may predict in vivo DIC risk. Furthermore, we identified a differentially expressed gene, DND microRNA-mediated repression inhibitor 1 (DND1), between the DOX-resistant and DOX-sensitive iPSC-CMs. Our results support the utilization of donor-specific iPSC-CMs in assessing interindividual differences in DIC. Further studies will encompass a large panel of donor-specific iPSC-CMs to identify potential novel molecular and genetic biomarkers for predicting DOX and other oncology drug-induced cardiotoxicity.

E7-mediated repression of miR-203 promotes LASP1-dependent proliferation in HPV-positive cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy, contributing to ∼5% of all human cancers worldwide, including most cervical cancer cases and a growing number of ano-genital and oral cancers. The major HPV viral oncogenes, E6 and E7, manipulate many host cellular pathways that promote cell proliferation and survival, predisposing infected cells to malignant transformation. Despite the availability of highly effective vaccines, there are still no specific anti-viral therapies targeting HPV or treatments for HPV-associated cancers. As such, a better understanding of viral-host interactions may allow the identification of novel therapeutic targets. Here, we demonstrate that the actin-binding protein LASP1 is upregulated in cervical cancer and significantly correlates with a poorer overall survival. In HPV positive cervical cancer, LASP1 depletion significantly inhibited proliferation in vitro , whilst having minimal effects in HPV negative cervical cancer cells. Furthermore, we show that the LASP1 SH3 domain is essential for LASP1-mediated proliferation in these cells. Mechanistically, we show that HPV E7 regulates LASP1 at the post-transcriptional level by repressing the expression of miR-203, which negatively regulated LASP1 mRNA levels by binding to its 3'UTR. Finally, we demonstrated that LASP1 expression is required for the growth of HPV positive cervical cancer cells in an in vivo tumourigenicity model. Together, these data demonstrate that HPV induces LASP1 expression to promote proliferation and survival role in cervical cancer, thus identifying a potential therapeutic target in these cancers.

Updated DPYD HapB3 haplotype structure and implications for pharmacogenomic testing.

The DPYD gene encodes dihydropyrimidine dehydrogenase, the rate-limiting enzyme for the metabolism of fluoropyrimidines 5-fluorouracil and capecitabine. Genetic variants in DPYD have been associated with altered enzyme activity, therefore accurate detection and interpretation is critical to predict metabolizer status for individualized fluoropyrimidine therapy. The most commonly observed deleterious variation is the causal variant linked to the previously described HapB3 haplotype, c.1129-5923C>G (rs75017182) in intron 10, which introduces a cryptic splice site. A benign synonymous variant in exon 11, c.1236G>A (rs56038477) is also linked to HapB3 and is commonly used for testing. Previously, these single-nucleotide polymorphisms (SNPs) have been reported to be in perfect linkage disequilibrium (LD); therefore, c.1236G>A is often utilized as a proxy for the function-altering intronic variant. Clinical genotyping of DPYD identified a patient who had c.1236G>A, but not c.1129-5923C>G, suggesting that these two SNPs may not be in perfect LD, as previously assumed. Additional individuals with c.1236G>A, but not c.1129-5923C>G, were identified in the Children's Mercy Data Warehouse and the All of Us Research Program version 7 cohort substantiating incomplete SNP linkage. Consequently, testing only c.1236G>A can generate false-positive results in some cases and lead to suboptimal dosing that may negatively impact patient therapy and prospect of survival. Our data show that DPYD genotyping should include the functional variant c.1129-5923C>G, and not the c.1236G>A proxy, to accurately predict DPD activity.

2023 American College of Rheumatology and American Association of Hip and Knee Surgeons Clinical Practice Guideline for the Optimal Timing of Elective Hip or Knee Arthroplasty for Patients With Symptomatic Moderate-to-Severe Osteoarthritis or Advanced Symptomatic Osteonecrosis With Secondary Arthritis for Whom Nonoperative Therapy Is Ineffective.

OBJECTIVE: To develop evidence-based consensus recommendations for the optimal timing of hip and knee arthroplasty to improve patient-important outcomes including, but not limited to, pain, function, infection, hospitalization, and death at 1 year for patients with symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis of the hip or knee who have previously attempted nonoperative therapy, and for whom nonoperative therapy was ineffective, and who have chosen to undergo elective hip or knee arthroplasty (collectively referred to as TJA). METHODS: We developed 13 clinically relevant population, intervention, comparator, outcomes (PICO) questions. After a systematic literature review, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to rate the quality of evidence (high, moderate, low, or very low), and evidence tables were created. A Voting Panel, including 13 physicians and patients, discussed the PICO questions until consensus was achieved on the direction (for/against) and strength (strong/conditional) of the recommendations. RESULTS: The panel conditionally recommended against delaying TJA to pursue additional nonoperative treatment including physical therapy, nonsteroidal antiinflammatory drugs, ambulatory aids, and intraarticular injections. It conditionally recommended delaying TJA for nicotine reduction or cessation. The panel conditionally recommended delay for better glycemic control for patients who have diabetes mellitus, although no specific measure or level was identified. There was consensus that obesity by itself was not a reason for delay, but that weight loss should be strongly encouraged, and the increase in operative risk should be discussed. The panel conditionally recommended against delay in patients who have severe deformity or bone loss, or in patients who have a neuropathic joint. Evidence for all recommendations was graded as low or very low quality. CONCLUSION: This guideline provides evidence-based recommendations regarding the optimal timing of TJA in patients who have symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis for whom nonoperative therapy was ineffective to improve patient-important outcomes, including pain, function, infection, hospitalization, and death at 1 year. We acknowledge that the evidence is of low quality primarily due to indirectness and hope future research will allow for further refinement of the recommendations.

2022 American College of Rheumatology Guideline for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis.

OBJECTIVE: The objective is to update recommendations for prevention and treatment of glucocorticoid-induced osteoporosis (GIOP) for patients with rheumatic or nonrheumatic conditions receiving >3 months treatment with glucocorticoids (GCs) ≥2.5 mg daily. METHODS: An updated systematic literature review was performed for clinical questions on nonpharmacologic, pharmacologic treatments, discontinuation of medications, and sequential therapy. Grading of Recommendations Assessment, Development and Evaluation approach was used to rate the certainty of evidence. A Voting Panel achieved ≥70% consensus on the direction (for or against) and strength (strong or conditional) of recommendations. RESULTS: For adults beginning or continuing >3 months of GC treatment, we strongly recommend as soon as possible after initiation of GCs, initial assessment of fracture risks with clinical fracture assessment, bone mineral density with vertebral fracture assessment or spinal x-ray, and Fracture Risk Assessment Tool if ≥40 years old. For adults at medium, high, or very high fracture risk, we strongly recommend pharmacologic treatment. Choice of oral or intravenous bisphosphonates, denosumab, or parathyroid hormone analogs should be made by shared decision-making. Anabolic agents are conditionally recommended as initial therapy for those with high and very high fracture risk. Recommendations are made for special populations, including children, people with organ transplants, people who may become pregnant, and people receiving very high-dose GC treatment. New recommendations for both discontinuation of osteoporosis therapy and sequential therapies are included. CONCLUSION: This guideline provides direction for clinicians and patients making treatment decisions for management of GIOP. These recommendations should not be used to limit or deny access to therapies.

2022 American College of Rheumatology Guideline for the Prevention and Treatment of Glucocorticoid-Induced Osteoporosis.

OBJECTIVE: The objective is to update recommendations for prevention and treatment of glucocorticoid-induced osteoporosis (GIOP) for patients with rheumatic or nonrheumatic conditions receiving >3 months treatment with glucocorticoids (GCs) ≥2.5 mg daily. METHODS: An updated systematic literature review was performed for clinical questions on nonpharmacologic, pharmacologic treatments, discontinuation of medications, and sequential therapy. Grading of Recommendations Assessment, Development and Evaluation approach was used to rate the certainty of evidence. A Voting Panel achieved ≥70% consensus on the direction (for or against) and strength (strong or conditional) of recommendations. RESULTS: For adults beginning or continuing >3 months of GC treatment, we strongly recommend as soon as possible after initiation of GCs, initial assessment of fracture risks with clinical fracture assessment, bone mineral density with vertebral fracture assessment or spinal x-ray, and Fracture Risk Assessment Tool if ≥40 years old. For adults at medium, high, or very high fracture risk, we strongly recommend pharmacologic treatment. Choice of oral or intravenous bisphosphonates, denosumab, or parathyroid hormone analogs should be made by shared decision-making. Anabolic agents are conditionally recommended as initial therapy for those with high and very high fracture risk. Recommendations are made for special populations, including children, people with organ transplants, people who may become pregnant, and people receiving very high-dose GC treatment. New recommendations for both discontinuation of osteoporosis therapy and sequential therapies are included. CONCLUSION: This guideline provides direction for clinicians and patients making treatment decisions for management of GIOP. These recommendations should not be used to limit or deny access to therapies.

2023 American College of Rheumatology and American Association of Hip and Knee Surgeons Clinical Practice Guideline for the Optimal Timing of Elective Hip or Knee Arthroplasty for Patients With Symptomatic Moderate-to-Severe Osteoarthritis or Advanced Symptomatic Osteonecrosis With Secondary Arthritis for Whom Nonoperative Therapy Is Ineffective.

OBJECTIVE: To develop evidence-based consensus recommendations for the optimal timing of hip and knee arthroplasty to improve patient-important outcomes including, but not limited to, pain, function, infection, hospitalization, and death at 1 year for patients with symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis of the hip or knee who have previously attempted nonoperative therapy, and for whom nonoperative therapy was ineffective, and who have chosen to undergo elective hip or knee arthroplasty (collectively referred to as TJA). METHODS: We developed 13 clinically relevant population, intervention, comparator, outcomes (PICO) questions. After a systematic literature review, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to rate the quality of evidence (high, moderate, low, or very low), and evidence tables were created. A Voting Panel, including 13 physicians and patients, discussed the PICO questions until consensus was achieved on the direction (for/against) and strength (strong/conditional) of the recommendations. RESULTS: The panel conditionally recommended against delaying TJA to pursue additional nonoperative treatment including physical therapy, nonsteroidal antiinflammatory drugs, ambulatory aids, and intraarticular injections. It conditionally recommended delaying TJA for nicotine reduction or cessation. The panel conditionally recommended delay for better glycemic control for patients who have diabetes mellitus, although no specific measure or level was identified. There was consensus that obesity by itself was not a reason for delay, but that weight loss should be strongly encouraged, and the increase in operative risk should be discussed. The panel conditionally recommended against delay in patients who have severe deformity or bone loss, or in patients who have a neuropathic joint. Evidence for all recommendations was graded as low or very low quality. CONCLUSION: This guideline provides evidence-based recommendations regarding the optimal timing of TJA in patients who have symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis for whom nonoperative therapy was ineffective to improve patient-important outcomes, including pain, function, infection, hospitalization, and death at 1 year. We acknowledge that the evidence is of low quality primarily due to indirectness and hope future research will allow for further refinement of the recommendations.

2023 American College of Rheumatology and American Association of Hip and Knee Surgeons Clinical Practice Guideline for the Optimal Timing of Elective Hip or Knee Arthroplasty for Patients With Symptomatic Moderate-to-Severe Osteoarthritis or Advanced Symptomatic Osteonecrosis With Secondary Arthritis for Whom Nonoperative Therapy Is Ineffective.

OBJECTIVE: To develop evidence-based consensus recommendations for the optimal timing of hip and knee arthroplasty to improve patient-important outcomes including, but not limited to, pain, function, infection, hospitalization, and death at 1 year for patients with symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis of the hip or knee who have previously attempted nonoperative therapy, and for whom nonoperative therapy was ineffective, and who have chosen to undergo elective hip or knee arthroplasty (collectively referred to as TJA). METHODS: We developed 13 clinically relevant population, intervention, comparator, outcomes (PICO) questions. After a systematic literature review, the Grading of Recommendations Assessment, Development and Evaluation (GRADE) approach was used to rate the quality of evidence (high, moderate, low, or very low), and evidence tables were created. A Voting Panel, including 13 physicians and patients, discussed the PICO questions until consensus was achieved on the direction (for/against) and strength (strong/conditional) of the recommendations. RESULTS: The panel conditionally recommended against delaying TJA to pursue additional nonoperative treatment including physical therapy, nonsteroidal antiinflammatory drugs, ambulatory aids, and intraarticular injections. It conditionally recommended delaying TJA for nicotine reduction or cessation. The panel conditionally recommended delay for better glycemic control for patients who have diabetes mellitus, although no specific measure or level was identified. There was consensus that obesity by itself was not a reason for delay, but that weight loss should be strongly encouraged, and the increase in operative risk should be discussed. The panel conditionally recommended against delay in patients who have severe deformity or bone loss, or in patients who have a neuropathic joint. Evidence for all recommendations was graded as low or very low quality. CONCLUSION: This guideline provides evidence-based recommendations regarding the optimal timing of TJA in patients who have symptomatic and radiographic moderate-to-severe osteoarthritis or advanced symptomatic osteonecrosis with secondary arthritis for whom nonoperative therapy was ineffective to improve patient-important outcomes, including pain, function, infection, hospitalization, and death at 1 year. We acknowledge that the evidence is of low quality primarily due to indirectness and hope future research will allow for further refinement of the recommendations.

A case of neonatal sweet syndrome associated with mevalonate kinase deficiency.

BACKGROUND: Sweet syndrome (SS), also known as acute febrile neutrophilic dermatosis, is an immunologic syndrome characterized by widespread neutrophilic infiltration. Histiocytoid Sweet syndrome (H-SS) is a histopathologic variant of SS. While SS most commonly occurs in adults, this case report discusses an infant patient who presented with H-SS. CASE PRESENTATION: Through a multidisciplinary approach, this patient was also found to have very early onset inflammatory bowel disease (VEO-IBD) and Mevalonate kinase-associated disease (MKAD). While prior case studies have characterized an association between VEO-IBD and MKAD, there is no literature describing the association of all three diagnoses this case: H-SS, VEO-IBD and MKAD. Initiation of canakinumab in this patient resulted in successful control of the disease. CONCLUSIONS: This case highlights the importance of a multidisciplinary approach to rare diagnoses, and collaboration during cases with significant diagnostic uncertainty.

Affimer-mediated locking of p21-activated kinase 5 in an intermediate activation state results in kinase inhibition.

Kinases are important therapeutic targets, and their inhibitors are classified according to their mechanism of action, which range from blocking ATP binding to covalent inhibition. Here, a mechanism of inhibition is highlighted by capturing p21-activated kinase 5 (PAK5) in an intermediate state of activation using an Affimer reagent that binds in the P+1 pocket. PAK5 was identified from a non-hypothesis-driven high-content imaging RNAi screen in urothelial cancer cells. Silencing of PAK5 resulted in reduced cell number, G1/S arrest, and enlargement of cells, suggesting it to be important in urothelial cancer cell line survival and proliferation. Affimer reagents were isolated to identify mechanisms of inhibition. The Affimer PAK5-Af17 recapitulated the phenotype seen with siRNA. Co-crystallization revealed that PAK5-Af17 bound in the P+1 pocket of PAK5, locking the kinase into a partial activation state. This mechanism of inhibition indicates that another class of kinase inhibitors is possible.