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Autosomal-recessive mutations inSPEG(striated muscle preferentially expressed protein kinase) have been linked to centronuclear myopathy. Loss of SPEG is associated with defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling in skeletal muscles. To elucidate the underlying molecular pathways, we have utilized multi-omics tools and analysis to obtain a comprehensive view of the complex biological processes. We identified that SPEG interacts with myospryn complex proteins (CMYA5, FSD2, RyR1), and SPEG deficiency results in myospryn complex abnormalities. In addition, transcriptional and protein profiles of SPEG-deficient muscle revealed defective mitochondrial function including aberrant accumulation of enlarged mitochondria on electron microscopy. Furthermore, SPEG regulates RyR1 phosphorylation at S2902, and its loss affects JPH2 phosphorylation at multiple sites. On analyzing the transcriptome, the most dysregulated pathways affected by SPEG deficiency included extracellular matrix-receptor interaction and peroxisome proliferator-activated receptors signaling, which may be due to defective triad and mitochondrial abnormalities. In summary, we have elucidated the critical role of SPEG in triad as it works closely with myospryn complex, phosphorylates JPH2 and RyR1, and demonstrated that its deficiency is associated with mitochondrial abnormalities. This study emphasizes the importance of using multi-omics techniques to comprehensively analyze the molecular anomalies of rare diseases.SynopsisWe have previously linked mutations inSPEG(striated preferentially expressed protein) with a recessive form of centronuclear myopathy and/or dilated cardiomyopathy and have characterized a striated muscle-specific SPEG-deficient mouse model that recapitulates human disease with disruption of the triad structure and calcium homeostasis in skeletal muscles. In this study, we applied multi-omics approaches (interactomic, proteomic, phosphoproteomic, and transcriptomic analyses) in the skeletal muscles of SPEG-deficient mice to assess the underlying pathways associated with the pathological and molecular abnormalities.SPEG interacts with myospryn complex proteins (CMYA5, FSD2, RyR1), and its deficiency results in myospryn complex abnormalities.SPEG regulates RyR1 phosphorylation at S2902, and its loss affects JPH2 phosphorylation at multiple sites.SPEGα and SPEGβ have different interacting partners suggestive of differential function.Transcriptome analysis indicates dysregulated pathways of ECM-receptor interaction and peroxisome proliferator-activated receptor signaling.Mitochondrial defects on the transcriptome, proteome, and electron microscopy, may be a consequence of defective calcium signaling.

Background and aimsWNT2B is a canonical Wnt ligand previously thought to be fully redundant with other Wnts in the intestinal epithelium. However, humans with WNT2B deficiency have severe intestinal disease, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.MethodsWe investigated the intestinal health ofWnt2bknock out (KO) mice. We assessed the impact of inflammatory challenge to the small intestine, using anti-CD3χ antibody, and to the colon, using dextran sodium sulfate (DSS). In addition, we generated human intestinal organoids (HIOs) from WNT2B-deficient human iPSCs for transcriptional and histological analyses.ResultsMice with WNT2B deficiency had significantly decreasedLgr5expression in the small intestine and profoundly decreased expression in the colon, but normal baseline histology. The small intestinal response to anti-CD3χ antibody was similar inWnt2bKO and wild type (WT) mice. In contrast, the colonic response to DSS inWnt2bKO mice showed an accelerated rate of injury, featuring earlier immune cell infiltration and loss of differentiated epithelium compared to WT. WNT2B-deficient HIOs showed abnormal epithelial organization and an increased mesenchymal gene signature.ConclusionWNT2B contributes to maintenance of the intestinal stem cell pool in mice and humans. WNT2B deficient mice, which do not have a developmental phenotype, show increased susceptibility to colonic injury but not small intestinal injury, potentially due to a higher reliance on WNT2B in the colon compared to the small intestine.WNT2B deficiency causes a developmental phenotype in human intestine with HIOs showing a decrease in their mesenchymal component and WNT2B-deficient patients showing epithelial disorganization.Data Transparency StatementAll RNA-Seq data will be available through online repository as indicated in Transcript profiling. Any other data will be made available upon request by emailing the study authors.

Supplementary Figure 2 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Supplementary Figure 1 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Recepteur d'origine nantais (RON) is a receptor tyrosine kinase closely related to c-Met. Both receptors are involved in cell proliferation, migration, and invasion, and there is evidence that both are deregulated in cancer. Receptor overexpression has been most frequently described, but other mechanisms can lead to the oncogenic activation of RON and c-Met. They include activating mutations or gene amplification for c-Met and constitutively active splicing variants for RON. We identified a novel inhibitor of RON and c-Met, compound I, and characterized its in vitro and in vivo activities. Compound I selectively and potently inhibited the kinase activity of RON and c-Met with IC50s of 9 and 4 nmol/L, respectively. Compound I inhibited hepatocyte growth factor–mediated and macrophage-stimulating protein–mediated signaling and cell migration in a dose-dependent manner. Compound I was tested in vivo in xenograft models that either were dependent on c-Met or expressed a constitutively active form of RON (RONΔ160 in HT-29). Compound I caused complete tumor growth inhibition in NIH3T3 TPR-Met and U-87 MG xenografts but showed only partial inhibition in HT-29 xenografts. The effect of compound I in HT-29 xenografts is consistent with the expression of the activating b-Raf V600E mutation, which activates the mitogen-activated protein kinase pathway downstream of RON. Importantly, tumor growth inhibition correlated with the inhibition of c-Met–dependent and RON-dependent signaling in tumors. Taken together, our results suggest that a small-molecule dual inhibitor of RON/c-Met has the potential to inhibit tumor growth and could therefore be useful for the treatment of patients with cancers where RON and/or c-Met are activated. [Cancer Res 2008;68(16):6680–7]

Supplementary Figure Legends 1-3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

Supplementary Figure 3 from Identification of a Novel Recepteur d'Origine Nantais/c-Met Small-Molecule Kinase Inhibitor with Antitumor Activity In vivo

The integration of genomic testing into clinical care enables the use of individualized approaches to the management of rare diseases. We describe the use of belzutifan, a potent and selective small-molecule inhibitor of the protein hypoxia-inducible factor 2α (HIF2α), in a patient with polycythemia and multiple paragangliomas (the Pacak-Zhuang syndrome). The syndrome was caused in this patient by somatic mosaicism for an activating mutation in EPAS1. Treatment with belzutifan led to a rapid and sustained tumor response along with resolution of hypertension, headaches, and long-standing polycythemia. This case shows the application of a targeted therapy for the treatment of a patient with a rare tumor-predisposition syndrome. (Funded by the Morin Family Fund for Pediatric Cancer and Alex's Lemonade Stand Foundation.).

Purpose To develop nomograms that predict the detection of clinically significant prostate cancer at diagnostic biopsy based on multiparametric prostate MRI (mpMRI), serum biomarkers, and patient clinicodemographic features. Materials and Methods Nomograms were developed from a cohort of biopsy-naïve men presenting to our 11-hospital system with a PSA of 2-20ng/mL who underwent pre-biopsy mpMRI from March 2018-June 2021 (n = 1494). The outcomes were the presence of clinically significant and high-grade prostate cancer (defined as ≥ GG2 [Grade Group 2] and ≥ GG3 prostate cancer, respectively). Using significant variables on multivariable logistic regression, individual nomograms were developed for men with PSA, % free PSA, or prostate health index (PHI) when available. The nomograms were both internally validated and evaluated in an independent cohort of 366 men presenting to our hospital system from July 2021-February 2022. Results 1031 of 1494 men (69%) underwent biopsy after initial evaluation with mpMRI, 493 (47.8%) of whom were found to have ≥ GG2 PCa, and 271 (26.3%) were found to have ≥ GG3 PCa. Age, race, highest PIRADS score, prostate health index (PHI) when available, % free PSA when available, and PSA density were significant predictors of ≥ GG2 and ≥ GG3 PCa on multivariable analysis and were used for nomogram generation. Accuracy of nomograms in both the training cohort and independent cohort were high, with areas under the curves (AUC) of ≥ 0.885 in the training cohort and ≥ 0.896 in the independent validation cohort. In our independent validation cohort, our model for ≥ GG2 prostate cancer with PHI saved 39.1% of biopsies (143/366) while only missing 0.8% of csPCa (1/124) with a biopsy threshold of 20% probability of csPCa. Conclusions Here we developed nomograms combining serum testing and mpMRI to help clinicians risk stratify patients with elevated PSA of 2-20ng/mL who are being considered for biopsy. Our nomograms are available at https://rossnm1.shinyapps.io/MynMRIskCalculator/ to aid with biopsy decisions.

Objectives: To quantify the volume and characterize the accuracy of men’s health-related content on TikTok and Instagram Methods: We conducted a search on both TikTok and Instagram for the terms: testosterone, erectile dysfunction, male infertility, semen retention, Peyronie’s disease, and vasectomy. The top 10 hashtags for each term were identified and used to estimate the total number of impressions for each term on each platform. We evaluated the top 40 posts for each topic on each platform and characterized the post by creator type, content type, and accuracy. Content accuracy was scored on a scale from one to five, with one being completely inaccurate information and five being completely accurate.Results: Overall, TikTok had more than 2.3 billion impressions and Instagran had more than 3 million posts across the men’s health topics. Semen retention had the most impressions on TikTok (1.2 billion) and posts on Instagram (1 million). Physicians created only a small portion of content comprising only 10.3% and 12.9% of total TikTok and Instagram posts, respectfully. Across all men’s health topics, the accuracy of content was poor with a score of 2.6 +/- 1.7 but physician posts were significantly more accurate than non-physician posts (4.2 vs 2.3, p < 0.001 respectfully).Conclusions: Men’s health content is popular on TikTok and Instagram but has a low level of overall accuracy. Given the higher accuracy of physician-created content, we recommend that physicians actively engage, on both individual and organizational levels, in social media to address misinformation

Striated preferentially expressed gene (SPEG), a member of the myosin light chain kinase family, is localized at the level of triad surrounding myofibrils in skeletal muscles. In humans, SPEG mutations are associated with centronuclear myopathy and cardiomyopathy. Using a striated muscle-specific Speg-knockout (KO) mouse model, we have previously shown that SPEG is critical for triad maintenance and calcium handling. Here, we further examined the molecular function of SPEG and characterized the effects of SPEG deficiency on triad and focal adhesion proteins. We used yeast two-hybrid assay, and identified desmin, an intermediate filament protein, to interact with SPEG and confirmed this interaction by co-immunoprecipitation. Using domain-mapping assay, we defined that Ig-like and fibronectin III domains of SPEG interact with rod domain of desmin. In skeletal muscles, SPEG depletion leads to desmin aggregates in vivo and a shift in desmin equilibrium from soluble to insoluble fraction. We also profiled the expression and localization of triadic proteins in Speg-KO mice using western blot and immunofluorescence. The amount of RyR1 and triadin were markedly reduced, whereas DHPRα1, SERCA1 and triadin were abnormally accumulated in discrete areas of Speg-KO myofibers. In addition, Speg-KO muscles exhibited internalized vinculin and β1 integrin, both of which are critical components of the focal adhesion complex. Further, β1 integrin was abnormally accumulated in early endosomes of Speg-KO myofibers. These results demonstrate that SPEG-deficient skeletal muscles exhibit several pathological features similar to those seen in MTM1 deficiency. Defects of shared cellular pathways may underlie these structural and functional abnormalities in both types of diseases.

Centronuclear myopathies (CNMs) are a subtype of congenital myopathies characterized by skeletal muscle weakness and an increase in the number of central myonuclei. SPEG (striated preferentially expressed protein kinase) has been identified as the sixth gene associated with CNM, and it has been shown that striated muscle-specific Speg-knockout (KO) mice have defective triad formation, abnormal excitation-contraction coupling, and calcium mishandling. The impact of SPEG deficiency on the survival and function of myogenic cells remains to be deciphered. In this study, the authors examined the overall population, proliferation, and differentiation of myogenic cells obtained from striated muscle-specific Speg-KO mice and compared them with wild-type (WT) controls. SPEG-deficient skeletal muscles contained fewer myogenic cells, which on further study demonstrated reduced proliferation and delayed differentiation compared with those from WT muscles. Regenerative response to skeletal muscle injury in Speg-KO mice was compared with that of WT mice, leading to the identification of similar abnormalities including fewer satellite cells, fewer dividing cells, and an increase in apoptotic cells in KO mice. Overall, these results reveal specific abnormalities in myogenic cell number and behavior associated with SPEG deficiency. Similar satellite cell defects have been reported in mouse models of MTM1- and DNM2-associated CNM, suggestive of shared underlying pathways.

Pathogenic variants in the SRCAP (SNF2-related CREBBP activator protein) gene, which encodes a chromatin-remodeling ATPase, cause neurodevelopmental disorders including Floating Harbor syndrome (FLHS). Here, we report the discovery of a de novo transposon insertion in SRCAP exon 13 from trio genome sequencing in a 28-year-old female with failure to thrive, developmental delay, mood disorder and seizure disorder. The insertion was a full-length (~2.8 kb), antisense-oriented SVA insertion relative to the SRCAP transcript, bearing a 5' transduction and hallmarks of target-primed reverse transcription. The 20-bp 5' transduction allowed us to trace the source SVA element to an intron of a long non-coding RNA on chromosome 12, which is highly expressed in testis. RNA sequencing and qRT-PCR confirmed significant depletion of SRCAP expression and low-level exon skipping in the proband. This case highlights a novel disease-causing structural variant and the importance of transposon analysis in a clinical diagnostic setting.

Striated preferentially expressed protein kinase (SPEG), a myosin light chain kinase, is mutated in centronuclear myopathy (CNM) and/or dilated cardiomyopathy. No precise therapies are available for this disorder, and gene replacement therapy is not a feasible option due to the large size of SPEG. We evaluated the potential of dynamin-2 (DNM2) reduction as a potential therapeutic strategy because it has been shown to revert muscle phenotypes in mouse models of CNM caused by MTM1, DNM2, and BIN1 mutations. We determined that SPEG-β interacted with DNM2, and SPEG deficiency caused an increase in DNM2 levels. The DNM2 reduction strategy in Speg-KO mice was associated with an increase in life span, body weight, and motor performance. Additionally, it normalized the distribution of triadic proteins, triad ultrastructure, and triad number and restored phosphatidylinositol-3-phosphate levels in SPEG-deficient skeletal muscles. Although DNM2 reduction rescued the myopathy phenotype, it did not improve cardiac dysfunction, indicating a differential tissue-specific function. Combining DNM2 reduction with other strategies may be needed to target both the cardiac and skeletal defects associated with SPEG deficiency. DNM2 reduction should be explored as a therapeutic strategy against other genetic myopathies (and dystrophies) associated with a high level of DNM2.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.

Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.

SLC25A46 (solute carrier family 25 member 46) mutations have been linked to various neurological diseases with recessive inheritance, including Leigh syndrome, optic atrophy, and lethal congenital pontocerebellar hypoplasia. SLC25A46 is expressed in the outer membrane of mitochondria, where it plays a critical role in mitochondrial dynamics. A deceased 7-month-old female infant was suspected to have Leigh syndrome. Clinical exome sequencing was non-diagnostic, but research reanalysis of the sequencing data identified two novel variants in SLC25A46: a missense (c.1039C>T, p.Arg347Cys; NM_138773, hg19) and a donor splice region variant (c.283+5G>A) in intron 1. Both variants were predicted to be damaging. Sanger sequencing of cDNA detected a single missense allele in the patient compared to control, and the SLC25A46 transcript levels were also reduced due to the splice region variant. Additionally, Western blot analysis of whole-cell lysate showed a decrease of SLC25A46 expression in proband fibroblasts, relative to control cells. Further, analysis of mitochondrial morphology revealed evidence of increased fragmentation of the mitochondrial network in proband fibroblasts, compared to control cells. Collectively, our findings suggest that these novel variants in SLC24A46, the donor splice one and the missense variant, are the cause of the neurological phenotype in this proband.

The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.

Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation. WO 2014201206, 2014] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (i...

Fellowship is an important part of postgraduate medical training as it facilitates advanced training in a subspecialty of interest. Internet is the most readily available and frequently used tool for acquiring information about the fellowship programs by residents worldwide. The aim of this study was to analyze the content of the websites of American and Canadian breast/women's imaging fellowship programs.The content of active Canadian and American breast/women's imaging fellowship websites was collected and analyzed in August 2019 using 27 different criteria in the categories of application process, recruitment, education, research, clinical work, and incentives. The fellowship program without a webpage were excluded from the study.Out of 76 active breast/women's imaging Radiology fellowship programs in the US and Canada, 75 had dedicated fellowship websites available for analysis. One program was excluded due to lack of a dedicated website. On average, websites showcased 11 of the 27 criteria (40.0%). The category with the least prevalent information was incentives especially career placement after completion of the fellowship (1/75, 1.3%). Majority of the programs around 80.0% (60/75) had adequate information about the application process and requirements. The mean number of schools satisfying the different groups of criteria differed (P0.01); more schools satisfied the application criteria (60.5/75; 79.6% ± 20.3%) than the incentives (12.8/75; 16.8% ± 8.2%) and recruitment (24.5/75; 32.2% ± 29.7%) criteria.Majority of the breast and women's imaging fellowship websites lack important information. Providing comprehensive online information about the program and application process may help the applicants to choose the best suited program for their academic needs and career progression.

Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel Na

Deregulation of the receptor tyrosine kinase mesenchymal epithelial transition factor (MET) has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of compound 23 (AMG 337), which demonstrates nanomolar inhibition of MET kinase activity, desirable preclinical pharmacokinetics, significant inhibition of MET phosphorylation in mice, and robust tumor growth inhibition in a MET-dependent mouse efficacy model.

Paid crowdsourcing platforms suffer from low-quality work and unfair rejections, but paradoxically, most workers and requesters have high reputation scores. These inflated scores, which make high-quality work and workers difficult to find, stem from social pressure to avoid giving negative feedback. We introduce Boomerang, a reputation system for crowdsourcing that elicits more accurate feedback by rebounding the consequences of feedback directly back onto the person who gave it. With Boomerang, requesters find that their highly-rated workers gain earliest access to their future tasks, and workers find tasks from their highly-rated requesters at the top of their task feed. Field experiments verify that Boomerang causes both workers and requesters to provide feedback that is more closely aligned with their private opinions. Inspired by a game-theoretic notion of incentive-compatibility, Boomerang opens opportunities for interaction design to incentivize honest reporting over strategic dishonesty.

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

The voltage-gated sodium channel Na

The voltage-gated sodium channel Na

Because of its strong genetic validation, Na

Several reports have recently emerged regarding the identification of heteroarylsulfonamides as Na

Human genetic evidence has identified the voltage-gated sodium channel Na

The impact of inhibitor depletion on the determination of shifted IC₅₀ (IC₅₀ determined after 30 minutes of preincubation with inhibitor) is examined. In addition, IC₅₀-shift data are analyzed using a mechanistic model that incorporates the processes of inhibitor depletion, as well as reversible and time-dependent inhibition. Anomalies such as a smaller-than-expected shift in IC₅₀ and even increases in IC₅₀ with preincubation were explained by the depletion of inhibitor during the preincubation. The IC₅₀-shift assay remains a viable approach to characterizing a wide range of reversible and time-dependent inhibitors. However, as with more traditional time-dependent inactivation methods, it is recommended that IC₅₀-shift experimental data be interpreted with some knowledge of the magnitude of inhibitor depletion. For the most realistic classification of time-dependent inhibitors using IC₅₀-shift methods, shifted IC₅₀ should be calculated using observed inhibitor concentrations at the end of the incubation rather than nominal inhibitor concentrations. Finally, a mechanistic model that includes key processes, such as competitive inhibition, enzyme inactivation, and inhibitor depletion, can be used to describe accurately the observed IC₅₀ and shifted IC₅₀ curves. For compounds showing an IC₅₀ fold shift >1.5 based on the observed inhibitor concentrations, reanalyzing the IC₅₀-shift data using the mechanistic model appeared to allow for reasonable estimation of Ki, KI, and kinact directly from the IC₅₀ shift experiments.

Human genetic evidence has identified the voltage-gated sodium channel NaV1.7 as an attractive target for the treatment of pain. We initially identified naphthalene sulfonamide 3 as a potent and selective inhibitor of NaV1.7. Optimization to reduce biliary clearance by balancing hydrophilicity and hydrophobicity (Log D) while maintaining NaV1.7 potency led to the identification of quinazoline 16 (AM-2099). Compound 16 demonstrated a favorable pharmacokinetic profile in rat and dog and demonstrated dose-dependent reduction of histamine-induced scratching bouts in a mouse behavioral model following oral dosing.