Unconventional insulins from predators and pathogens.

Insulin and its related peptides are found throughout the animal kingdom, in which they serve diverse functions. This includes regulation of glucose homeostasis, neuronal development and cognition. The surprising recent discovery that venomous snails evolved specialized insulins to capture fish demonstrated the nefarious use of this hormone in nature. Because of their streamlined role in predation, these repurposed insulins exhibit unique characteristics that have unraveled new aspects of the chemical ecology and structural biology of this important hormone. Recently, insulins were also reported in other venomous predators and pathogenic viruses, demonstrating the broader use of insulin by one organism to manipulate the physiology of another. In this Review, we provide an overview of the discovery and biomedical application of repurposed insulins and other hormones found in nature and highlight several unique insights gained from these unusual compounds.

Symmetric and asymmetric receptor conformation continuum induced by a new insulin.

Cone snail venoms contain a wide variety of bioactive peptides, including insulin-like molecules with distinct structural features, binding modes and biochemical properties. Here, we report an active humanized cone snail venom insulin with an elongated A chain and a truncated B chain, and use cryo-electron microscopy (cryo-EM) and protein engineering to elucidate its interactions with the human insulin receptor (IR) ectodomain. We reveal how an extended A chain can compensate for deletion of B-chain residues, which are essential for activity of human insulin but also compromise therapeutic utility by delaying dissolution from the site of subcutaneous injection. This finding suggests approaches to developing improved therapeutic insulins. Curiously, the receptor displays a continuum of conformations from the symmetric state to a highly asymmetric low-abundance structure that displays coordination of a single humanized venom insulin using elements from both of the previously characterized site 1 and site 2 interactions.

Treatment of rectosigmoid endometriosis by laparoscopic reverse submucosal dissection (LRSD): The Sydney partial thickness discoid excision technique.

BACKGROUND: Laparoscopic reverse submucosal dissection (LRSD) is a standardised surgical technique for removal of rectosigmoid endometriosis which optimises the anatomical dissection plane for excision of endometriotic nodules. AIM: This cohort study assesses the outcomes of the first cohort of women treated by LRSD, for deeply infiltrating rectosigmoid endometriosis. MATERIALS AND METHODS: Primary outcomes assessed were complication rate as defined by the Clavien-Dindo system, and completion of the planned LRSD. Secondary outcomes include mucosal breach, specimen margin involvement, length of hospital admission, and a comparison of pre-operative and post-operative pain, bowel function and quality of life surveys. These included the Endometriosis Health Profile Questionnaire (EHP-30), the Knowles-Eccersley-Scott Symptom Questionnaire (KESS) and the Wexner scale. RESULTS: Of 19 patients treated, one required a segmental resection. The median length of hospital admission was two days (range 1-5) and no post-operative complications occurred. Median pain visual analogue scales (scale 0-10) were higher prior to surgery (dysmenorrhoea 9.0, dyspareunia 7.5, dyschezia 9.0, pelvic pain 6.0) compared to post-surgical median scores (dysmenorrhoea 5.0, dyspareunia 4.0, dyschezia 2.0, pelvic pain 4.0) at a median of six months (range 4-32). Quality of life studies suggested improvement following surgery with pre-operative median EHP-30 and KESS scores (EHP-30: 85 (5-106), KESS score 9 (0-20)) higher than post-operative scores (EHP-30: 48.5 (0-80), KESS score: 3 (0-19)). CONCLUSION: This series highlights the feasibility of LRSD with low associated morbidity as a progression of partial thickness discoid excision (rectal shaving) for the treatment of rectosigmoid deep infiltrating endometriosis.

From Natural Insulin to Designed Analogs: A Chemical Biology Exploration.

Since its discovery in 1921, insulin has been at the forefront of scientific breakthroughs. From its amino acid sequencing to the revelation of its three-dimensional structure, the progress in insulin research has spurred significant therapeutic breakthroughs. In recent years, protein engineering has introduced innovative chemical and enzymatic methods for insulin modification, fostering the development of therapeutics with tailored pharmacological profiles. Alongside these advances, the quest for self-regulated, glucose-responsive insulin remains a holy grail in the field. In this article, we highlight the pivotal role of chemical biology in driving these innovations and discuss how it continues to shape the future trajectory of insulin research.

Supramolecular Protein Stabilization with Zwitterionic Polypeptide-Cucurbit[7]uril Conjugates.

Protein aggregation is an obstacle for the development of new biopharmaceuticals, presenting challenges in shipping and storage of vital therapies. Though a variety of materials and methods have been explored, the need remains for a simple material that is biodegradable, nontoxic, and highly efficient at stabilizing protein therapeutics. In this work, we investigated zwitterionic polypeptides prepared using a rapid and scalable polymerization technique and conjugated to a supramolecular macrocycle host, cucurbit[7]uril, for the ability to inhibit aggregation of model protein therapeutics insulin and calcitonin. The polypeptides are based on the natural amino acid methionine, and zwitterion sulfonium modifications were compared to analogous cationic and neutral structures. Each polymer was end-modified with a single cucurbit[7]uril macrocycle to afford supramolecular recognition and binding to terminal aromatic amino acids on proteins. Only conjugates prepared from zwitterionic structures of sufficient chain lengths were efficient inhibitors of insulin aggregation and could also inhibit aggregation of calcitonin. This polypeptide exhibited no cytotoxicity in human cells even at concentrations that were five-fold of the intended therapeutic regime. We explored treatment of the zwitterionic polypeptides with a panel of natural proteases and found steady biodegradation as expected, supporting eventual clearance when used as a protein formulation additive.

Serine-mediated hydrazone ligation displaying insulin-like peptides on M13 phage pIII.

Phage display has emerged as a tool for the discovery of therapeutic antibodies and proteins. However, the effective display and engineering of structurally complex proteins, such as insulin, pose significant challenges due to the sequence of insulin, which is composed of two peptide chains linked by three disulfide bonds. In this study, we developed a new approach for the display of insulin-like peptides on M13 phage pIII, employing N-terminal serine-mediated hydrazone ligation. The insulin-displaying phage retains the biological binding affinity of human insulin. To address the viability loss after ligation, we introduced a trypsin-cleavable spacer on pIII, enabling insulin-displayed phage library selection. This method offers a general pathway for the display of structurally complex proteins on pIII, enhancing the practicality of selecting chemically modified phage libraries and opening avenues for the engineering of new insulin analogs for the treatment of diabetes by using phage display.

Superior Hypogastric Plexus Nerve Block in Minimally Invasive Gynecology: A Randomized Controlled Trial.

STUDY OBJECTIVE: To assess the efficacy of a superior hypogastric plexus nerve block in reducing opioid requirements in the first 24 hours after minimally invasive gynecologic surgery. DESIGN: Patient-blinded randomized controlled trial. SETTING: Single-center academic institution (Sydney Women's Endosurgery Centre). Two surgeons administering the blocks in their own surgeries. PATIENTS: Patients undergoing either laparoscopic or robot-assisted laparoscopic hysterectomy or myomectomy for benign indications. INTERVENTIONS: Ropivacaine 10 mL (0.75%) infiltrated into the retroperitoneal space overlying the superior hypogastric plexus vs control of no block given at the completion of surgery. MEASUREMENTS AND MAIN RESULTS: The primary outcome was the total opioid use in the first 24 hours after surgery, measured in morphine milligram equivalents (MME). Standardized fentanyl patient-controlled analgesia was given to all patients in the trial. The secondary outcome was pain measured on a visual analog scale (1 to 10) at 1, 2, 6, 12, and 24 hours after surgery. Fifty patients out of 56 approached for the study participated in, and completed, the study (89.2%). The patients were randomized over a 5-month period, March 2020 to July 2020. A total of 27 patients were randomized to receive a nerve block, and 23 were randomized to the control. There was a difference of -21.8 MME in the block group compared with the no-block group (95% confidence interval [CI], -38.2 to -5.5; p = .008). This correlated to a 38% reduction in opioid use in the block group. The mean opioid use in the patients in the block group was 33.1 MME (95% CI, 24.2-41.9) and in those in the no-block group 54.9 MME (95% CI, 40.7-69.1). For the block group, opioid use ranged from 1.0 to 76.5 MME, with an interquartile range of 37 (14-51). For the control group, the range was 7.5 to 113.5 MME, with a higher interquartile range of 60 (28-88). Pairwise comparisons of the mean pain scores over the 24 hours showed a lower pain score with a nerve block of 1.8 (95% CI, 1.5-2.1) compared with a no-block score of 2.6 (95% CI, 2.3-2.9) No adverse effects of local anesthetic toxicity, nerve injury, or bowel/vascular injury were noted in any patient. CONCLUSION: A superior hypogastric plexus nerve block is a simple technique for reducing postoperative opioid requirements and pain in the first 24 hours after minimally invasive gynecologic surgery.

Rectal Shaving for Bowel Endometriosis by Laparoscopic Reverse Submucosal Dissection for Easier, Safer and More Complete Excision of Disease.

STUDY OBJECTIVE: To demonstratefull-thickness excision of the affected muscularis along the submucosal plane. DESIGN: Stepwise demonstration of LRSD technique with narrated video footage. SETTING: LRSD takes advantage of the submucosal layer of the bowel wall and uses it as an easier line of excision for rectal endometriosis compared with the very difficult traditional line of excision of irregular disease-muscularis interface. The expansion of the submucosal layer by the injection separates the affected muscularis away from the mucosa, making it safer to excise the lesion with less chance of entering the bowel lumen. Excision of disease is more complete with LRSD because the full-thickness excision of the muscularis layer includes the healthy deep muscularis, which will form the disease-free deep excision margin. INTERVENTION: This video will highlight anatomic and technical aspects of LRSD including the following key steps: 1. Mobilization of diseased bowel segment 2. Submucosal injection 3. Circumferential incision of the muscularis 4. Submucosal dissection along the submucosal plane 5. Bowel wall integrity test 6. Muscularis defect repair CONCLUSION: Rectal shaving by LRSD appears to be easier, safer, and more complete in excision of bowel endometriosis than the classical rectal shaving technique. This modification requires further evaluation to confirm its potential in the surgical management of rectosigmoid deep infiltrative endometriosis.

Removing the large uterus without morcellation - The Colpo-V incision for specimen extraction at hysterectomy.

BACKGROUND: Hysterectomy is the most commonly performed benign gynaecological surgery. Recently, the rates of minimally invasive hysterectomy have fallen due to the banning of mechanical morcellation techniques that rendered minimal invasive gynaecology surgeons unable to extract large uteri from the relatively small colpotomy incisions. AIMS: This study aims to share our experience in utilising Colpo-V incision to remove large uterine specimens transvaginally and report its success and complication rates to promote a minimal invasive approach in patients with large uteri without the need to perform large abdominal incisions or transabdominal morcellation. METHODS: This is a prospective case series study in which women with large uteri and|or narrow vaginal canal underwent total laparoscopic hysterectomy and a subsequent posterior vaginal wall incision (Colpo-V) to facilitate the intact extraction of the uterus through the vagina. Patients were seen in the clinic six weeks after the surgery for post-operative assessment and documentation of late complications. RESULTS: Seventeen women underwent the procedure, and the intact extraction of the specimen was successful in 16 out of the 17 cases (94%). No major complications were encountered during or after the procedure. CONCLUSION: Colpo-V incision is a simple and effective technique for the intact extraction of larger uterine specimens at hysterectomy.

Transvaginal Ultrasound Can Accurately Predict the American Society of Reproductive Medicine Stage of Endometriosis Assigned at Laparoscopy.

STUDY OBJECTIVE: To evaluate the diagnostic accuracy of transvaginal ultrasound in predicting a laparoscopic, surgically assigned, revised American Society of Reproductive Medicine (ASRM) endometriosis stage. DESIGN: A multicenter, retrospective, diagnostic accuracy study. SETTING: The patients visited 1 of 2 academic gynecologic ultrasound units and underwent laparoscopy led by 1 of 6 surgeons in metropolitan Sydney, Australia, between 2016 and 2018. PATIENTS: Patients with suspected endometriosis (n = 204). INTERVENTIONS: Ultrasound followed by laparoscopy. MEASUREMENTS AND MAIN RESULTS: Surgical cases were identified. The preoperative ultrasound report and surgical operative notes were each used to retrospectively assign an ASRM score and stage. The breakdown of surgical findings was as follows: ASRM 0 (i.e., no endometriosis), 24/204 (11.8%); ASRM 1, 110/204 (53.9%); ASRM 2, 22/204 (10.8%); ASRM 3, 16/204 (7.8%); ASRM 4, 32 204 (15.7%). The overall accuracy of ultrasound in predicting the surgical ASRM stage was as follows: ASRM 1, 53.4%; ASRM 2, 93.8%; ASRM 3, 89.7%; ASRM 4, 93.1%; grouped ASRM 0, 1, and 2, 94.6%; and grouped ASRM 3 and 4 of 94.6%. Ultrasound had better test performance in higher disease stages. When the ASRM stages were dichotomized, ultrasound had sensitivity and specificity of 94.9% and 93.8%, respectively, for ASRM 0, 1, and 2 and of 93.8% and 94.9%, respectively, for ASRM 3 and 4. CONCLUSION: Ultrasound has high accuracy in predicting the mild, moderate, and severe ASRM stages of endometriosis and can accurately differentiate between stages when ASRM stages are dichotomized (nil/minimal/mild vs moderate/severe). This can have major positive implications on patient triaging at centers of excellence in minimally invasive gynecology for advanced-stage endometriosis.

Shaving for Bowel Endometriosis.

STUDY OBJECTIVE: To demonstrate laparoscopic shaving of deeply infiltrative endometriosis affecting the rectosigmoid colon, with particular emphasis on the anatomic and technical aspects of the procedure. DESIGN: Stepwise demonstration of the technique with narrated video footage. SETTING: Intestinal involvement in deep endometriosis is estimated to occur in 8% to 12% of patients, with 90% of occurrences being located in the colorectal segment. Deep endometriosis of the rectosigmoid is defined as endometriosis involving the muscular layer of the bowel wall, usually >5 mm deep, thus excluding superficial lesions that only affect the serosal layer. In cases in which medical therapy is unsatisfactory, rectosigmoid deep endometriosis can be surgically managed by 3 recognized surgical techniques: (1) rectal shaving, (2) disc excision, and (3) segmental resection. There are helpful recommendations for different approaches on the basis of the characteristics of the lesion, including the size, length, depth of invasion, involved rectal circumference, and number of lesions, among other factors [1]. Rectal shaving is well suited for smaller lesions, typically <3 cm, and involves "shaving" the lesion in the affected muscular layer of the bowel wall off the mucosa, ideally without entering the bowel lumen. It is associated with lower rates of perioperative complications and lower probability of long-term postoperative bladder and bowel dysfunctions [2]. INTERVENTIONS: This video demonstrates and highlights the anatomic and technical aspects of the following important steps of the rectal shaving procedure: (1) suspension of ovaries; (2) mobilization of the diseased segment of the rectum; (3) shaving of the lesions, with pertinent comments at different stages of nodule excision; (4) checking for the integrity of the bowel wall; and (5) suture of the muscularis defect after excision of the lesions from the muscularis layer of the bowel. CONCLUSION: Compared with other alternatives, shaving for bowel endometriosis is a more conservative procedure with lower rates of perioperative complications, and it is less likely to result in long-term bladder and bowel dysfunctions. Therefore, shaving is preferable and recommended for appropriate lesions.

Display of Single-Chain Insulin-like Peptides on a Yeast Surface.

Insulin and insulin-like peptides play a pivotal role in a wide variety of cellular and physiological events, including energy storage, proliferation, aging, and differentiation. Variants of insulin and insulin-like peptides may therefore be probes for studying the insulin signaling pathway and therapeutic candidates for treating metabolic diseases. Here, we report a method for genetically displaying single-chain insulin-like peptides on the surface of Saccharomyces cerevisiae strain DY1632. Using a previously reported single-chain insulin analogue, SCI-57, as a model, we demonstrate that nearly 70% of yeast binds to insulin receptor (IR), suggesting that SCI-57 is folded correctly and maintains its IR binding property. Furthermore, the interaction between displayed SCI-57 and IR can be weakened using increasing concentrations of native insulin as a soluble competitor, suggesting that the interaction is insulin-dependent. We further applied this methodology to three other single-chain insulin analogues with various lengths and confirmed their interactions with IR. In summary, we successfully displayed a number of insulin-like peptides on a yeast surface and demonstrated insulin-dependent interactions with IR. This method may, therefore, be used for construction of libraries of insulin-like peptides to select for chemical probes or therapeutic molecules.

Synthesis of hydrophobic insulin-based peptides using a helping hand strategy.

The introduction of solid-phase peptide synthesis in the 1960s improved the chemical synthesis of both the A- and B-chains of insulin and insulin analogs. However, the subsequent elaboration of the synthetic peptides to generate active hormones continues to be difficult and complex due in part to the hydrophobicity of the A-chain. Over the past decade, several groups have developed different methods to enhance A-chain solubility. Two of the most popular methods are use of isoacyl dipeptides, and the attachment of an A-chain C-terminal pentalysine tag with a base-labile 4-hydroxymethylbenzoic acid linker. These methods have proven effective but can be limited in scope depending on the peptide sequence of a specific insulin. Herein we describe an auxiliary approach to enhance the solubility of insulin-based peptides by incorporating a tri-lysine tag attached to a cleavable Fmoc-Ddae-OH linker. Incorporation of this linker, or "helping hand", on the N-terminus greatly improved the solubility of chicken insulin A-chain, which is analogous to human insulin, and allowed for coupling of the insulin A- and B-chain via directed disulfide bond formation. After formation of the insulin heterodimer, the linker and tag could be easily removed using a hydrazine buffer (pH 7.5) to obtain an overall 12.6% yield based on A-chain. This strategy offers an efficient method to enhance the solubility of hydrophobic insulin-based peptides as well as other traditionally difficult peptides.

Diagnostic Accuracy and Reproducibility of Predicting Cul-de-Sac Obliteration by General Gynaecologists and Minimally Invasive Gynaecologic Surgeons.

OBJECTIVE: Knowledge of rectouterine cul-de-sac state and consistent classification among surgeons are important in the surgical management of women with endometriosis. The objective of this study was to determine the diagnostic accuracy and interobserver and intraobserver agreement among general gynaecologists (GGs) and minimally invasive gynaecologic surgeons (MIGSs) in the prediction of cul-de-sac obliteration at off-line analysis of laparoscopic videos. METHODS: Five GGs and five MIGSs viewed 33 prerecorded laparoscopic video sets off-line to determine cul-de-sac obliteration state (non-obliterated, partially obliterated, or completely obliterated) on two occasions (at least 7days apart). Diagnostic accuracy and interobserver and intraobserver agreement were evaluated. RESULTS: The interobserver agreements for all 10 observers for the description of cul-de-sac state ranged from fair to substantial agreement, with moderate overall agreement. MIGSs had slightly higher within-group interobserver agreement compared with GGs. MIGSs achieved overall almost perfect intraobserver agreement compared with substantial agreement for GGs. The accuracy, sensitivity, specificity, positive predictive value, and negative predictive value for MIGSs classifying the cul-de-sac state were 83.9%, 88.5%, 88.5%, 89.2%, 92.0%, and 84.7%, respectively, whereas for GGs, they were 79.1%, 79.4%, 88.1%, 89.9%, and 76.1%, respectively. CONCLUSION: Diagnostic accuracy and interobserver and intraobserver agreement for cul-de-sac obliteration state classification is acceptable in both groups. MIGSs had greater diagnostic accuracy and exhibited high interobserver and intraobserver agreement, a finding suggesting that their advanced training makes them more reliable in cul-de-sac obliteration assessment. Partial cul-de-sac obliteration was the most commonly incorrectly diagnosed state, thus implying that partial obliteration is not well understood.

Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast.

Long-term patient-reported outcomes after laparoscopic Burch colposuspension.

BACKGROUND: The negative media attention surrounding vaginal mesh procedures has seen a rise in demand for minimally invasive non-mesh options for the treatment of stress urinary incontinence (SUI). The laparoscopic Burch colposuspension (LBC) is a non-mesh alternative to synthetic midurethral slings (MUS) with similar short-term outcomes. However, long-term outcomes are not well established. AIMS: To evaluate the long-term outcomes of LBC for treatment of SUI in women. MATERIAL AND METHODS: One hundred and fifty-one cases of LBC were performed by a single surgeon over two private hospital settings between January 2010 and January 2016. Follow-up subjective outcomes were obtained in 137 cases (90.7%) utilising standardised questionnaires. Primary outcome was successful treatment of SUI, defined as subjective cure or significant improvement of stress incontinence symptoms. Secondary outcomes included new-onset or worsened symptoms of overactive bladder (OAB), voiding dysfunction, prolapse, and perioperative complications. RESULTS: One hundred and thirty-seven patients were analysed with a mean follow-up of 50.6 months (range: 13-89 months). Primary outcome of successful treatment was achieved in 90.5% of women. New-onset or worsened symptoms of OAB was reported in 10.2%, with a further 8.8% of women experiencing symptomatic voiding dysfunction. Sixteen patients (11.7%) reported new-onset or worsening symptoms of prolapse. There were no major surgical complications. CONCLUSIONS: LBC is a safe and effective long-term treatment for SUI, with low failure rates and minimal adverse outcomes. It is a suitable alternative for women with contraindications to mesh or those having concomitant laparoscopic procedures.

Glucose-responsive insulin activity by covalent modification with aliphatic phenylboronic acid conjugates.

Since its discovery and isolation, exogenous insulin has dramatically changed the outlook for patients with diabetes. However, even when patients strictly follow an insulin regimen, serious complications can result as patients experience both hyperglycemic and hypoglycemic states. Several chemically or genetically modified insulins have been developed that tune the pharmacokinetics of insulin activity for personalized therapy. Here, we demonstrate a strategy for the chemical modification of insulin intended to promote both long-lasting and glucose-responsive activity through the incorporation of an aliphatic domain to facilitate hydrophobic interactions, as well as a phenylboronic acid for glucose sensing. These synthetic insulin derivatives enable rapid reversal of blood glucose in a diabetic mouse model following glucose challenge, with some derivatives responding to repeated glucose challenges over a 13-h period. The best-performing insulin derivative provides glucose control that is superior to native insulin, with responsiveness to glucose challenge improved over a clinically used long-acting insulin derivative. Moreover, continuous glucose monitoring reveals responsiveness matching that of a healthy pancreas. This synthetic approach to insulin modification could afford both long-term and glucose-mediated insulin activity, thereby reducing the number of administrations and improving the fidelity of glycemic control for insulin therapy. The described work is to our knowledge the first demonstration of a glucose-binding modified insulin molecule with glucose-responsive activity verified in vivo.

Application of Thiol-yne/Thiol-ene Reactions for Peptide and Protein Macrocyclizations.

The application of thiol-yne/thiol-ene reactions to synthesize mono- and bicyclic-stapled peptides and proteins is reported. First, a thiol-ene-based peptide-stapling method in aqueous conditions was developed. This method enabled the efficient stapling of recombinantly expressed coil-coiled proteins. The resulting stapled protein demonstrated higher stability in its secondary structure than the unstapled version. Furthermore, a thiol-yne coupling was performed by using an α,ω-diyne to react with two cysteine residues to synthesize a stapled peptide with two vinyl sulfide groups. The stapled peptide could further react with another biscysteine peptide to yield a bicyclic stapled peptide with enhanced properties. For example, the cell permeability of a stapled peptide was further increased by appending an oligoarginine cell-penetrating peptide. The robustness and versatility of thiol-yne/thiol-ene reactions that can be applied to both synthetic and expressed peptides and proteins were demonstrated.

Kinase-Independent Small-Molecule Inhibition of JAK-STAT Signaling.

Phenotypic cell-based screening is a powerful approach to small-molecule discovery, but a major challenge of this strategy lies in determining the intracellular target and mechanism of action (MoA) for validated hits. Here, we show that the small-molecule BRD0476, a novel suppressor of pancreatic ß-cell apoptosis, inhibits interferon-gamma (IFN-γ)-induced Janus kinase 2 (JAK2) and signal transducer and activation of transcription 1 (STAT1) signaling to promote ß-cell survival. However, unlike common JAK-STAT pathway inhibitors, BRD0476 inhibits JAK-STAT signaling without suppressing the kinase activity of any JAK. Rather, we identified the deubiquitinase ubiquitin-specific peptidase 9X (USP9X) as an intracellular target, using a quantitative proteomic analysis in rat ß cells. RNAi-mediated and CRISPR/Cas9 knockdown mimicked the effects of BRD0476, and reverse chemical genetics using a known inhibitor of USP9X blocked JAK-STAT signaling without suppressing JAK activity. Site-directed mutagenesis of a putative ubiquitination site on JAK2 mitigated BRD0476 activity, suggesting a competition between phosphorylation and ubiquitination to explain small-molecule MoA. These results demonstrate that phenotypic screening, followed by comprehensive MoA efforts, can provide novel mechanistic insights into ostensibly well-understood cell signaling pathways. Furthermore, these results uncover USP9X as a potential target for regulating JAK2 activity in cellular inflammation.