Proteolysis-targeting chimeras with reduced off-targets.

Proteolysis-targeting chimeras (PROTACs) are molecules that induce proximity between target proteins and E3 ligases triggering target protein degradation. Pomalidomide, a widely used E3 ligase recruiter in PROTACs, can independently degrade other proteins, including zinc-finger (ZF) proteins, with vital roles in health and disease. This off-target degradation hampers the therapeutic applicability of pomalidomide-based PROTACs, requiring development of PROTAC design rules that minimize off-target degradation. Here we developed a high-throughput platform that interrogates off-target degradation and found that reported pomalidomide-based PROTACs induce degradation of several ZF proteins. We generated a library of pomalidomide analogues to understand how functionalizing different positions of the phthalimide ring, hydrogen bonding, and steric and hydrophobic effects impact ZF protein degradation. Modifications of appropriate size on the C5 position reduced off-target ZF degradation, which we validated through target engagement and proteomics studies. By applying these design principles, we developed anaplastic lymphoma kinase oncoprotein-targeting PROTACs with enhanced potency and minimal off-target degradation.

A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules.

The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in a demand for inhibitory anti-CRISPR molecules. The small-molecule inhibitor discovery platforms available at present are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity and require high concentrations of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Here we report a high-throughput discovery pipeline consisting of a fluorescence resonance energy transfer-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentrations and targets all catalytic steps. We applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9 that is twofold more potent than other inhibitors identified to date. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA-repair pathway or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases.

Bifunctional modalities for repurposing protein function.

Nature takes advantage of induced proximity to perform various functions. Taking inspiration from nature, we can also trigger desired biological processes using bifunctional small molecules that artificially induce proximity. For example, bifunctional small molecules have been designed to trigger the ubiquitin-dependent proteasomal degradation of intracellular proteins. Now, recent classes of bifunctional compounds have been developed to degrade extracellular targets, membrane proteins, damaged organelles, and RNA by recruiting alternative degradation pathways. In addition to inducing degradation, bifunctional modalities can change phosphorylation and glycosylation states to evoke a biological response. In this review, we highlight recent advances in these innovative classes of compounds that build on a rich history of chemical inducers of dimerization. We anticipate that more bifunctional molecules, which induce or remove posttranslational modifications, to endow neo-functionalities will emerge.

Online Marketing of Ephedra Weight Loss Supplements: Labeling and Marketing Compliance with the U.S. Food and Drug Administration Ban on Ephedra.

Objective: To characterize dietary supplements marketed online as "ephedra-containing or ephedra-like products" for weight management and to assess labeling/marketing compliance with the ban on the sale of ephedrine alkaloids. Materials and Methods: This cross-sectional study assessed websites selling ephedra-like supplements using the search term "buy ephedra." For each website, the first three featured products were characterized by evaluating the label for (1) Ephedra sp. or its alkaloid content, (2) serving size, (3) other ingredients, (4) directions, (5) side effects, (6) reported interactions, (7) recommendation to consult a health care provider, (8) recommendation to use with diet and exercise, and (9) Food and Drug Administration (FDA) disclaimer. Results: Thirty-six (71%) of the first 51 websites evaluated sold at least one weight loss product. A total of 105 products were assessed, 93 had labeling with 10 (11%) in possible violation of the ephedra ban. Five were labeled as containing ephedrine or ephedrine hydrochloride, two reported containing ephedrine alkaloids, and two reported containing unidentified Ephedra sp. not formulated as an extract; one reported containing Ma Huang. Sixty-seven (72%) products listed caffeine with a daily serving size averaging 400 mg. Other ingredients with stimulant properties include green tea, yohimbe, and phenylethylamine. Conclusions: Nearly 20% of websites sold weight loss products that potentially violated the 2004 ban of ephedra alkaloids. Ephedrine, unidentified Ephedra sp. not formulated as an extract, and Ma Huang were labeled as present in 11% of products evaluated. Incomplete reporting of adverse effects and drug interactions was common.

Phosphorylation-Inducing Chimeric Small Molecules.

Small molecules have been classically developed to inhibit enzyme activity; however, new classes of small molecules that endow new functions to enzymes via proximity-mediated effect are emerging. Phosphorylation (native or neo) of any given protein-of-interest can alter its structure and function, and we hypothesized that such modifications can be accomplished by small molecules that bring a kinase in proximity to the protein-of-interest. Herein, we describe phosphorylation-inducing chimeric small molecules (PHICS), which enable two example kinases-AMPK and PKC-to phosphorylate target proteins that are not otherwise substrates for these kinases. PHICS are formed by linking small-molecule binders of the kinase and the target protein, and exhibit several features of a bifunctional molecule, including the hook-effect, turnover, isoform specificity, dose and temporal control of phosphorylation, and activity dependent on proximity (i.e., linker length). Using PHICS, we were able to induce native and neo-phosphorylations of BRD4 by AMPK or PKC. Furthermore, PHICS induced a signaling-relevant phosphorylation of the target protein Bruton's tyrosine kinase in cells. We envision that PHICS-mediated native or neo-phosphorylations will find utility in basic research and medicine.

Managing Nonoperable Intracranial Bleeding Associated With Apixaban: A Series of 2 Cases.

OBJECTIVE: To report 2 cases of nonoperable intracranial bleeding associated with apixaban managed by 3-factor prothrombin complex concentrate (PCC3). CASE SUMMARIES: Case 1 presented with a 1.3-cm left parieto-occipital hemorrhage and a thin subdural hematoma (SDH) on the left tentorium of the brain about 6 hours after his last dose of apixaban. Case 2 presented with a 4-mm left parafalcine SDH with time of most recent apixaban dose unknown. The patients received 24.9 to 25.5 U/kg of PCC3 with none to 1 U fresh frozen plasma (FFP) and demonstrated minimal or no progression in lesions measured by repeat computed tomography (CT) after treatment. One patient was discharged to a skilled nursing facility after 8 days; the other patient was discharged to home after 18 days. DISCUSSION: Apixaban has no specific antidote. Current bleeding management strategies are based on expert opinion. The risks and benefits for differing strategies are unclear, and little clinical experience for managing apixaban-associated intracranial bleeding has been reported to date. These cases describe the clinical use of PCC3 to manage parieto-occipital and subdural hemorrhage associated with apixaban in events not requiring surgical intervention. CONCLUSION: In these 2 cases, 25 U/kg PCC3, with none to one unit FFP, ceased apixaban-associated intracranial bleeding without apparent thrombogenic complications.

Evaluation of the effect of torsemide on warfarin dosage requirements.

Background According to drug interaction databases, torsemide may potentiate the effects of warfarin. Evidence for this drug-drug interaction, however, is conflicting and the clinical significance is unknown. Objective The aim of this study is to evaluate the impact of torsemide initiation on warfarin dosage requirements. Setting This study was conducted at the Veterans Affairs Healthcare System in San Diego, California. Method A retrospective cohort study was conducted using Veterans Affairs data from patients who were converted from bumetanide to torsemide between March 2014 and July 2014. Patients were also prescribed and taking warfarin during the observation period. Warfarin dosage requirements were evaluated to determine if any changes occurred within the first 3 months of starting torsemide. Main outcome measure The primary outcome was the average weekly warfarin dose before and after torsemide initiation. Results Eighteen patients met study inclusion criteria. The weekly warfarin dose before and after initiation of torsemide was not significantly different (34 ± 15 and 34 ± 13 mg, p > 0.05). Of those eighteen patients, only two experienced elevations in INR that required a decrease in warfarin dosage after torsemide initiation. Between those two patients, dosage reductions ranged from 5.3 to 18%. Conclusion These results indicated that most patients did not require any warfarin dosage adjustments after torsemide was initiated. The potential for interaction, however, still exists. While empiric warfarin dosage adjustments are not recommended when initiating torsemide, increased monitoring is warranted to minimize the risk of adverse effects.

The Most Commonly Dispensed Prescription Medications Among Pregnant Women Enrolled in the U.S. Medicaid Program.

OBJECTIVE: To characterize the 20 most common prescription medications and the 10 most common prescription medications classified in the former U.S. Food and Drug Administration categories D or X dispensed to pregnant women enrolled in the U.S. Medicaid program. METHODS: We conducted a cohort study of 1,106,757 pregnant women with live births using 2000-2007 Medicaid Analytic eXtract data. We used outpatient pharmacy records to identify medication dispensings and reported the proportion of pregnancies that were dispensed at least one prescription medication. Maternal age and race and ethnicity-stratified estimates were compared using prevalence ratios and 95% confidence intervals (CIs). RESULTS: During pregnancy, 82.5% of the cohort had a dispensing for one or more prescription medication. The most commonly dispensed medications during pregnancy included nitrofurantoin (21.6%), metronidazole (19.4%), amoxicillin (18.0%), azithromycin (16.9%), and promethazine (13.5%). Proportions were highest among younger women for several medications; eg, nitrofurantoin (23.9% compared with 15.4%; prevalence ratio 1.55, CI 1.52-1.58), metronidazole (20.7% compared with 12.0%; prevalence ratio 1.73, CI 1.69-1.77), and azithromycin (21.1% compared with 11.0%; prevalence ratio 1.93, CI 1.89-1.97) were more common among women younger than 20 years than among women aged 35 years or older. Proportions were highest among white women with some exceptions; eg, compared with white women, metronidazole was more common among black women (29.8% compared with 14.4%; prevalence ratio 2.07, CI 2.05-2.09). Excluding fertility treatments, 42.0% had at least one dispensing for a D or X medication during pregnancy. Codeine (11.9%) and hydrocodone (10.2%) were the most common D medications. CONCLUSION: Medications used to treat infections were the most commonly dispensed prescription medications. Dispensing of commonly used prescription medications during pregnancy varied by maternal age and race-ethnicity. LEVEL OF EVIDENCE: II.

Contraceptive use in female recipients of a solid-organ transplant.

CONTEXT: Women of reproductive age account for more than one-third of all solid-organ transplant recipients and are advised against becoming pregnant for 1 to 2 years after their surgeries. The risks posed to the woman, the transplanted organ, and the fetus underscore the importance of systems to ensure that patients receive counseling on family planning, including return to fertility, contraceptive use, and when pregnancy can be safely considered, and highly effective methods of contraception. OBJECTIVE: To investigate use of contraceptives among women after solid-organ transplant and to identify opportunities to improve care. DESIGN: A cross-sectional survey study. SETTING: An urban academic medical center. PATIENTS: Women 18 to 50 years old who have received a kidney, pancreas, and/or liver transplant within the past 1 to 24 months. INTERVENTION: Participants completed self-administered questionnaires regarding their menstrual pattern, pregnancy history, contraceptive use before and after transplant, and counseling on family planning issues. MAIN OUTCOME MEASURES: Contraceptive use. RESULTS: The most common contraceptive method used in both the year preceding transplant and the year after transplant was condoms. Participants desired more counseling on highly effective contraceptive methods, such as intrauterine devices. Participants would like to receive contraceptive counseling from an obstetrician/gynecologist or transplant care team provider. CONCLUSION: Female recipients of solid-organ transplants want more counseling on highly effective methods of contraception.