Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase.

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.

Retraction notice to "Endophthalmitis Occurring after Cataract Surgery: Outcomes of More Than 480 000 Cataract Surgeries, Epidemiologic Features, and Risk Factors" (Ophthalmology. 2016;123:295-301).

This article has been retracted: please see Elsevier policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). The editors wish to note that concerns were raised regarding coding errors in the data set that formed the basis of this study. Patient record numbers were found to be duplicated, so that the number of endophthalmitis cases was unclear as was the associated treatment, and the number of unique patients estimated to be far less than the 480,000 reported. Upon review of the information provided, Ophthalmology has determined the dataset to be flawed with unverifiable elements from which reliable conclusions cannot be drawn, and therefore has made the decision to issue a retraction of the manuscript.

Effect of phosphodiesterase (1B, 2A, 9A and 10A) inhibitors on central nervous system cyclic nucleotide levels in rats and mice.

Phosphodiesterase (PDE) inhibition has been broadly investigated as a target for a wide variety of indications including central nervous system (CNS) disorders. Cyclic nucleotide (cNT) changes within associated tissues may serve as a biomarker of PDE inhibition. We recently developed robust sample harvesting and bioanalytical methods to quantify cNT levels in rodent brain and cerebrospinal fluid (CSF). Herein, we report on the application of those methods to study rodent species-specific and rodent brain region-specific cNT changes following individual or concomitant PDE inhibitor administration. Male Sprague Dawley (Crl:CD® [SD]) rats were dosed subcutaneously (sc) with a PDE1B inhibitor (DNS-0056), a PDE2A inhibitor (PF-05180999), a PDE9A inhibitor (PF-4447943), and a PDE10A inhibitor (MP10), each at a single dose of 10 or 30 mg/kg, or concomitantly with all 4 inhibitors at 10 mg/kg each. Male Carworth Farms (Crl:CF1 ®[CF-1]) mice were dosed intraperitoneally (ip) with the four individual inhibitors at a single dose of 10 mg/kg or concomitantly with all 4 inhibitors at 10 mg/kg each. The doses studied are generally adequate for affecting measurable cNT levels in the tissues of interest and were thereby chosen for this investigation. Measured 3',5'-cyclic adenosine monophosphate (cAMP) changes were generally statistically insignificant in the brain, striatum and CSF after administration of the aforementioned PDE inhibitors. However, the levels of 3',5'-cyclic guanosine monophosphate (cGMP) increased in both rat and mouse striatum (2.2-, 2.1- and 1.7-fold and 6.4-, 2.8- and 1.7-fold, respectively) after PDE2A, 9A, and 10A inhibitor dosing. In all cases, the cNT changes followed the same trend in the brain, striatum and CSF after PDE inhibitor dosing and dose response was observed in rats. Concomitant treatment with PDE1B, PDE2A, PDE9A and PDE10A inhibitors resulted in a 4.4- and 36.7-fold increase of cGMP in rat and mouse striatum. The drug exposures after concomitant treatment were also higher than in the individual inhibitor-treated animals. cGMP enhancement observed could be due to synergistic effects, though an additive effect of the combined inhibitor concentrations may also contribute.

A surrogate analyte-based liquid chromatography-tandem mass spectrometry method for the determination of endogenous cyclic nucleotides in rat brain.

A robust high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) assay was developed and qualified for the measurement of cyclic nucleotides (cNTs) in rat brain tissue. Stable isotopically labeled 3',5'-cyclic adenosine-13C5 monophosphate (13C5-cAMP) and 3',5'-cyclic guanosine-13C,15N2 monophosphate (13C15N2-cGMP) were used as surrogate analytes to measure endogenous 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP). Pre-weighed frozen rat brain samples were rapidly homogenized in 0.4M perchloric acid at a ratio of 1:4 (w/v). Following internal standard addition and dilution, the resulting extracts were analyzed using negative ion mode electrospray ionization LC-MS/MS. The calibration curves for both analytes ranged from 5 to 2000ng/g and showed excellent linearity (r2>0.996). Relative surrogate analyte-to-analyte LC-MS/MS responses were determined to correct concentrations derived from the surrogate curves. The intra-run precision (CV%) for 13C5-cAMP and 13C15N2-cGMP was below 6.6% and 7.4%, respectively, while the inter-run precision (CV%) was 8.5% and 5.8%, respectively. The intra-run accuracy (Dev%) for 13C5-cAMP and 13C15N2-cGMP was <11.9% and 10.3%, respectively, and the inter-run Dev% was <6.8% and 5.5%, respectively. Qualification experiments demonstrated high analyte recoveries, minimal matrix effects and low autosampler carryover. Acceptable frozen storage, freeze/thaw, benchtop, processed sample and autosampler stability were shown in brain sample homogenates as well as post-processed samples. The method was found to be suitable for the analysis of rat brain tissue cAMP and cGMP levels in preclinical biomarker development studies.

Endophthalmitis Occurring after Cataract Surgery: Outcomes of More Than 480 000 Cataract Surgeries, Epidemiologic Features, and Risk Factors.

PURPOSE: To report the incidence of endophthalmitis after senile cataract surgery and to describe the epidemiology and main risk factors. DESIGN: Retrospective, single-center, cross-sectional descriptive study. PARTICIPANTS: Patients who underwent cataract surgery in Farabi Eye Hospital from 2006 through 2014. METHODS: All patients were evaluated retrospectively to compare risk factors, epidemiologic factors, and prophylaxis methods related to endophthalmitis. Patient records were used to gather the data. MAIN OUTCOME MEASURES: Epidemiologic factors, systemic diseases, other ocular pathologic characteristics, complications during the surgery, technique of cataract surgery, intraocular lens type, method of antibiotic prophylaxis, surgeon experience, vitreous culture, and vision outcome were evaluated in these patients. RESULTS: One hundred twelve endophthalmitis cases among 480 104 operations reported, equaling an incidence of 0.023%. Patients with diabetes mellitus (14.3%) and of older age (mean age, 81 years), perioperative communication with the vitreous (17.9%), extracapsular cataract surgery procedure (11%), and surgery on the left eye (58.9% vs. 41.1% for right eye; P = 0.03) showed a statistically significant association with endophthalmitis. Short-term treatment with topical or systemic preoperative antibiotics or postoperative subconjunctival injection was associated with a 40% to 50% reduced odds of endophthalmitis compared with no prophylaxis (P = 0.2). No cases of endophthalmitis were observed among the 25 920 patients who received intracameral cefuroxime, suggesting that this approach to antibiotic prophylaxis may be far more effective than traditional topical or subconjunctival approaches. CONCLUSIONS: The incidence of endophthalmitis after cataract surgery in our center was 0.023%, comparable with that of other previously published international studies. Older rural patients with immune suppressive diseases, such as diabetes mellitus, are particularly more prone to endophthalmitis. Vitreous loss at the time of surgery was associated with a significantly increased risk. Whereas antibiotic prophylaxis overall showed a 40% to 50% reduction in risk, intracameral cefuroxime was 100% effective in preventing endophthalmitis in this series.

Accuracy of 3 imaging modalities for evaluation of the posterior lens capsule in traumatic cataract.

PURPOSE: To compare the accuracy of 3 imaging modalities for preoperative evaluation of the posterior lens capsule in traumatic cataract. SETTING: Farabi Eye Hospital, Tehran, Iran. DESIGN: Case series. METHODS: The study comprised eyes with traumatic cataract opaque enough to prevent visualization of the posterior lens capsule on slitlamp examination. To detect posterior lens capsule rupture before surgery, imaging was performed with 20 MHz echography (Eye Cubed), anterior segment optical coherence tomography (AS-OCT) (Visante model 1000), and Scheimpflug imaging (Pentacam). All patients subsequently had cataract extraction, and the intraoperative findings of the posterior lens capsule were compared with the preoperative findings of the imaging modalities. RESULTS: The study enrolled 21 eyes of 21 patients (20 men, 1 woman) with a mean age of 31.5 years ± 1.45 (SD). The nature of trauma was blunt (5 eyes) or sharp (16 eyes). To detect posterior lens capsule rupture, the sensitivity and specificity were, respectively, 80% and 86% for 20 MHz echography, 71% and 77% for AS-OCT, and 62% and 57% for Scheimpflug imaging (95% confidence intervals: sensitivity, 30.00-90.32; specificity, 54.81-92.95). Insufficient resolution for posterior lens capsule evaluation occurred in 33.3% cases for AS-OCT and 57.1% cases for Scheimpflug imaging. The accuracy of 20 MHz echography, AS-OCT, and Scheimpflug imaging was 76.1%, 61.9%, and 42.9%, respectively. CONCLUSION: In the evaluation of the posterior lens capsule in eyes with traumatic cataract, 20 MHz echography had higher accuracy than AS-OCT and Scheimpflug imaging. FINANCIAL DISCLOSURE: No author has a financial or proprietary interest in any material or method mentioned.

Evaluation of posterior lens capsule by 20-MHz ultrasound probe in traumatic cataract.

PURPOSE: To investigate the accuracy of echography with a 20-MHz probe for evaluation of posterior lens capsule in traumatic cataract before surgery. DESIGN: Prospective interventional case series. METHODS: This study consisted of 43 eyes with traumatic cataract that were scheduled to undergo surgery. In all cases, cataract was dense enough to prevent visualization of the posterior lens capsule. Echography was performed using a 20-MHz probe to detect rupture of the posterior lens capsule. All patients subsequently underwent cataract extraction and intraoperative findings of the posterior lens capsule were compared with the preoperative echographic findings. RESULTS: This study included 43 eyes of 43 patients (38 men and 5 women) with a mean age of 35.6 ± 15.3 years (range, 4-68 years). The trauma was either blunt (4 eyes) or sharp (39 eyes); there was closed globe injury in 2 eyes and open globe injury in 41 eyes. By 20-MHz echography, posterior border of the crsytalline lens was clearly visualized in all 43 eyes. By 20-MHz echographic imaging, rupture of the posterior lens capsule was identified in 17 eyes (39.5%). During cataract surgery, it was noted that 14 eyes (32.6%) had rupture of the posterior lens capsule. Sensitivity, specificity, positive predictive value, and negative predictive value were 93%, 86%, 76%, and 96%, respectively, for 20-MHz echography to detect rupture of the posterior lens capsule. Also, the positive likelihood ratio, negative likelihood ratio, and odds ratio were 6.7, 0.08, and 81, respectively. CONCLUSION: Echography with 20-MHz probe is an accurate imaging modality for detection of posterior lens capsule rupture in traumatic cataract preoperatively. This technique helps ophthalmologists have an appropriate surgical plan before operating.

Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators.

Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.

Intravitreal bevacizumab with or without triamcinolone for refractory diabetic macular edema; a placebo-controlled, randomized clinical trial.

PURPOSE: To evaluate the effect of three intravitreal injections of bevacizumab (IVB) alone or combined with triamcinolone (IVT) in the first injection for treatment of refractory diabetic macular edema (DME). METHODS: In this prospective, placebo-controlled, randomized clinical trial, 115 eyes of 101 patients with refractory DME were included. Subjects were randomly assigned to one of the three study arms: 1) three injections of IVB (1.25 mg/0.05 ml) at 6-week intervals, 2) combined IVB and IVT (1.25 mg/0.05 ml and 2 mg/0.05 ml respectively) followed by two injections of IVB at 6-week intervals, and 3) sham injection (control group). The primary outcome measure was change in central macular thickness (CMT). Secondary outcome measures were change in best-corrected logMAR visual acuity (BCVA ) and incidence of potential adverse events. RESULTS: Central macular thickness was reduced significantly in both the IVB and IVB/IVT groups. At week 24, CMT change compared to the baseline was -95.7 microm (95% CI, -172.2 to -19.26) in the IVB group, -92.1 microm (95% CI, -154.4 to -29.7) in the IVB/IVT group, and 34.9 microm (95% CI, 7.9 to 61.9) in the control group. There was a significant difference between the IVB and control groups (P = 0.012) and between the IVB/IVT and control groups (P = 0.022). Improvement of BCVA was initiated at weeks 6 and 12 in the IVB/IVT and IVB groups respectively. In terms of BCVA change compared to the baseline at 24 weeks, the differences between the IVB and control groups (P = 0.01) and also between the IVB/IVT and control groups (P = 0.006) were significant. No significant differences were detected in the changes of CMT and BCVA between the IVB and IVB/IVT groups (P = 0.99). Anterior chamber reaction was noticed in eight (19.5%) and seven (18.9%) eyes respectively in the IVB and IVB/IVT groups the day after injection, and it resolved with no sequel. Elevation of IOP occurred in three eyes (8.1%) in the IVB/IVT group. CONCLUSION: Three consecutive intravitreal injections of bevacizumab had a beneficial effect on refractory DME in terms of CMT reduction and BCVA improvement. Addition of triamcinolone in the first injection seemed to induce earlier visual improvement; however, it did not show any significant additive effect later during follow-up.

Identification of glutathione-derived metabolites from an IP receptor antagonist.

The metabolic fate of three aromatic carboxylic acid analogs under evaluation as prostaglandin I2-preferring receptor antagonists was studied. The initial analog with unsubstituted phenyl groups was subject to a complex set of aromatic oxidative biotransformations. By introduction of one or two fluorines, these pathways were inhibited. All three analogs were metabolized to a wide variety of carboxylic acid conjugates. Among these were several conjugates formed via secondary metabolism and oxidation of acyl glutathione intermediates. Two of the structure classes, represented by the S-methyl-N-cysteinylglycine conjugate and the N-cysteinylglycine disulfide conjugates, have been described only rarely in the literature. The related S-oxide of the S-methyl-N-cysteinylglycine conjugate and the N,S-bis-acyl derivative of cysteinylglycine are here described for the first time as conjugate metabolites of carboxylic drugs.

Comparison of in vitro P-glycoprotein screening assays: recommendations for their use in drug discovery.

The ATP-dependent drug efflux pump P-glycoprotein (P-gp) affects the absorption and disposition of many compounds. P-gp may also play role in clinically significant drug-drug interactions. Therefore, it is important to find potential substrates or inhibitors of P-gp early in the drug discovery process. To identify compounds that interact with this transporter, several P-gp assays were validated and compared by testing a set of 28 reference compounds, including inhibitors of cytochrome P450 3A4 (CYP3A4). The assays included in silico predictions, inhibition assays (based on cellular uptake of rhodamine-123 or calcein AM), and functional assays (ATPase activity assay and transcellular transport assay, the latter for a subset of compounds). In addition, species differences were studied in an indirect fluorescence indicator screening assay and test systems expressing porcine, mouse, or human P-gp. Our results suggest that several P-gp assays should be used in combination to classify compounds as substrates or inhibitors of P-gp. Recommendations are given on screening strategies which can be applied to different phases of the drug discovery and development process.