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.

Azetidine-based selective glycine transporter-1 (GlyT1) inhibitors with memory enhancing properties.

A strategy to conformationally restrain a series of GlyT1 inhibitors identified potent analogs that exhibited slowly interconverting rotational isomers. Further studies to address this concern led to a series of azetidine-based inhibitors. Compound 26 was able to elevate CSF glycine levels in vivo and demonstrated potency comparable to Bitopertin in an in vivo rat receptor occupancy study. Compound 26 was subsequently shown to enhance memory in a Novel Object Recognition (NOR) behavioral study after a single dose of 0.03 mg/kg, and in a contextual fear conditioning (cFC) study after four QD doses of 0.01-0.03 mg/kg.

Rapid identification of a novel phosphodiesterase 7B tracer for receptor occupancy studies using LC─MS/MS.

Phosphodiesterase 7B (PDE7B) inhibition has been considered as a therapeutic target for the treatment of several neurological disorders. Currently, there are no radio-labeled tracers available to determine receptor occupancy (RO) of this target. Developing such a tracer could greatly facilitate the identification of viable PDE7B inhibitors. In the current study, a liquid chromatography tandem mass spectrometry (LC─MS/MS) method was utilized to evaluate the brain distribution of unlabeled tracer candidates following intravenous micro-dosing. This novel approach resulted in an accelerated identification of a potential novel RO tracer for PDE7B. The identified molecule, Compound 30, showed reasonable target-tissue specificity (striatum/cerebellum ratio of 2.2) and suitable uptake (0.25% of the injected dose/g brain tissue) as demonstrated in rats dosed with the unlabeled compound. Compound 30 was subsequently labeled with tritium (3H). In vitro characterization of 3H-Compound 30 demonstrated that this compound possessed a high target affinity with a subnanomolar Kd (0.8 nM) and a Bmax of 58 fmol/mg of protein using rat brain homogenate. Intravenous microdosing of 3H-Compound 30 showed preferential binding in the rat striatum, consistent with the mRNA distribution of PDE7B. In vitro displacement study with other structurally distinct PDE7B target-specific inhibitors using rat brain homogenate indicated that 3H-Compound 30 is an ideal tracer for Ki analysis. This is the first report of a preclinical tracer for PDE7B. With further characterization, Compound 30 may ultimately show the appropriate properties required to be further developed as a PDE7B PET ligand for clinical studies.

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.

Target Engagement of a Phosphodiesterase 2A Inhibitor Affecting Long-Term Memory in the Rat.

Inhibition of phosphodiesterase 2A (PDE2A) has been proposed as a potential approach to enhance cognitive functioning and memory through boosting intracellular cGMP/cAMP and enhancing neuroplasticity in memory-related neural circuitry. Previous preclinical studies demonstrated that PDE2A inhibitors could reverse N-methyl-D-aspartate receptor antagonist (5S,10R)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine or ketamine-induced memory deficit. Here, we report that the potent and selective PDE2A inhibitor 4-(1-azetidinyl)-7-methyl-5-[1-methyl-5-[5-(trifluoromethyl)-2-pyridinyl]-1H-pyrazol-4-yl]-imidazo[5,1-f][1,2,4]triazine (PF-05180999) enhances long-term memory in a contextual fear conditioning model in the rat at the oral dose of 0.3 mg/kg. Target engagement at this efficacious dose was explored using in vivo autoradiography. Converse to the results of a decrease of PDE2A binding (target occupancy) by the PDE2A inhibitor, a paradoxical increase (up to 40%) in PDE2A binding was detected. However, a typical target occupancy curve could be generated by PF-05180999 at much higher doses. In vitro experiments using recombinant PDE2A protein or rat brain homogenate that contains native PDE2A protein demonstrated that increased cGMP after initial PDE2A inhibition could be responsible for the activation of PDE2A enzyme via allosteric binding to the GAF-B domain, leading to positive cooperativity of the dormant PDE2A enzymes. Our results suggest that when evaluating target engagement of PDE2A inhibitors for memory disorder in clinical setting with occupancy assays, the efficacious dose may not fall on the typical receptor/target curve. On the contrary, an increase in PDE2A tracer binding is likely seen. Our results also suggest that when evaluating target occupancy of enzymes, potential regulation of enzyme activities should be considered.

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.

Design and Synthesis of Novel and Selective Glycine Transporter-1 (GlyT1) Inhibitors with Memory Enhancing Properties.

We report here the identification and optimization of a novel series of potent GlyT1 inhibitors. A ligand design campaign that utilized known GlyT1 inhibitors as starting points led to the identification of a novel series of pyrrolo[3,4- c]pyrazoles amides (21-50) with good in vitro potency. Subsequent optimization of physicochemical and in vitro ADME properties produced several compounds with promising pharmacokinetic profiles. In vivo inhibition of GlyT1 was demonstrated for select compounds within this series by measuring the elevation of glycine in the cerebrospinal fluid (CSF) of rats after a single oral dose of 10 mg/kg. Ultimately, an optimized lead, compound 46, demonstrated in vivo efficacy in a rat novel object recognition (NOR) assay after oral dosing at 0.1, 1, and 3 mg/kg.

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.

Discovery of Selective Phosphodiesterase 1 Inhibitors with Memory Enhancing Properties.

A series of potent thienotriazolopyrimidinone-based PDE1 inhibitors was discovered. X-ray crystal structures of example compounds from this series in complex with the catalytic domain of PDE1B and PDE10A were determined, allowing optimization of PDE1B potency and PDE selectivity. Reduction of hERG affinity led to greater than a 3000-fold selectivity for PDE1B over hERG. 6-(4-Methoxybenzyl)-9-((tetrahydro-2H-pyran-4-yl)methyl)-8,9,10,11-tetrahydropyrido[4',3':4,5]thieno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidin-5(6H)-one was identified as an orally bioavailable and brain penetrating PDE1B enzyme inhibitor with potent memory-enhancing effects in a rat model of object recognition memory.

Design and Synthesis of Novel and Selective Phosphodiesterase 2 (PDE2a) Inhibitors for the Treatment of Memory Disorders.

A series of potent and selective [1,2,4]triazolo[1,5-a]pyrimidine PDE2a inhibitors is reported. The design and improvement of the binding properties of this series was achieved using X-ray crystal structures in conjunction with careful analysis of electronic and structural requirements for the PDE2a enzyme. One of the lead compounds, compound 27 (DNS-8254), was identified as a potent and highly selective PDE2a enzyme inhibitor with favorable rat pharmacokinetic properties. Interestingly, the increased potency of compound 27 was facilitated by the formation of a halogen bond with the oxygen of Tyr827 present in the PDE2a active site. In vivo, compound 27 demonstrated significant memory enhancing effects in a rat model of novel object recognition. Taken together, these data suggest that compound 27 may be a useful tool to explore the pharmacology of selective PDE2a inhibition.

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.

Clinical characteristics and causality of eye lid laceration in iran.

OBJECTIVE: The aim of this study was to assess the etiological characteristics and visual outcomes of ocular trauma with more attention to eyelid laceration. METHODS: In a cross-sectional study, 98 cases of isolated traumatic eyelid laceration were consecutively studied and its epidemiology, etiology and association with visual outcome were evaluated. The findings of this study could be used to develop healthcare related precautions and work place safety recommendations. RESULTS: Of the 98 patients included in the study, men exhibited a greater vulnerability and they were mostly aged around 29 years old. In terms of the place of trauma, 40 (43.5%) cases occurred in the street, 27 cases (29.3%) occurred at home, and 17 cases (18.5%) occurred at the workplace, while 4 cases (4.3%) happened in entertaining environments like parks. For 3 patients (3.3%), the incident took place at a public pathway, and in 1 patient (1.1%), the case occurred at school. As the most common cause of trauma, 41 patients (42.3%) had an object hit their eyes. In addition, assaults were a major cause of injury. The right eye and the upper lid were also the most common sites of injuries. Although no blindness occurred due to trauma causing eyelid laceration, the visual outcomes were correlated with severity of the incident defined based on the presence of open globe injuries. CONCLUSION: This study could possibly highlight the risk factors of eyelid laceration and provide the healthcare community with the essential recommendations regarding the safety precautions in dangerous settings, including daily / routine work places.

A controlled study of amniotic membrane transplantation for acute Pseudomonas keratitis.

OBJECTIVE: To evaluate the efficacy of amniotic membrane transplantation (AMT) to improve the outcomes of acute Pseudomonas keratitis as compared with a control group. DESIGN: Prospective interventional case series with retrospective controls. PARTICIPANTS: We studied 14 eyes with Pseudomonas keratitis as the AMT group and 11 eyes with Pseudomonas keratitis as the control group. METHODS: Eyes in the AMT group were treated with antibiotic therapy followed by single-layer AMT at 2 to 3 days. Eyes in the control group received only antibiotic therapy. Patients were followed for 11.1 ± 2.4 months. RESULTS: In the AMT group, pain significantly decreased from a mean score of 2.4 ± 0.5 preoperatively to 1.1 ± 0.9 at day 2 postoperatively (p < 0.001). Corneal epithelial defects healed completely within 13.2 ± 2.6 days in the AMT group compared with 15.5 ± 3.4 days in the control group (p = 0.07). At final follow-up visits, the sizes of corneal opacity and deep neovascularization were not different between the 2 groups. However, the mean score for density of the corneal opacity was significantly less in the AMT group compared with the control group (2.1 ± 0.4 vs 2.5 ± 0.7, respectively, p = 0.04). Although the best corrected visual acuity using hard contact lenses was not different between the 2 groups, uncorrected visual acuity was better in the AMT group (0.45 ± 0.22 logMAR) than in the control group (0.71 ± 0.32 logMAR, p = 0.03). No patient in either group developed significant corneal thinning or perforation. CONCLUSIONS: AMT in acute Pseudomonas keratitis was associated with immediate pain relief, less density of the final corneal opacity, and better uncorrected visual acuity at the final follow-up visit.

Discovery of potential antipsychotic agents possessing pro-cognitive properties.

Current antipsychotic drug therapies for schizophrenia have limited efficacy and are notably ineffective at addressing the cognitive deficits associated with this disorder. The present study was designed to develop effective antipsychotic agents that would also ameliorate the cognitive deficits associated with this disease. In vitro studies comprised of binding and functional assays were utilized to identify compounds with the receptor profile that could provide both antipsychotic and pro-cognitive features. Antipsychotic and cognitive models assessing in vivo activity of these compounds included locomotor activity assays and novel object recognition assays. We developed a series of potential antipsychotic agents with a novel receptor activity profile comprised of muscarinic M(1) receptor agonism in addition to dopamine D(2) antagonism and serotonin 5-HT(2A) inverse agonism. Like other antipsychotic agents, these compounds reverse both amphetamine and dizocilpine-induced hyperactivity in animals. In addition, unlike other antipsychotic drugs, these compounds demonstrate pro-cognitive actions in the novel object recognition assay. The dual attributes of antipsychotic and pro-cognitive actions distinguish these compounds from other antipsychotic drugs and suggest that these compounds are prototype molecules in the development of novel pro-cognitive antipsychotic agents.