A High-Throughput Fluorescence Polarization-Based Assay for the SH2 Domain of STAT4.

The signal transducer and activation of transcription (STAT) proteins are a family of Src homology 2 (SH2) domain-containing transcription factors. The family member STAT4 is a mediator of IL-12 signalling and has been implicated in the pathogenesis of multiple autoimmune diseases. The activity of STAT4 requires binding of phosphotyrosine-containing motifs to its SH2 domain. Selective inhibitors of the STAT4 SH2 domain have not been published to date. Here, we present a fluorescence polarization-based assay for the identification of inhibitors of the STAT4 SH2 domain. The assay is based on the interaction between the STAT4 SH2 domain and the fluorophore-labelled peptide 5-carboxyfluorescein-GpYLPQNID (Kd = 34 ± 4 nM). The assay is stable with respect to DMSO concentrations of up to 10% and incubation times of at least 8 h. The Z'-value of 0.85 ± 0.01 indicates that the assay is suited for use in high-throughput screening campaigns aimed at identifying new therapeutic modalities for the treatment of autoimmune diseases.

Efficacy and safety at 6 months of the XEN implant for the management of open angle glaucoma.

The purpose of this study is to evaluate the efficacy and complications of the XEN implant as a solo procedure or in association with cataract surgery in patients with open angle glaucoma (OAG). All patients who received a XEN implant between June 2017 and June 2018 were included in the study. The primary and secondary outcomes were: the reduction of the intraocular pressure (IOP) at 6 months postoperatively, the decrease of the glaucoma medications 6 months after surgery, the clinical success rate (eyes (%) achieving ≥20% IOP reduction on the same or fewer medications without secondary surgical intervention), the frequency and type of postoperative interventions as well as the complication rate. We included one hundred and seven eyes from 97 patients with primary OAG (79%), or secondary OAG (21%). Seventy-seven patients (72%) received a standalone XEN implantation and 30 (28%) underwent XEN implantation combined with phacoemusification. The IOP decreased from 20.4 mm Hg ± 6.4 preoperatively to 15.4 mm Hg ± 5.3 six months after the surgery, which represented a reduction of 24.5% (P = 1.4.10-7). It was associated with a lowering of glaucoma medications from 2.8 ± 1.0 preoperatively to 0.6 ± 1.0 six months postoperatively (P = 3.6.10-34). The clinical success rate was 67.2% six months after the surgery. The most frequent complications were: IOP spikes >30 mmHg (16.8%), improper position or angled drain (14.0%) and transient minimal hyphema (<1 week) (11.2%). During the follow-up, the needling was required in 34.6% of cases and a total of 10 eyes (9.4%) required a new glaucoma surgery. To conclude XEN implantation appears to be an effective short- and mid-term surgical technique to control IOP in OAG with a low risk of complication. However postoperative maneuvers were frequently required to maintain efficiency.

Comparative Effectiveness and Tolerance of Subliminal Subthreshold Transscleral Cyclophotocoagulation With a Duty Factor of 25% Versus 31.3% for Advanced Glaucoma.

PRéCIS:: Subliminal subthreshold transscleral cyclophotocoagulation (SS-TSCPC) with duty cycles 25% and 31.3% seems to be an effective approach to reduce intraocular pressure (IOP) in glaucoma that is refractory to medical management. OBJECTIVE: The objective of this study was to compare the effectiveness and the tolerance of SS-TSCPC with a duty cycle of 25% versus 31.3% with Supra 810 nm Subliminal Quantel Medical laser stimulation for advanced glaucoma. MATERIALS AND METHODS: This was a retrospective, single-center, comparative case series of patients treated by SS-TSCPC between January 2017 and July 2017. The diagnostic and inclusion criteria were patients with advanced and refractory glaucoma, defined as IOP >21 mm Hg on maximal tolerated medical therapy with or without previous glaucoma surgical procedures, a minimum follow-up of 12 months, and patients who refused or were poor candidates for additional filtering surgery or implantation of glaucoma drainage devices. The primary endpoint was surgical success defined as an IOP of 6 to 21 mm Hg or a reduction of IOP by 20% from baseline without an increase in glaucoma medication from baseline. The secondary endpoints were the mean IOP and best visual corrected acuity best-corrected visual acuity at 12 months after surgery, retreatment outcomes, glaucoma medications, and complications such as inflammation, uveitis, cataract, mydriasis, and phthisis. RESULTS: Forty eyes of 32 patients were included: 20 eyes were subjected to SS-TSCPC with 31.3% duty cycle and 20 eyes with a 25% duty cycle. The surgical success of the TSCPC 12 months after the first procedure was better in the 31.3% duty cycle group (83.5%) than in the 25% duty cycle group (65%). The most common complications were inflammation (50%, 1 mo after surgery) and IOP spikes (increase in IOP of >25% from baseline within 1 mo of laser) in both groups. Inflammation was higher in the 31.3% duty cycle group. CONCLUSIONS: SS-TSCPC at 31.3% and 25% duty cycle seems to be an effective approach to reduce IOP in glaucoma that is refractory to medical management. SS-TSCPC at 31.3% duty cycle is more effective than the 25% duty cycle SS-TSCPC. However, the 31.3% duty cycle SS-TSCPC induces more inflammation than the 25% duty cycle SS-TSCPC. Each procedure should be considered on a case by case basis.

Argon Laser Iridoplasty For Plateau Iris Syndrome: Long-Term Outcomes of 48 Eyes.

PRECIS: Argon laser peripheral iridoplasty (ALPI) was performed on 48 eyes with plateau iris syndrome (PIS). Indentation gonioscopy was used to monitor the opening of the iridocorneal angle. Mean intraocular pressure (IOP) at 5 years decreased from 15.9 to 14.4 mm Hg. None of the eyes required trabeculectomy. PURPOSE: The purpose of this article was to report the long-term outcomes of ALPI for PIS. MATERIALS AND METHODS: A retrospective chart review was performed on all patients with PIS treated with ALPI from 2001 to 2012. The study included 48 eyes from 28 patients with PIS after peripheral iridotomy, with a follow-up of at least 5 years. Patients with advanced glaucoma requiring initial surgical treatment (pathologic discs and IOP above the target IOP despite medical treatment) were excluded. The primary outcomes were the effect on the number of IOP medications, and the need for complementary selective laser trabeculoplasty (SLT) or surgery (trabeculectomy and/or phacoemulsification). Secondary outcomes were optic nerve head changes and adverse events. RESULTS: The mean IOP statistically decreased after ALPI (15.91±2.62 vs. 14.35±2.18 mm Hg, P>0.001). The mean number of IOP-lowering medications statistically increased after ALPI (0.81±0.94 vs. 1.2±1.04, P>0.001). Mean follow-up was 92.4±26.5 months. At the end of the follow-up, 12 (25%) eyes had no medications, 20 (42%) had 1 medication, 11 (23%) had 2 medications, 3 (6%) had 3 medications, and 2 (4%) had 4. Ten (21%) eyes underwent SLT, 6 (10%) underwent phacoemulsification, and no trabeculectomy was necessary during follow-up. The change in cup to disc ratio from pre-ALPI to latest follow-up was not statistically significant, and no adverse events were reported. CONCLUSIONS: ALPI is relatively effective and safe to prevent angle-closure glaucoma and avoid trabeculectomy in patients with PIS. This procedure often helps to control IOP, although SLT and additional medical treatments are frequently necessary to maintain the target IOP.

The STAT5b Linker Domain Mediates the Selectivity of Catechol Bisphosphates for STAT5b over STAT5a.

STAT family proteins are important mediators of cell signaling and represent therapeutic targets for the treatment of human diseases. Most STAT inhibitors target the protein-protein interaction domain, the SH2 domain, but specificity for a single STAT protein is often limited. Recently, we developed catechol bisphosphates as the first inhibitors of STAT5b demonstrated to exhibit a high degree of selectivity over the close homologue STAT5a. Here, we show that the amino acid in position 566 of the linker domain, not the SH2 domain, is the main determinant of specificity. Arg566 in wild-type STAT5b favors tight binding of catechol bisphosphates, while Trp566 in wild-type STAT5a does not. Amino acid 566 also determines the affinity for a tyrosine-phosphorylated peptide derived from the EPO receptor for STAT5a and STAT5b, demonstrating the functional relevance of the STAT5 linker domain for the adjacent SH2 domain. These results provide the first demonstration that a residue in the linker domain can determine the affinity of nonpeptidic small-molecule inhibitors for the SH2 domain of STAT proteins. We propose targeting the interface between the SH2 domain and linker domain as a novel design approach for the development of potent and selective STAT inhibitors. In addition, our data suggest that the linker domain could contribute to the enigmatically divergent biological functions of the two STAT5 proteins.

Synthesis and biochemical evaluation of highly enantiomerically pure (R,R)- and (S,S)-alexidine.

Alexidine is in everyday human use as oral disinfectant and contact lens disinfectant. It is used as a mixture of stereoisomers. Since all of alexidine's known biological targets are chiral, the biological activity of any of its chiral stereoisomers could be significantly higher than that of the mixture of stereoisomers. This makes a synthetic methodology for obtaining the individual enantiomers of the chiral diastereoisomer highly desirable. Here, we describe the first synthesis of both enantiomers of alexidine in high enantiomeric purity, and demonstrate their activity against the protein-protein interaction between the anti-apoptotic protein Bcl-xL and the pro-apoptotic protein Bak.

Phosphopeptides with improved cellular uptake properties as ligands for the polo-box domain of polo-like kinase 1.

Human polo-like kinase 1 (Plk1) is involved in cell proliferation and overexpressed in a broad variety of different cancer types. Due to its crucial role in cancerogenesis Plk1 is a potential target for diagnostic and therapeutic applications. Peptidic ligands can specifically interact with the polo-box domain (PBD) of Plk1, a C-terminal located phosphoepitope binding motif. Recently, phosphopeptide MQSpTPL has been identified as ligand with high binding affinity. However, a radiolabeled version of this peptide showed only insufficient cellular uptake. The present study investigated peptide dimers consisting of PBD-targeting phosphopeptide MQSpTPL and a cell-penetrating peptide (CPP) moiety. The new constructs demonstrate superior uptake in different cancer cell-lines compared to the phosphopeptide alone. Furthermore, we could demonstrate binding of phosphopeptide-CPP dimers to PBD of Plk1 making the compounds interesting leads for the development of molecular probes for imaging Plk1 in cancer.

Serendipitous alkylation of a Plk1 ligand uncovers a new binding channel.

We obtained unanticipated synthetic byproducts from alkylation of the δ(1) nitrogen (N3) of the histidine imidazole ring of the polo-like kinase-1 (Plk1) polo-box domain (PBD)-binding peptide PLHSpT. For the highest-affinity byproduct, bearing a C(6)H(5)(CH(2))(8)- group, a Plk1 PBD cocrystal structure revealed a new binding channel that had previously been occluded. An N-terminal PEGylated version of this peptide containing a hydrolytically stable phosphothreonyl residue (pT) bound the Plk1 PBD with affinity equal to that of the non-PEGylated parent but showed markedly less interaction with the PBDs of the two closely related proteins Plk2 and Plk3. Treatment of cultured cells with this PEGylated peptide resulted in delocalization of Plk1 from centrosomes and kinetochores and in chromosome misalignment that effectively induced mitotic block and apoptotic cell death. This work provides insights that might advance efforts to develop Plk1 PBD-binding inhibitors as potential Plk1-specific anticancer agents.

Selective targeting of disease-relevant protein binding domains by O-phosphorylated natural product derivatives.

Phosphorylation-dependent protein binding domains are crucially important for intracellular signaling pathways and thus highly relevant targets in chemical biology. By screening of chemical libraries against 12 structurally diverse phosphorylation-dependent protein binding domains, we have identified fosfosal and dexamethasone-21-phosphate as selective inhibitors of two antitumor targets: the SH2 domain of the transcription factor STAT5b and the substrate-binding domain of the peptidyl-prolyl isomerase Pin1, respectively. Both compounds are phosphate prodrugs with documented clinical use as anti-inflammatory agents in humans and were discovered with a high hit rate from a small subgroup within the screening library. Our study indicates O-phosphorylation of appropriately preselected natural products or natural product derivatives as a generally applicable strategy for the identification of non-reactive and non-peptidic ligands of phosphorylation-dependent protein binding domains. Moreover, our data indicate that it would be advisable to monitor the bioactivities of clinically used prodrugs in their uncleaved state against phosphorylation-dependent protein binding domains.

A novel direct screening method for alkyl glucoside production by glucosidases expressed in E. coli in 96-well plates.

The present work describes the development of a novel direct screening method, assayed in 96-well format, for evaluation of enzymatic alkyl glycoside production in a hexanol-water two-phase system. Alkyl glycosides are surfactants with a range of applications and with good biodegradability and low toxicity. Enzymatic synthesis makes it possible to prepare beta-d-glucopyranosides with high purity. In the developed screening assay, hexyl-beta-d-glucopyranoside was chosen as a model product to be synthesised by reversed hydrolysis in a water-hexanol two-phase system. In a first step the model product is produced by glucosidases expressed in E. coli cells in 96-deep-well plates. After phase separation, the hexyl-beta-d-glucopyranoside in the organic phase is degraded enzymatically and the released glucose detected spectrophotometrically at 405nm utilizing peroxidase/glucose oxidase, and the reagent 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). The aqueous phase is used to monitor hydrolysis of p-NPG at 405nm, allowing use of a ratio of the two assays to compensate for expression differences. The complete method was used for comparison of two different beta-glucosidases, classified under glycoside hydrolase family 1 and 3, respectively, showing a significant difference in their ability to synthesise hexyl-beta-d-glucopyranoside by reversed hydrolysis.

Development of high-throughput assays based on fluorescence polarization for inhibitors of the polo-box domains of polo-like kinases 2 and 3.

The serine/threonine kinases Plk1, Plk2, and Plk3 harbor a protein-protein interaction domain dubbed polo-box domain (PBD). Recently, the inhibition of the PBD of the cancer target Plk1 has been successfully explored as an alternative to the inhibition of the kinase by ATP-competitive ligands. However, because the PBDs of Plk1, Plk2, and Plk3 have very similar optimal binding motifs, absolute specificity for the PBD of Plk1 over the PBDs of Plk2 and Plk3 may also represent a big challenge for a small molecule. To aid in the activity profiling of Plk PBD inhibitors, and to identify selective small molecules that will reveal the cellular consequences of inhibiting the PBDs of Plk2 and Plk3, we have developed high-throughput assays based on fluorescence polarization against the PBDs of Plk2 and Plk3. The assays are stable with regard to time and 10% dimethyl sulfoxide and have Z' values > or = 0.7, making them well-suited for high-throughput screening. Moreover, our data provide insights into the binding preferences of the PBDs of Plk2 and Plk3.