Identification of MK-5710 ((8aS)-8a-methyl-1,3-dioxo-2-[(1S,2R)-2-phenylcyclo- propyl]-N-(1-phenyl-1H-pyrazol-5-yl)hexahydro-imidazo[1,5-a]pyrazine-7(1H)-carboxamide), a potent smoothened antagonist for use in Hedgehog pathway dependent malignancies, part 2

The Hedgehog (Hh-) signaling pathway is a key developmental pathway which gets reactivated in many human tumors, and smoothened (Smo) antagonists are emerging as novel agents for the treatment of malignancies dependent on the Hh-pathway, with the most advanced compounds demonstrating encouraging results in initial clinical trials. A novel series of potent bicyclic hydantoin Smo antagonists was reported in the preceding article, these have been resolved, and optimized to identify potent homochiral derivatives with clean off-target profiles and good pharmacokinetic properties in preclinical species. While showing in vivo efficacy in mouse allograft models, unsubstituted bicyclic tetrahydroimidazo[1,5-a]pyrazine-1,3(2H,5H)-diones were shown to epimerize in plasma. Alkylation of the C-8 position blocks this epimerization, resulting in the identification of MK-5710 (47) which was selected for further development.     

Crystal structures of the active and alloxanthine-inhibited forms of xanthine dehydrogenase from Rhodobacter capsulatus.

Xanthine dehydrogenase (XDH), a complex molybdo/iron-sulfur/flavoprotein, catalyzes the oxidation of hypoxanthine to xanthine followed by oxidation of xanthine to uric acid with concomitant reduction of NAD+. The 2.7 A resolution structure of Rhodobacter capsulatus XDH reveals that the bacterial and bovine XDH have highly similar folds despite differences in subunit composition. The NAD+ binding pocket of the bacterial XDH resembles that of the dehydrogenase form of the bovine enzyme rather than that of the oxidase form, which reduces O(2) instead of NAD+. The drug allopurinol is used to treat XDH-catalyzed uric acid build-up occurring in gout or during cancer chemotherapy. As a hypoxanthine analog, it is oxidized to alloxanthine, which cannot be further oxidized but acts as a tight binding inhibitor of XDH. The 3.0 A resolution structure of the XDH-alloxanthine complex shows direct coordination of alloxanthine to the molybdenum via a nitrogen atom. These results provide a starting point for the rational design of new XDH inhibitors.     

Invasive Mold Infections in Patients with Chronic Lymphoproliferative Disorders

Purpose of the Review

This review summarizes data about epidemiology, treatment, and risk factors for invasive fungal infections (IFI) in patients affected by chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and indolent non Hodgkin lymphoma (iNHL).

Recent Findings

Despite advances in the prognosis and treatment of hematological malignancies in recent years, susceptibility to infection remains a significant challenge to patient care. A large amount of data regarding patients with acute leukemias have been published while little information is available on incidence of IFI in chronic lymphoproliferative disorders (CLD).

Summary

The overall incidence of IFI in CLL patients is reported from 1.3 to 7.8% and the main risk factors are related to disease status (high-risk in relapsed/refractory disease), number of previous chemotherapy regimens, and Ig levels.In MM, most of the IFI occurred during refractory or progressive disease. The rate of IFI ranges from 0.5 to 12.3%. Neutropenia is the main risk factor in MM and risk seems to be related to its duration and severity. The overall incidence of IFI in iNHL ranges from 0.5 to 4% and the most important risk factors are disease status (high-risk in relapsed/refractory and advance stage disease) and type of treatment (high-risk for steroid administration, intensive chemotherapy with prolonged neutropenia, use of monoclonal antibodies and purine analogs).

     

Association of genetic variants rs641153 (CFB), rs2230199 (C3), and rs1410996 (CFH) with age-related macular degeneration in a Brazilian population

This study aimed to investigate the association among genetic variants of the complement pathway CFB R32Q (rs641153), C3 R102G (rs2230199), and CFH (rs1410996) with age-related macular degeneration (AMD) in a sample of the Brazilian population. In a case-control study, 484 AMD patients were classified according to the clinical age-related maculopathy grading system (CARMS) and compared to 479 unrelated controls. The genetic variants rs1410996 of complement H (CFH), rs641153 of complement factor B (CFB), and rs2230199 of complement 3 (C3) were evaluated through polymerase chain reaction (PCR) and direct sequencing. The associations between single nucleotide polymorphisms (SNPs) and AMD, adjusted by age, were assessed by using logistic regression models. A statistically significant association was observed between AMD risk and rs2230199 variant with an OR of 2.01 (P  = 0.0002) for CG individuals compared to CC individuals. Regarding the comparison of advanced AMD versus the control group, the OR was 2.12 (P = 0.0036) for GG versus AA genotypes for rs1410996 variant. Similarly, the OR for rs2230199 polymorphism was 2.3034 (P  = 5.47^e-05) when comparing CG individuals to CC carriers. In contrast, the rs641153 variant showed a significant protective effect against advanced AMD for GA versus GG genotype (OR = 0.4406; P  = 0.0019). When comparing wet AMD versus controls, a significant association was detected for rs1410996 variant (OR = 2.16; P  = 0.0039) comparing carriers of the homozygous GG versus AA genotype, as well as in the comparisons of GG (OR = 3.0713; P  = 0.0046) and CG genotypes (OR = 2.2249; P  = 0.0002) versus CC genotype for rs2230199 variant, respectively. The rs641153 variant granted a significant protective effect against wet AMD for GA versus GG genotypes (OR = 0.4601; P  = 0.0044). Our study confirmed the risk association between rs2230199 and rs1410996 variants and AMD, and the protective role against AMD for rs641153 variant.     

Design and synthesis of 4,5,6,7‐tetrahydro‐1H‐1,2‐diazepin‐7‐one derivatives as a new series of Phosphodiesterase 4 (PDE4) inhibitors

Phosphodiesterase 4 (PDE4) inhibitors have attractive therapeutic potential in respiratory, inflammatory, metabolic and CNS disorders. The present work details the design, chemical exploration and biological profile of a novel PDE4 inhibitor chemotype. A diazepinone ring was identified as an under-represented heterocyclic system fulfilling a set of PDE4 structure-based design hypotheses. Rapid exploration of the structure activity relationships for the series was enabled by robust and scalable two/three-steps parallel chemistry protocols. The resulting compounds demonstrated PDE4 inhibitory activity in cell free and cell-based assays comparable to the Zardaverine control used, suggesting potential avenues for their further development.     

Crystal Structure of the FeS Cluster–Containing Nucleotide Excision Repair Helicase XPD

DNA damage recognition by the nucleotide excision repair pathway requires an initial step identifying helical distortions in the DNA and a proofreading step verifying the presence of a lesion. This proofreading step is accomplished in eukaryotes by the TFIIH complex. The critical damage recognition component of TFIIH is the XPD protein, a DNA helicase that unwinds DNA and identifies the damage. Here, we describe the crystal structure of an archaeal XPD protein with high sequence identity to the human XPD protein that reveals how the structural helicase framework is combined with additional elements for strand separation and DNA scanning. Two RecA-like helicase domains are complemented by a 4Fe4S cluster domain, which has been implicated in damage recognition, and an α-helical domain. The first helicase domain together with the helical and 4Fe4S-cluster–containing domains form a central hole with a diameter sufficient in size to allow passage of a single stranded DNA. Based on our results, we suggest a model of how DNA is bound to the XPD protein, and can rationalize several of the mutations in the human XPD gene that lead to one of three severe diseases, xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy.     

Parallel alternating sliding knots are effective for ligation of mesenteric arteries during resection and anastomosis of the equine jejunum

Background

In literature only one article describes and compares methods of achieving hemostasis in equine mesenteric arteries during jejunal resection and anastomosis, and most textbooks favor ligating-dividing mechanical devices. The latter method cannot always be used, not least because the devices are expensive and in some cases even contra-indicated. Various types of knots, including sliding knots, are widely used to provide hemostasis in laparoscopy.The objective of this study was to compare a triple ligature for mesenteric vessels composed of three sliding knots with a triple ligature composed of a modified transfixing and two surgeon’s knots.

Methods

Portions of jejunum with associated mesenteric vessels were collected from 12 horses at a local abattoir. These were divided into 24 specimens containing five mesenteric arteries each. Each artery was closed with a triple ligature. In group A, a surgeon’s knot was used to tie the ligatures (two circumferential and one modified transfixing) while in group B all ligatures (three circumferential) were tied with a parallel alternating sliding knot. Both groups were divided ino two subgroups depending on suture material used (multifilament or monofilament suture material). Time to perform ligatures for every specimen were recorded and compared between groups.After closure, arteries were cannulated and intraluminal pressures were increased until ligature failure. Leaking pressures were recorded and compared between groups.

Results

Ligation of mesenteric arteries was significantly faster to perform with sliding knots than with surgeon’s knots, both with monofilament and multifilament suture material. With multifilament suture material, the leaking pressure of sliding knot ligatures was significantly higher than that of surgeon’s knot ligatures. With monofilament suture, there were no statistically significant differences in leaking pressure between ligature methods. Both ligating methods were stronger with monofilament suture material than with multifilament suture material.

Conclusions

Regardless of the ligature used, monofilament suture material performed better than multifilament suture material to achieve hemostatic knots. Independently of the suture material, the sliding knot is comparable or better than the surgeon’s knot in providing hemostasis, and is faster to perform.

     

Mycoplasma agalactiae MAG_5040 is a Mg2+-Dependent,Sugar-Nonspecific SNase Recognised by the Host Humoral Response during Natural Infection

In this study the enzymatic activity of Mycoplasma agalactiae MAG_5040, a magnesium-dependent nuclease homologue to the staphylococcal SNase was characterized and its antigenicity during natural infections was established. A UGA corrected version of MAG_5040, lacking the region encoding the signal peptide, was expressed in Escherichia coli as a GST fusion protein. Recombinant GST-MAG_5040 exhibits nuclease activity similar to typical sugar-nonspecific endo- and exonucleases, with DNA as the preferred substrate and optimal activity in the presence of 20 mM MgCl₂ at temperatures ranging from 37 to 45°C. According to in silico analyses, the position of the gene encoding MAG_5040 is consistently located upstream an ABC transporter, in most sequenced mycoplasmas belonging to the Mycoplasma hominis group. In M. agalactiae, MAG_5040 is transcribed in a polycistronic RNA together with the ABC transporter components and with MAG_5030, which is predicted to be a sugar solute binding protein by 3D modeling and homology search. In a natural model of sheep and goats infection, anti-MAG_5040 antibodies were detected up to 9 months post infection. Taking into account its enzymatic activity, MAG_5040 could play a key role in Mycoplasma agalactiae survival into the host, contributing to host pathogenicity. The identification of MAG_5040 opens new perspectives for the development of suitable tools for the control of contagious agalactia in small ruminants.