Discovery of subtype selective muscarinic receptor antagonists as alternatives to atropine using in silico pharmacophore modeling and virtual screening methods

Muscarinic acetylcholine receptors (mAChRs) have five known subtypes which are widely distributed in both the peripheral and central nervous system for regulation of a variety of cholinergic functions. Atropine is a well known muscarinic subtype non-specific antagonist that competitively inhibits acetylcholine (ACh) at postganglionic muscarinic sites. Atropine is used to treat organophosphate (OP) poisoning and resulting seizures in the warfighter because it competitively inhibits acetylcholine (ACh) at the muscarinic cholinergic receptors. ACh accumulates due to OP inhibition of acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh. However, atropine produces several unwanted side-effects including dilated pupils, blurred vision, light sensitivity, and dry mouth. To overcome these side-effects, our goal was to find an alternative to atropine that emphasizes M1 (seizure prevention) antagonism but has minimum M2 (cardiac) and M3 (e.g., eye) antagonism so that an effective less toxic medical countermeasure may be developed to protect the warfighter against OP and other chemical warfare agents (CWAs). We adopted an in silico pharmacophore modeling strategy to develop features that are characteristics of known M1 subtype-selective compounds and used the model to identify several antagonists by screening an in-house (WRAIR-CIS) compound database. The generated model for the M1 selectivity was found to contain two hydrogen bond acceptors, one aliphatic hydrophobic, and one ring aromatic feature distributed in a 3D space. From an initial identification of about five hundred compounds, 173 compounds were selected through principal component and cluster analyses and in silico ADME/Toxicity evaluations. Next, these selected compounds were evaluated in a subtype-selective in vitro radioligand binding assay. Twenty eight of the compounds showed antimuscarinic activity. Nine compounds showed specificity for M1 receptors and low specificity for M3 receptors. The pK_i values of the compounds range from 4.5 to 8.5 nM in comparison to a value of 8.7 nM for atropine. 2-(diethylamino)ethyl 2,2-diphenylpropanoate (ZW62841) was found have the best desired selectivity. None of the newly found compounds were previously reported to exhibit antimuscarinic specificity. Both theoretical and experimental results are presented.     

Uncovering species boundaries in the Neotropical ant complex Ectatomma ruidum (Ectatomminae) under the presence of nuclear mitochondrial paralogues

Nuclear mitochondrial (mt) paralogues (numts) are non‐functional fragments of mtDNA integrated into the nuclear genome that can overestimate the number of species in analyses based on mtDNA sequences. As numts have relatively slow mutation rates, they can pass undetected by conventional procedures such as inspecting for internal stop codons, indels or apparent polymorphism in chromatograms. Species boundaries based on mtDNA markers therefore require a thorough assessment of numts, especially in insects, where this phenomenon appears to be relatively frequent. Ectatomma ruidum is a widely distributed Neotropical ant species that is distributed from northern Mexico to northern Brazil. Previous behavioural and molecular evidence suggests that this species actually represents a composite taxon. Here we assessed the species boundaries in E. ruidum based on two mt (COI, cyt b) and one nuclear (H3) marker, as well as on external morphology. Ancient and recent mt paralogues were detected in several specimens, although pre‐PCR dilution of DNA template helped to recover most of the mt orthologues. Based on the congruence found between our species delineation obtained from the mt genealogies and the discriminated morphospecies, we propose that E. ruidum is actually composed of at least three species. Two of these species have a wide geographical distribution in the Neotropics, whereas the remaining one was restricted to localities situated near the Pacific coast in south‐east Mexico. We also found extensive intra‐ and interspecific variation in the barcoding locus. Moreover, the nuclear evidence suggests the existence of hybrids between two of these species in Oaxaca, south‐east Mexico. This study agrees with previous studies of other closely related animal taxa, which have revealed a complex evolutionary history and overlooked species diversity in the latter region.     

Tumor-Stroma Mechanics Coordinate Amino Acid Availability to Sustain Tumor Growth and Malignancy

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Direct Nanoscale Characterization of Deep Levels in AgCuInGaSe2 Using Electron Energy‐Loss Spectroscopy in the Scanning Transmission Electron Microscope

A new experimental framework for the characterization of defects in semiconductors is demonstrated. Through the direct, energy‐resolved correlation of three analytical techniques spanning six orders of magnitude in spatial resolution, a critical mid‐bandgap electronic trap level (E_V + 0.56 eV) within Ag_0.2Cu_0.8In_1?xGa_xSe₂ is traced to its nanoscale physical location and chemical source. This is achieved through a stepwise, site‐specific correlated characterization workflow consisting of device‐scale (≈1 mm²) deep level transient spectroscopy (DLTS) to survey the traps present, scanning probe–based DLTS (scanning‐DLTS) for mesoscale‐resolved (hundreds of nanometers) mapping of the target trap state's spatial distribution, and scanning transmission electron microscope based electron energy‐loss spectroscopy (STEM‐EELS) and X‐ray energy‐dispersive spectroscopy for nanoscale energy‐, structure, and chemical‐resolved investigation of the defect source. This first demonstration of the direct observation of sub‐bandgap defect levels via STEM‐EELS, combined with the DLTS methods, provides strong evidence that the long‐suspected Cu_In/Ga substitutional defects are indeed the most likely source of the E_V + 0.56 eV trap state and serves as a key example of this approach for the fundamental identification of defects within semiconductors, in general.     

Synthesis and biological evaluation of PSMA-targeting paclitaxel conjugates

Prostate cancer (PC) is the second most commonly occurring cancer in men. Conventional chemotherapy has wide variety of disadvantages such as high systemic toxicity and low selectivity. Targeted drug delivery is a promising approach to decrease side effects of therapy. Prostate specific membrane antigen (PSMA) is overexpressed in prostate cancer cells while low level of expression is observed in normal cells. In this study we describe the development of Glu-urea-Lys based PSMA-targeting conjugates with paclitaxel. A series of new PSMA targeting conjugates with paclitaxel was designed and synthesized. The cytotoxicity of conjugates was evaluated against prostate (LNCaP, 22Rv1 and PC-3) and non-prostate (Hek293T, VA13, A549 and MCF-7) cell lines. The most promising conjugate 21 was examined in vivo using 22Rv1 xenograft mice model. It demonstrated good efficiency comparable with paclitaxel, while reduced toxicity. 3D molecular docking study was also performed to understand underlying mechanism of binding and further optimization of the linker substructure and conjugates structure for improving the target affinity. These conjugates may be useful for further design of novel PSMA targeting delivery systems for PC.