A practical Java tool for small-molecule compound appraisal

The increased use of small-molecule compound screening by new users from a variety of different academic backgrounds calls for adequate software to administer, appraise, analyse and exchange information obtained from screening experiments. While software and spreadsheet solutions exist, there is a need for software that can be easily deployed and is convenient to use.The Java application cApp addresses this need and aids in the handling and storage of information on small-molecule compounds. The software is intended for the appraisal of compounds with respect to their physico-chemical properties, analysis in relation to adherence to likeness rules as well as recognition of pan-assay interference components and cross-linking with identical entries in the PubChem Compound Database. Results are displayed in a tabular form in a graphical interface, but can also be written in an HTML or PDF format. The output of data in ASCII format allows for further processing of data using other suitable programs. Other features include similarity searches against user-provided compound libraries and the PubChem Compound Database, as well as compound clustering based on a MaxMin algorithm.cApp is a personal database solution for small-molecule compounds which can handle all major chemical formats. Being a standalone software, it has no other dependency than the Java virtual machine and is thus conveniently deployed. It streamlines the analysis of molecules with respect to physico-chemical properties and drug discovery criteria; cApp is distributed under the GNU Affero General Public License version 3 and available from http://www.structuralchemistry.org/pcsb/. To download cApp, users will be asked for their name, institution and email address. A detailed manual can also be downloaded from this site, and online tutorials are available at http://www.structuralchemistry.org/pcsb/capp.php

ω-Conotoxin GVIA mimetics that bind and inhibit neuronal Cav2.2 ion channels

The neuronal voltage-gated N-type calcium channel (Cav2.2) is a validated target for the treatment of neuropathic pain. A small library of anthranilamide-derived ω-Conotoxin GVIA mimetics bearing the diphenylmethylpiperazine moiety were prepared and tested using three experimental measures of calcium channel blockade. These consisted of a 125I-ω-conotoxin GVIA displacement assay, a fluorescence-based calcium response assay with SH-SY5Y neuroblastoma cells, and a whole-cell patch clamp electrophysiology assay with HEK293 cells stably expressing human Cav2.2 channels. A subset of compounds were active in all three assays. This is the first time that compounds designed to be mimics of ω-conotoxin GVIA and found to be active in the 125I-ω-conotoxin GVIA displacement assay have also been shown to block functional ion channels in a dose-dependent manner

Metabolomics and lipidomics reveal perturbation of sphingolipid metabolism by a novel anti-trypanosomal 3-(oxazolo[4,5-b]pyridine-2-yl)anilide

Introduction: Trypanosoma brucei is the causative agent of human African trypanosomiasis, which is responsible for thousands of deaths every year. Current therapies are limited and there is an urgent need to develop new drugs. The anti-trypanosomal compound, 3-(oxazolo[4,5-b]pyridine-2-yl)anilide (OXPA), was initially identified in a phenotypic screen and subsequently optimized by structure–activity directed medicinal chemistry. It has been shown to be non-toxic and to be active against a number of trypanosomatid parasites. However, nothing is known about its mechanism of action. Objective: Here, we have utilized an untargeted metabolomics approach to investigate the biochemical effects and potential mode of action of this compound in T. brucei. Methods: Total metabolite extracts were analysed by HILIC-chromatography coupled to high resolution mass spectrometry. Results: Significant accumulation of ceramides was observed in OXPA-treated T. brucei. To further understand drug-induced changes in lipid metabolism, a lipidomics method was developed which enables the measurement of hundreds of lipids with high throughput and precision. The application of this LC–MS based approach to cultured bloodstream-form T. brucei putatively identified over 500 lipids in the parasite including glycerophospholipids, sphingolipids and fatty acyls, and confirmed the OXPA-induced accumulation of ceramides. Labelling with BODIPY-ceramide further confirmed the ceramide accumulation following drug treatment. Conclusion: These findings clearly demonstrate perturbation of ceramide metabolism by OXPA and indicate that the sphingolipid pathway is a promising drug target in T. brucei.No Full Tex

Ponatinib (AP24534) inhibits MEKK3-KLF signaling and prevents formation and progression of cerebral cavernous malformations.

Cerebral cavernous malformation (CCM) is a common cerebrovascular disease that can occur sporadically or be inherited. They are major causes of stroke, cerebral hemorrhage, and neurological deficits in the younger population. Loss-of-function mutations in three genes, CCM1, CCM2, and CCM3, have been identified as the cause of human CCMs. Currently, no drug is available to treat CCM disease. Hyperactive mitogen-activated protein kinase kinase Kinase 3 (MEKK3) kinase signaling as a consequence of loss of CCM genes is an underlying cause of CCM lesion development. Using a U.S. Food and Drug Administration-approved kinase inhibitor library combined with virtual modeling and biochemical and cellular assays, we have identified a clinically approved small compound, ponatinib, that is capable of inhibiting MEKK3 activity and normalizing expression of downstream kruppel-like factor (KLF) target genes. Treatment with this compound in neonatal mouse models of CCM can prevent the formation of new CCM lesions and reduce the growth of already formed lesions. At the ultracellular level, ponatinib can normalize the flattening and disorganization of the endothelium caused by CCM deficiency. Collectively, our study demonstrates ponatinib as a novel compound that may prevent CCM initiation and progression in mouse models through inhibition of MEKK3-KLF signaling

Structure-function analysis of the AMPK activator SC4 and identification of a potent pan AMPK activator

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a primary cellular energy sensor and central regulator of energy homeostasis. Activating skeletal muscle AMPK with small molecule drugs improves glucose uptake and provides an opportunity for new strategies to treat type 2 diabetes and insulin resistance, with recent genetic and pharmacological studies indicating the α2β2γ1 isoform combination as the heterotrimer complex primarily responsible. With the goal of developing α2β2-specific activators, here we perform structure/function analysis of the 2-hydroxybiphenyl group of SC4, an activator with tendency for α2-selectivity that is also capable of potently activating β2 complexes. Substitution of the LHS 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes resulted in two AMPK agonists, MSG010 and MSG011, which did not display α2-selectivity when screened against a panel of AMPK complexes. By radiolabel kinase assay, MSG010 and MSG011 activated α2β2γ1 AMPK with one order of magnitude greater potency than the pan AMPK activator MK-8722. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4 and the potential importance of an interaction between the SC4 2-hydroxyl group and α2-Lys31 for directing α2-selectivity. MSG011 induced robust AMPK signalling in mouse primary hepatocytes and commonly used cell lines, and in most cases this occurred in the absence of changes in phosphorylation of the kinase activation loop residue α-Thr172, a classical marker of AMP-induced AMPK activity. These findings will guide future design of α2β2-selective AMPK activators, that we hypothesise may avoid off-target complications associated with indiscriminate activation of AMPK throughout the body

Just Click Here : A Brief Glance at Absurd Electronic Contracts and the Law Failing to Protect Consumers

As e-commerce explodes around the world, consumers’ rights have been left behind. Before the completion of virtually every transaction on the Internet, the onus is placed on consumers to read and agree to an onslaught of terms and conditions. Often hidden in the middle of this extremely lengthy list of terms are massive exemption and limitation of liability clauses that deny consumers most if not all of their rights as “equal” trading partners. The common law principle that all onerous clauses in a contract need to be brought to the attention of the consumer for them to be binding seems to have been lost with the invention of the “click here to agree” button for signing online contracts. As the courts in Canada have not provided clear guidance on this issue thus far, other means must be pursued in order to protect consumers from the near-tyrannical control of unencumbered electronic standard form contracts in e-commerce. This paper will describe the principle of sufficiency of notice as it applies to paper contracts, and then contrast it with the newer jurisprudence that has refused to apply the principle to electronic contracts. The reasons for the refusal will be explored, followed by an examination of why the principle of sufficiency of notice needs to be applied and strengthened to respond to the increasingly onerous provisions hidden in electronic contracts. Finally, some other options for achieving the goal of consumer protection from hidden onerous clauses will be briefly explored. These other options include introducing stiffer consumer protection legislation domestically, the creation of international treaties, developing voluntary standards of contracting, and relying on Internet self-regulation

Health Locus of Control and Assimilation of Cervical Cancer Information in Deaf Women

This study assessed the relationship between Deaf women's internal health locus of control (IHLC) and their cervical cancer knowledge acquisition and retention. A blind, randomized trial evaluated Deaf women's (N = 130) baseline cancer knowledge and knowledge gained and retained from an educational intervention, in relation to their IHLC. The Multidimensional Health Locus of Control scales measured baseline IHLC, and a cervical cancer knowledge survey evaluated baseline to post-intervention knowledge change. Women's IHLC did not significantly predict greater cervical cancer knowledge at baseline or over time. IHLC does not appear to be a characteristic that must be considered when creating Deaf women's cancer education programs

Crystallographic and electrophilic fragment screening of the SARS-CoV-2 main protease

COVID-19, caused by SARS-CoV-2, lacks effective therapeutics. Additionally, no antiviral drugs or vaccines were developed against the closely related coronavirus, SARS-CoV-1 or MERS-CoV, despite previous zoonotic outbreaks. To identify starting points for such therapeutics, we performed a large-scale screen of electrophile and non-covalent fragments through a combined mass spectrometry and X-ray approach against the SARS-CoV-2 main protease, one of two cysteine viral proteases essential for viral replication. Our crystallographic screen identified 71 hits that span the entire active site, as well as 3 hits at the dimer interface. These structures reveal routes to rapidly develop more potent inhibitors through merging of covalent and non-covalent fragment hits; one series of low-reactivity, tractable covalent fragments were progressed to discover improved binders. These combined hits offer unprecedented structural and reactivity information for on-going structure-based drug design against SARS-CoV-2 main protease

Biochemical and structural characterization of mycobacterial aspartyl-tRNA synthetase AspS, a promising TB drug target.

The human pathogen Mycobacterium tuberculosis is the causative agent of pulmonary tuberculosis (TB), a disease with high worldwide mortality rates. Current treatment programs are under significant threat from multi-drug and extensively-drug resistant strains of M. tuberculosis, and it is essential to identify new inhibitors and their targets. We generated spontaneous resistant mutants in Mycobacterium bovis BCG in the presence of 10× the minimum inhibitory concentration (MIC) of compound 1, a previously identified potent inhibitor of mycobacterial growth in culture. Whole genome sequencing of two resistant mutants revealed in one case a single nucleotide polymorphism in the gene aspS at 535GAC>535AAC (D179N), while in the second mutant a single nucleotide polymorphism was identified upstream of the aspS promoter region. We probed whole cell target engagement by overexpressing either M. bovis BCG aspS or Mycobacterium smegmatis aspS, which resulted in a ten-fold and greater than ten-fold increase, respectively, of the MIC against compound 1. To analyse the impact of inhibitor 1 on M. tuberculosis AspS (Mt-AspS) activity we over-expressed, purified and characterised the kinetics of this enzyme using a robust tRNA-independent assay adapted to a high-throughput screening format. Finally, to aid hit-to-lead optimization, the crystal structure of apo M. smegmatis AspS was determined to a resolution of 2.4 Å

Shaping a screening file for maximal lead discovery efficiency and effectiveness: elimination of molecular redundancy

High Throughput Screening (HTS) is a successful strategy for finding hits and leads that have the opportunity to be converted into drugs. In this paper we highlight novel computational methods used to select compounds to build a new screening file at Pfizer and the analytical methods we used to assess their quality. We also introduce the novel concept of molecular redundancy to help decide on the density of compounds required in any region of chemical space in order to be confident of running successful HTS campaigns