Small molecule screening in context: Lipid-catalyzed amyloid formation

Islet Amyloid Polypeptide (IAPP) is a 37-residue hormone cosecreted with insulin by the β-cells of the pancreas. Amyloid fiber aggregation of IAPP has been correlated with the dysfunction and death of these cells in type II diabetics. The likely mechanisms by which IAPP gains toxic function include energy independent cell membrane penetration and induction of membrane depolarization. These processes have been correlated with solution biophysical observations of lipid bilayer catalyzed acceleration of amyloid formation. Although the relationship between amyloid formation and toxicity is poorly understood, the fact that conditions promoting one also favor the other suggests related membrane active structural states. Here, a novel high throughput screening protocol is described that capitalizes on this correlation to identify compounds that target membrane active species. Applied to a small library of 960 known bioactive compounds, we are able to report identification of 37 compounds of which 36 were not previously reported as active toward IAPP fiber formation. Several compounds tested in secondary cell viability assays also demonstrate cytoprotective effects. It is a general observation that peptide induced toxicity in several amyloid diseases (such as Alzhiemer’s and Parkinson’s) involves a membrane bound, preamyloid oligomeric species. Our data here suggest that a screening protocol based on lipid-catalyzed assembly will find mechanistically informative small molecule hits in this subclass of amyloid diseases.     

The current state of validated small molecules inhibiting SARS-CoV-2 nonstructural proteins

The current COVID-19 outbreak has had a profound influence on public health and daily life. Despite all restrictions and vaccination programs, COVID-19 still can lead to fatality due to a lack of COVID-19-specific treatments. A number of studies have demonstrated the feasibility to develop therapeutics by targeting underlying components of the viral proteome. Here we reviewed recently developed and validated small molecule inhibitors of SARS-CoV-2’s nonstructural proteins. We described the validation level of identified compounds specific for SARS-CoV-2 in the presence of in vitro and in vivo supporting data. The mechanisms of pharmacological activity, as well as approaches for developing improved SARS-CoV-2 NSP inhibitors have been emphasized.     

Creating Novel Activated Factor XI Inhibitors through Fragment Based Lead Generation and Structure Aided Drug Design

Activated factor XI (FXIa) inhibitors are anticipated to combine anticoagulant and profibrinolytic effects with a low bleeding risk. This motivated a structure aided fragment based lead generation campaign to create novel FXIa inhibitor leads. A virtual screen, based on docking experiments, was performed to generate a FXIa targeted fragment library for an NMR screen that resulted in the identification of fragments binding in the FXIa S1 binding pocket. The neutral 6-chloro-3,4-dihydro-1H-quinolin-2-one and the weakly basic quinolin-2-amine structures are novel FXIa P1 fragments. The expansion of these fragments towards the FXIa prime side binding sites was aided by solving the X-ray structures of reported FXIa inhibitors that we found to bind in the S1-S1’-S2’ FXIa binding pockets. Combining the X-ray structure information from the identified S1 binding 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment and the S1-S1’-S2’ binding reference compounds enabled structure guided linking and expansion work to achieve one of the most potent and selective FXIa inhibitors reported to date, compound 13, with a FXIa IC₅₀ of 1.0 nM. The hydrophilicity and large polar surface area of the potent S1-S1’-S2’ binding FXIa inhibitors compromised permeability. Initial work to expand the 6-chloro-3,4-dihydro-1H-quinolin-2-one fragment towards the prime side to yield molecules with less hydrophilicity shows promise to afford potent, selective and orally bioavailable compounds.     

Molecular Basis for Benzodiazepine Agonist Action at the Type 1 Cholecystokinin Receptor

Understanding the molecular basis of drug action can facilitate development of more potent and selective drugs. Here, we explore the molecular basis for action of a unique small molecule ligand that is a type 1 cholecystokinin (CCK) receptor agonist and type 2 CCK receptor antagonist, GI181771X. We characterize its binding utilizing structurally related radioiodinated ligands selective for CCK receptor subtypes that utilize the same allosteric ligand-binding pocket, using wild-type receptors and chimeric constructs exchanging the distinct residues lining this pocket. Intracellular calcium assays were performed to determine biological activity. Molecular models for docking small molecule agonists to the type 1 CCK receptor were developed using a ligand-guided refinement approach. The optimal model was distinct from the previous antagonist model for the same receptor and was mechanistically consistent with the current mutagenesis data. This study revealed a key role for Leu^7.39 that was predicted to interact with the isopropyl group in the N1 position of the benzodiazepine that acts as a “trigger” for biological activity. The molecular model was predictive of binding of other small molecule agonists, effectively distinguishing these from 1065 approved drug decoys with an area under curve value of 99%. The model also selectively enriched for agonist compounds, with 130 agonists identified by ROC analysis when seeded in 2175 non-agonist ligands of the type 1 CCK receptor (area under curve 78%). Benzodiazepine agonists in this series docked in consistent pose within this pocket, with a key role played by Leu^7.39, whereas the role of this residue was less clear for chemically distinct agonists.     

The translocator protein ligands as mitochondrial functional modulators for the potential anti-Alzheimer agents

Small molecule modulators of mitochondrial function have been attracted much attention in recent years due to their potential therapeutic applications for neurodegenerative diseases. The mitochondrial translocator protein (TSPO) is a promising target for such compounds, given its involvement in the formation of the mitochondrial permeability transition pore in response to mitochondrial stress. In this study, we performed a ligand-based pharmacophore design and virtual screening, and identified a potent hit compound, 7 (VH34) as a TSPO ligand. After validating its biological activity against amyloid-β (Aβ) induced mitochondrial dysfunction and in acute and transgenic Alzheimer’s disease (AD) model mice, we developed a library of analogs, and we found two most active compounds, 31 and 44, which restored the mitochondrial membrane potential, ATP production, and cell viability under Aβ-induced mitochondrial toxicity. These compounds recovered learning and memory function in acute AD model mice with improved pharmacokinetic properties.     

Discovery of novel triple helical DNA intercalators by an integrated virtual and actual screening platform

Virtual Screening is an increasingly attractive way to discover new small molecules with potential medicinal value. We introduce a novel strategy that integrates use of the molecular docking software Surflex with experimental validation by the method of competition dialysis. This integrated approach was used to identify ligands that selectively bind to the triplex DNA poly(dA)-[poly(dT)]₂. A library containing ~2 million ligands was virtually screened to identify compounds with chemical and structural similarity to a known triplex intercalator, the napthylquinoline MHQ-12. Further molecular docking studies using compounds with high structural similarity resulted in two compounds that were then demonstrated by competition dialysis to have a superior affinity and selectivity for the triplex nucleic acid than MHQ-12. One of the compounds has a different chemical backbone than MHQ-12, which demonstrates the ability of this strategy to ‘scaffold hop’ and to identify small molecules with novel binding properties. Biophysical characterization of these compounds by circular dichroism and thermal denaturation studies confirmed their binding mode and selectivity. These studies provide a proof-of-principle for our integrated screening strategy, and suggest that this platform may be extended to discover new compounds that target therapeutically relevant nucleic acid morphologies.     

2-(4-Chlorophenyl)-2-oxoethyl 4-benzamidobenzoate derivatives,a novel class of SENP1 inhibitors: Virtual screening,synthesis and biological evaluation

Prostate cancer is one of the most prevalent types of malignant cancers in men and has a high mortality rate among all male cancers. Previous studies have demonstrated that Sentrin/SUMO-specific protease 1 (SENP1) plays an important role in the occurrence and development of prostate cancer, and has been identified as a novel drug target for development of small molecule drugs against prostate cancer. In this paper, we used virtual screening and docking to identify compound J5 as a novel lead compound inhibiting SENP1, from SPECS library. We further investigated the SAR (structure–activity relationship) of the benzoate substituent of compound J5, and discovered compounds 8d and 8e as better small molecule inhibitors of SENP1. Both compounds are the high potent SENP1 small molecule inhibitors discovered up to date, and further lead optimization may lead to a series of novel anti-SENP1 agents. Further SAR studies are in process and will be reported in due course.     

Positive allosteric modulators of lecithin: Cholesterol acyltransferase adjust the orientation of the membrane-binding domain and alter its spatial free energy profile

Lecithin:cholesterol acyltransferase protein (LCAT) promotes the esterification reaction between cholesterol and phospholipid-derived acyl chains. Positive allosteric modulators have been developed to treat LCAT deficiencies and, plausibly, also cardiovascular diseases in the future. The mechanism of action of these compounds is poorly understood. Here computational docking and atomistic molecular dynamics simulations were utilized to study the interactions between LCAT and the activating compounds. Results indicate that all drugs bind to the allosteric binding pocket in the membrane-binding domain in a similar fashion. The presence of the compounds in the allosteric site results in a distinct spatial orientation and sampling of the membrane-binding domain (MBD). The MBD’s different spatial arrangement plausibly affects the lid’s movement from closed to open state and vice versa, as suggested by steered molecular dynamics simulations.     

Identification of a Novel Drug Lead That Inhibits HCV Infection and Cell-to-Cell Transmission by Targeting the HCV E2 Glycoprotein

Hepatitis C Virus (HCV) infects 200 million individuals worldwide. Although several FDA approved drugs targeting the HCV serine protease and polymerase have shown promising results, there is a need for better drugs that are effective in treating a broader range of HCV genotypes and subtypes without being used in combination with interferon and/or ribavirin. Recently, two crystal structures of the core of the HCV E2 protein (E2c) have been determined, providing structural information that can now be used to target the E2 protein and develop drugs that disrupt the early stages of HCV infection by blocking E2’s interaction with different host factors. Using the E2c structure as a template, we have created a structural model of the E2 protein core (residues 421–645) that contains the three amino acid segments that are not present in either structure. Computational docking of a diverse library of 1,715 small molecules to this model led to the identification of a set of 34 ligands predicted to bind near conserved amino acid residues involved in the HCV E2: CD81 interaction. Surface plasmon resonance detection was used to screen the ligand set for binding to recombinant E2 protein, and the best binders were subsequently tested to identify compounds that inhibit the infection of Huh-7 cells by HCV. One compound, 281816, blocked E2 binding to CD81 and inhibited HCV infection in a genotype-independent manner with IC50’s ranging from 2.2 µM to 4.6 µM. 281816 blocked the early and late steps of cell-free HCV entry and also abrogated the cell-to-cell transmission of HCV. Collectively the results obtained with this new structural model of E2c suggest the development of small molecule inhibitors such as 281816 that target E2 and disrupt its interaction with CD81 may provide a new paradigm for HCV treatment.     

Design,synthesis, and biological evaluation of a small molecule oral agonist of the glucagon-like-peptide-1 receptor

An absolute or relative deficiency of pancreatic β-cells mass and functionality is a crucial pathological feature common to type 1 diabetes mellitus and type 2 diabetes mellitus. Glucagon-like-peptide-1 receptor (GLP1R) agonists have been the focus of considerable research attention for their ability to protect β-cell mass and augment insulin secretion with no risk of hypoglycemia. Presently commercially available GLP1R agonists are peptides that limit their use due to cost, stability, and mode of administration. To address this drawback, strategically designed distinct sets of small molecules were docked on GLP1R ectodomain and compared with previously known small molecule GLP1R agonists. One of the small molecule PK2 (6-((1-(4-nitrobenzyl)-1H-1,2,3-triazol-4-yl)methyl)-6H-indolo[2,3-b]quinoxaline) displays stable binding with GLP1R ectodomain and induces GLP1R internalization and increasing cAMP levels. PK2 also increases insulin secretion in the INS-1 cells. The oral administration of PK2 protects against diabetes induced by multiple low-dose streptozotocin administration by lowering high blood glucose levels. Similar to GLP1R peptidic agonists, treatment of PK2 induces β-cell replication and attenuate β-cell apoptosis in STZ-treated mice. Mechanistically, this protection was associated with decreased thioredoxin-interacting protein expression, a potent inducer of diabetic β-cell apoptosis and dysfunction. Together, this report describes a small molecule, PK2, as an orally active nonpeptidic GLP1R agonist that has efficacy to preserve or restore functional β-cell mass.     

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020⁽¹⁾. Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs⁽²⁾. However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD^(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients⁽⁶⁾, but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill’s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson’s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.     

Esophageal Adenocarcinoma and Its Rare Association with Barrett’s Esophagus in Henan,China

Incidence of esophageal adenocarcinoma (EAC) has increased sharply in Western Europe and United States over the past three decades. Nearly all cases of EAC in the west are thought to be associated with Barrett’s esophagus (BE) at the time of diagnosis. Regions in the Henan province of China have one of world’s highest incidences of esophageal cancer, yet recent temporal trends in the relative rates of EAC with respect to esophageal squamous-cell carcinoma (ESCC), as well as its association with Barrett’s esophagus (BE), have not been reported. In this report, we present large-scale longitudinal clinical and histological data on 5401 esophageal cancers (EC) patients diagnosed during the recent 10-year period (2002–2011) at Henan Cancer Hospital, China. All 217 esophageal adenocarcinoma (EAC) patients from these 5401 EC patients were examined to better understand the relationship between Barrett’s esophagus (BE) and EAC. We found that EAC was relatively rare and accounted for approximately 5% of all esophageal cancers each year during 2002–2011. There is no evidence of significant temporal trends in the rate of EAC relative to ESCC. Only 10 out of 217 (4.6%) EAC cases were detected to have any evidence of Barrett’s esophagus. This result raises the possibility of a different etiological basis for EAC in China motivating more detailed epidemiological, clinical and molecular characterization of EAC in China in order to better understand the neoplastic development of EAC.     

Analysis of the Spatial Organization of Molecules with Robust Statistics

One major question in molecular biology is whether the spatial distribution of observed molecules is random or organized in clusters. Indeed, this analysis gives information about molecules’ interactions and physical interplay with their environment. The standard tool for analyzing molecules’ distribution statistically is the Ripley’s K function, which tests spatial randomness through the computation of its critical quantiles. However, quantiles’ computation is very cumbersome, hindering its use. Here, we present an analytical expression of these quantiles, leading to a fast and robust statistical test, and we derive the characteristic clusters’ size from the maxima of the Ripley’s K function. Subsequently, we analyze the spatial organization of endocytic spots at the cell membrane and we report that clathrin spots are randomly distributed while clathrin-independent spots are organized in clusters with a radius of , which suggests distinct physical mechanisms and cellular functions for each pathway.     

Identification of Small Molecule Activators of BMP Signaling

Bone Morphogenetic Proteins (BMPs) are morphogens that play a major role in regulating development and homeostasis. Although BMPs are used for the treatment of bone and kidney disorders, their clinical use is limited due to the supra-physiological doses required for therapeutic efficacy causing severe side effects. Because recombinant BMPs are expensive to produce, small molecule activators of BMP signaling would be a cost-effective alternative with the added benefit of being potentially more easily deliverable. Here, we report our efforts to identify small molecule activators of BMP signaling. We have developed a cell-based assay to monitor BMP signaling by stably transfecting a BMP-responsive human cervical carcinoma cell line (C33A) with a reporter construct in which the expression of luciferase is driven by a multimerized BMP-responsive element from the Id1 promoter. A BMP-responsive clone C33A-2D2 was used to screen a bioactive library containing ∼5,600 small molecules. We identified four small molecules of the family of flavonoids all of which induced luciferase activity in a dose-dependent manner and ventralized zebrafish embryos. Two of the identified compounds induced Smad1, 5 phosphorylation (P-Smad), Id1 and Id2 expression in a dose-dependent manner demonstrating that our assays identified small molecule activators of BMP signaling.     

Role of Molecular Electronic Properties of Some Novel Antimalarial Cyclic Peroxy Ketals in Relation to Potency and Potential Toxicity

Calculated molecular electronicproperties of 20 recently reported cyclic peroxy ketals have been rationalized with their in vitro antimalarial activity with the overall goal to guide the design for safer and effective peroxide-containing antimalarial agents. Stereoelectronicproperties were calculated on the optimized geometry of each compound using the ab initio 3-21G* quantum chemical basis set. Potency appears to be related to electronic properties rather than structural properties such as bond lengths and bond angles though an aliphatic cyclic ring seems to be a structural requirement for potent activity. Electronic properties such as differences in molecular polarity, in electrostatic potential profiles about the peroxide bond and the aromatic ring, in peroxide bond strength, and in the LUMO orbital energy of the molecules are all associated with potency. The three-dimensionalisopotential profile beyondthe van der Waals surface at –10 kcal/mol for the more potent analogs has a distinct large negative potential region by the aromatic ring extending to the methoxy moiety, suggesting a site for initial recognition interaction with the receptor away from the peroxide bond. The HOMO and LUMO isodensity surfaces for all molecules are located on the aromatic ring. The peroxide bond strength of the compounds is around 100 kcal/mol greater than the peroxide-containing clinically used antimalarial artemisinin compounds. In addition, density of the peroxy ketals also appears related to potent activity. The above features of the cyclic peroxy ketals are consistent with these compounds being less potent than the artemisinin compounds, but at the same time are less likely to be as neurotoxic as the artemisinins.     

Morphological Characterization of small,dumpy, and long Phenotypes in Caenorhabditis elegans

The determinant factors of an organism’s size during animal development have been explored from various angles but remain partially understood. In Caenorhabditis elegans, many genes affecting cuticle structure, cell growth, and proliferation have been identified to regulate the worm’s overall morphology, including body size. While various mutations in those genes directly result in changes in the morphological phenotypes, there is still a need for established, clear, and distinct standards to determine the apparent abnormality in a worm’s size and shape. In this study, we measured the body length, body width, terminal bulb length, and head size of mutant worms with reported Dumpy (Dpy), Small (Sma) or Long (Lon) phenotypes by plotting and comparing their respective ratios of various parameters. These results show that the Sma phenotypes are proportionally smaller overall with mild stoutness, and Dpy phenotypes are significantly stouter and have disproportionally small head size. This study provides a standard platform for determining morphological phenotypes designating and annotating mutants that exhibit body shape variations, defining the morphological phenotype of previously unexamined mutants.