STAT3 inhibitory activity of naphthoquinones isolated from Tabebuia avellanedae

The extract of Tabebuia avellanedae has been used as a folk medicine, and the various biological activities of T. avellanedae have been extensively studied. However, few studies have reported which natural products play a role in their biological effects. In this study, we evaluated representative naphthoquinones isolated from T. avellanedae and found that furanonaphthoquinones were the key structures required to exhibit STAT3 phosphorylation inhibitory activities. Our SAR analysis indicated that removal of a hydroxyl group enhanced the STAT3 phosphorylation inhibitory activity. In addition, the combined results of a mobility shift assay, SH2 domain binding assay, and docking simulation by Autodock 4.2.6 suggested that (S)-5-hydroxy-2-(1-hydroxyethyl)naphtho[2,3-b]furan-4,9-dione (1) could directly bind to the hinge region of STAT3.     

Identification of Compounds with Anti-Proliferative Activity against Trypanosoma brucei brucei Strain 427 by a Whole Cell Viability Based HTS Campaign

Human African Trypanosomiasis (HAT) is caused by two trypanosome sub-species, Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense. Drugs available for the treatment of HAT have significant issues related to difficult administration regimes and limited efficacy across species and disease stages. Hence, there is considerable need to find new alternative and less toxic drugs. An approach to identify starting points for new drug candidates is high throughput screening (HTS) of large compound library collections. We describe the application of an Alamar Blue based, 384-well HTS assay to screen a library of 87,296 compounds against the related trypanosome subspecies, Trypanosoma brucei brucei bloodstream form lister 427. Primary hits identified against T.b. brucei were retested and the IC₅₀ value compounds were estimated for T.b. brucei and a mammalian cell line HEK293, to determine a selectivity index for each compound. The screening campaign identified 205 compounds with greater than 10 times selectivity against T.b. brucei. Cluster analysis of these compounds, taking into account chemical and structural properties required for drug-like compounds, afforded a panel of eight compounds for further biological analysis. These compounds had IC₅₀ values ranging from 0.22 µM to 4 µM with associated selectivity indices ranging from 19 to greater than 345. Further testing against T.b. rhodesiense led to the selection of 6 compounds from 5 new chemical classes with activity against the causative species of HAT, which can be considered potential candidates for HAT early drug discovery. Structure activity relationship (SAR) mining revealed components of those hit compound structures that may be important for biological activity. Four of these compounds have undergone further testing to 1) determine whether they are cidal or static in vitro at the minimum inhibitory concentration (MIC), and 2) estimate the time to kill.     

Discovery of substituted 4-anilino-2-(2-pyridyl)pyrimidines as a new series of apoptosis inducers using a cell- and caspase-based high throughput screening assay. Part 1: structure-activity relationships of the 4-anilino group

A series of 4-anilino-2-(2-pyridyl)pyrimidines has been discovered as a new class of potent inducers of apoptosis using a cell-based HTS assay. Compound 5a was found to arrest T47D cells in G2/M and induced apoptosis. SAR studies showed that a small and electron-donating group at the meta-position of the anilino ring is important for activity. A 20-fold increase in potency, from hit compound 4-(3-methoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5a) to lead compound 4-(2,5-dimethoxyanilino)-2-(2-pyridinyl)-6-(trifluoromethyl)pyrimidine (5l), was obtained through the SAR studies. Compound 5l is highly active with an EC50 value of 18 nM in the caspase activation assay in T47D breast cells. Interestingly, 5a and other meta-mono-substituted compounds were active against T47D cells but were not active against H1299 and HT29 cells, while 5l and other 2,5-disubstituted compounds were active against all the three cells. In a tubulin polymerization assay, compound 5l inhibited tubulin polymerization with an IC50 value of < 0.5 microM, while 5a was not active up to 50 microM.     

Discovery and structure-activity relationship of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a new series of potent apoptosis inducers

We report the discovery and SAR study of a series of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as potent inducers of apoptosis. N-(3-Acetylphenyl)-2,3-dihydro-1H-cyclopenta[b]quinolin-9-amine (2) was discovered through our cell- and caspase-based HTS assays as an inducer of apoptosis. Compound 2 is active against cancer cells derived from several human solid tumors, with EC(50) values ranging from 400 to 700 nM. SAR study of hit 2 led to the discovery of N-phenyl-1H-pyrazolo[3,4-b]quinolin-4-amines as a novel series of potent apoptosis inducers, with 1,3-dimethyl-N-(4-propionylphenyl)-1H-pyrazolo[3,4-b]quinolin-amine (6b) having EC(50) values ranging from 30 to 70 nM in cancer cells. These compounds also demonstrated potent activity in the cell growth inhibition assay, with GI(50) values of 16-42 nM for compound 6b.     

Discovery and SAR of indole-2-carboxylic acid benzylidene-hydrazides as a new series of potent apoptosis inducers using a cell-based HTS assay

A series of indole-2-carboxylic acid benzylidene-hydrazides has been identified as a new class of potent apoptosis inducers through a novel cell-based caspase HTS assay. The screening hit, 5-chloro-3-methyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (3a), was found to arrest T47D cells in G(2)/M and to induce apoptosis as measured by the flow cytometric analysis assay. A SAR study was carried out by modification of the substitutions on the indole and benzene rings. Substitution at the 3-position of the indole ring was found to be important for apoptotic activity. A 20-fold increase of apoptotic activity was achieved from screening hit 3a to 5-methyl-3-phenyl-indole-2-carboxylic acid (4-methylbenzylidene)-hydrazide (9a) and 5-chloro-3-phenyl-indole-2-carboxylic acid (4-nitrobenzylidene)-hydrazide (9b), with EC(50) value of 0.1microM in the caspase activation assay in T47D breast cancer cells. Compound 9b also was found to be highly active in a standard growth inhibition assay with a GI(50) value of 0.9microM in T47D cells. Compound 3a and its analogs were found to inhibit tubulin polymerization, which is the most probable primary mechanism of action of these compounds.     

Further delineation of hydrophobic binding sites in dopamine D2/D3 receptors for N-4 substituents on the piperazine ring of the hybrid template 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol

Here we report a structure–activity relationship (SAR) study of analogues of 5/7-{[2-(4-aryl-piperazin-1-yl)-ethyl]-propyl-amino}-5,6,7,8-tetrahydro-naphthalen-2-ol. Our SAR is focused on introduction of various substitutions in the piperazine ring of the hybrid template. The goal behind this study is to delineate the nature of the binding pocket for N-aryl substitution in the piperazine ring by observing the effect of various hydrophobic and other heteroaromatic substitutions on binding affinity (K_i), as measured with tritiated spiperone and HEK-293 cells expressing either D₂ or D₃ receptors. Functional activity of selected compounds was assessed with the GTPγS binding assay. Compound 8d was the most selective for the D₃ receptor in the spiperone binding assay. An interesting similarity in binding affinity was observed between isoquinoline derivative D-301 and the 2-substituted pyridine derivative 8d, suggesting the importance of relative spatial relationships between the N-atom of the ligand and the molecular determinants of the binding pocket in D₂/D₃ receptors. Functional activity assays demonstrated high potency and selectivity of (+)-8a and (?)-28b (D₂/D₃ (ratio of EC₅₀): 105 and 202, respectively) for the D₃ receptor and both compounds were more selective compared to the reference drug ropinirole (D₂/D₃ (ratio of EC₅₀): 29.5).     

Discovery of novel N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides as potent RORγt inhibitors

Novel series of N-(5-(arylcarbonyl)thiazol-2-yl)amides and N-(5-(arylcarbonyl)thiophen-2-yl)amides were discovered as potent retinoic acid receptor-related orphan receptor-gamma-t (RORγt) inhibitors. SAR studies of the RORγt HTS hit 6a led to identification of thiazole ketone amide 8h and thiophene ketone amide 9g with high binding affinity and inhibitory activity of Th17 cell differentiation. Compound 8h showed in vivo efficacy in both mouse experimental autoimmune encephalomyelitis (EAE) and collagen induced arthritis (CIA) models via oral administration.     

Discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-phenyl-(E)-2,3,6,7-tetrahydro-1,4-thiazepines as a new series of apoptosis inducers using a cell- and caspase-based HTS assay

We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.     

Phenyl substituted 3-hydroxypyridin-2(1H)-ones: Inhibitors of influenza A endonuclease

Inhibition of the endonuclease activity of influenza RNA-dependent RNA polymerase is recognized as an attractive target for the development of new agents for the treatment of influenza infection. Our earlier study employing small molecule fragment screening using a high-resolution crystal form of pandemic 2009 H1N1 influenza A endonuclease domain (PA_N) resulted in the identification of 5-chloro-3-hydroxypyridin-2(1H)-one as a bimetal chelating ligand at the active site of the enzyme. In the present study, several phenyl substituted 3-hydroxypyridin-2(1H)-one compounds were synthesized and evaluated for their ability to inhibit the endonuclease activity as measured by a high-throughput fluorescence assay. Two of the more potent compounds in this series, 16 and 18, had IC₅₀ values of 11 and 23 nM in the enzymatic assay, respectively. Crystal structures revealed that these compounds had distinct binding modes that chelate the two active site metal ions (M1 and M2) using only two chelating groups. The SAR and the binding mode of these 3-hydroxypyridin-2-ones provide a basis for developing a new class of anti-influenza drugs.     

Discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers

We report the discovery of N-((benzo[d][1,3]dioxol-5-yl)methyl)-6-phenylthieno[3,2-d]pyrimidin-4-amine (2a) as an apoptosis inducer using our proprietary cell- and caspase-based ASAP HTS assay, and SAR study of HTS hit 2a which led to the discovery of 4-anilino-N-methylthieno[3,2-d]pyrimidines and 4-anilino-N-methylthieno[2,3-d]pyrimidines as potent apoptosis inducers. Compounds 5d and 5e were the most potent with EC₅₀ values of 0.008 and 0.004 μM in T47D human breast cancer cells, respectively. Compound 5d was found to be highly active in the MX-1 breast cancer model. Functionally, compounds 5d and 5e both induced apoptosis through inhibition of tubulin polymerization.     

Ligand-based design,synthesis, computational insights,and in vitro studies of novel N-(5-Nitrothiazol-2-yl)-carboxamido derivatives as potent inhibitors of SARS-CoV-2 main protease

The global outbreak of the COVID-19 pandemic provokes scientists to make a prompt development of new effective therapeutic interventions for the battle against SARS-CoV-2. A new series of N-(5-nitrothiazol-2-yl)-carboxamido derivatives were designed and synthesised based on the structural optimisation principle of the SARS-CoV Mpro co-crystallized WR1 inhibitor. Notably, compound 3b achieved the most promising anti-SARS-CoV-2 activity with an IC₅₀ value of 174.7 µg/mL. On the other hand, compounds 3a, 3b, and 3c showed very promising SARS-CoV-2 Mpro inhibitory effects with IC₅₀ values of 4.67, 5.12, and 11.90 µg/mL, respectively. Compound 3b docking score was very promising (−6.94 kcal/mol) and its binding mode was nearly similar to that of WR1. Besides, the molecular dynamics (MD) simulations of compound 3b showed its great stability inside the binding pocket until around 40 ns. Finally, a very promising SAR was concluded to help to design more powerful SARS-CoV-2 Mpro inhibitors shortly.     

High-Throughput Screening (HTS) and Hit Validation to Identify Small Molecule Inhibitors with Activity against NS3/4A proteases from Multiple Hepatitis C Virus Genotypes

Development of drug-resistant mutations has been a major problem with all currently developed Hepatitis C Virus (HCV) NS3/4A inhibitors, including the two FDA approved drugs, significantly reducing the efficacy of these inhibitors. The high incidence of drug-resistance mutations and the limited utility of these inhibitors against only genotype 1 highlight the need for novel, broad-spectrum HCV therapies. Here we used high-throughput screening (HTS) to identify low molecular weight inhibitors against NS3/4A from multiple genotypes. A total of 40,967 compounds from four structurally diverse molecular libraries were screened by HTS using fluorescence-based enzymatic assays, followed by an orthogonal binding analysis using surface plasmon resonance (SPR) to eliminate false positives. A novel small molecule compound was identified with an IC₅₀ value of 2.2 µM against the NS3/4A from genotype 1b. Mode of inhibition analysis subsequently confirmed this compound to be a competitive inhibitor with respect to the substrate, indicating direct binding to the protease active site, rather than to the allosteric binding pocket that was discovered to be the binding site of a few recently discovered small molecule inhibitors. This newly discovered inhibitor also showed promising inhibitory activity against the NS3/4As from three other HCV genotypes, as well as five common drug-resistant mutants of genotype 1b NS3/4A. The inhibitor was selective for NS3 from multiple HCV genotypes over two human serine proteases, and a whole cell lysate assay confirmed inhibitory activity in the cellular environment. This compound provides a lead for further development of potentially broader spectrum inhibitors.     

A Fluorescence-Based High Throughput Screen for the Transporter Associated with Antigen Processing

The transporter associated with antigen processing (TAP) is essential for antigen presentation by major histocompatibility complex (MHC) class I molecules. Traditional methods used to analyze peptide transport mediated by TAP require radioactive labeling of peptides and time-consuming manipulation of Concanavalin A-Sepharose. Drug discovery research requires rapid and reliable evaluation of large number of samples for bioactivity. To meet these requirements a nonradioactive, HTS assay for peptide transport activity of TAP has been developed. The radioactive label in the traditional assays has been replaced by a fluorescent label without compromising the transport efficiency of labeled peptide or the sensitivity of the assay. The use of multiscreen filtration plates has facilitated higher throughput and eliminated the centrifugation steps used in traditional TAP assays. The HTS assay shows similar kinetic characteristics as compared to the traditional assay. The HTS assay has been adapted on a Quadratrade mark 96-320 96-channel pipetting station (Tomtec, Hamden, CT) by optimizing time course, dose response of TAP to peptides and adenosine triphosphate (ATP), signal/noise ratio, reproducibility, and reagent stability. This HTS system has been utilized to screen a multiplexed compound library with a maximum of throughput 17,600 compounds per week.     

Evolution of novel tricyclic CRTh2 receptor antagonists from a (E)-2-cyano-3-(1H-indol-3-yl)acrylamide scaffold

(E)-2-(3-(3-((3-Bromophenyl)amino)-2-cyano-3-oxoprop-1-en-1-yl)-1H-indol-1-yl)acetic acid (1) was discovered in a HTS campaign for CRTh2 receptor antagonists. An SAR around this hit could be established and representatives with interesting activity profiles were obtained. Ring closing tactics to convert this hit series into a novel 2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole based CRTh2 receptor antagonist series is presented.