Development of a time-resolved fluorescence resonance energy transfer ultra-high throughput screening assay targeting SYK and FCER1G interaction.

The spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) interaction has a major role in the normal innate and adaptive immune responses, but dysregulation of this interaction is implicated in several human diseases, including autoimmune disorders, hematological malignancies, and Alzheimer's Disease. Development of small molecule chemical probes could aid in studying this pathway both in normal and aberrant contexts. Herein, we describe the miniaturization of a time-resolved fluorescence resonance energy transfer (TR-FRET) assay to measure the interaction between SYK and FCER1G in a 1536-well ultrahigh throughput screening (uHTS) format.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting the SMAD4-SMAD3-DNA complex.

The transforming growth factor-beta (TGFβ) signaling pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small-molecule inhibitors specifically targeting SMAD4, the downstream master regulator of the TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGFβ signaling.

A Novel 7-[1,2,4]Triazolo[3,4-]thiadiazine-based Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Directed toward Treatment of Cystic Fibrosis.

Cystic fibrosis (CF) is an autosomal genetic disorder caused by disrupted anion transport in epithelial cells lining tissues in the human airways and digestive system. While cystic fibrosis transmembrane conductance regulator (CFTR) modulator compounds have provided transformative improvement in CF respiratory function, certain patients exhibit marginal clinical benefit or detrimental effects or have a form of the disease not approved or unlikely to respond using CFTR modulation. We tested hit compounds from a 300,000-drug screen for their ability to augment CFTR transepithelial transport alone or in combination with the FDA-approved CFTR potentiator ivacaftor (VX-770).

Constitutively Synergistic Multiagent Drug Formulations Targeting MERTK, FLT3, and BCL-2 for Treatment of AML.

Purpose: Although high-dose, multiagent chemotherapy has improved leukemia survival rates, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. The development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need.

Methods: The dual MERTK/FLT3 inhibitor MRX-2843 and BCL-2 family protein inhibitors were screened in high-throughput against a panel of AML and MLL-rearranged precursor B-cell ALL (infant ALL) cell lines.

Development of constitutively synergistic nanoformulations to enhance chemosensitivity in T-cell leukemia.

Advances in multiagent chemotherapy have led to recent improvements in survival for patients with acute lymphoblastic leukemia (ALL); however, a significant fraction do not respond to frontline chemotherapy or later relapse with recurrent disease, after which long-term survival rates remain low. To develop new, effective treatment options for these patients, we conducted a series of high-throughput combination drug screens to identify chemotherapies that synergize in a lineage-specific manner with MRX-2843, a small molecule dual MERTK and FLT3 kinase inhibitor currently in clinical testing for treatment of relapsed/refractory leukemias and solid tumors. Using experimental and computational approaches, we found that MRX-2843 synergized strongly-and in a ratio-dependent manner-with vincristine to inhibit both B-ALL and T-ALL cell line expansion.

A multiplexed time-resolved fluorescence resonance energy transfer ultrahigh-throughput screening assay for targeting SMAD4-SMAD3-DNA complex.

The signaling pathway of transforming growth factor-beta (TGFβ) plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small molecule inhibitors specifically targeting SMAD4, the downstream master regulator of TGFβ pathway, would offer an alternative approach with significant therapeutic potential for anti-TGF-β signaling.

Nicardipine is a putative EED inhibitor and has high selectivity and potency against chemoresistant prostate cancer in preclinical models.

Background: It is imperative to develop novel therapeutics to overcome chemoresistance, a significant obstacle in the clinical management of prostate cancer (PCa) and other cancers.

Methods: A phenotypic screen was performed to identify novel inhibitors of chemoresistant PCa cells. The mechanism of action of potential candidate(s) was investigated using in silico docking, and molecular and cellular assays in chemoresistant PCa cells.

Bromocriptine monotherapy overcomes prostate cancer chemoresistance in preclinical models.

Chemoresistance is a major obstacle in the clinical management of metastatic, castration-resistant prostate cancer (PCa). It is imperative to develop novel strategies to overcome chemoresistance and improve clinical outcomes in patients who have failed chemotherapy. Using a two-tier phenotypic screening platform, we identified bromocriptine mesylate as a potent and selective inhibitor of chemoresistant PCa cells.

Constitutively synergistic multiagent drug formulations targeting MERTK, FLT3, and BCL-2 for treatment of AML.

Although high-dose, multi-agent chemotherapy has improved leukemia survival rates in recent years, treatment outcomes remain poor in high-risk subsets, including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) in infants. Development of new, more effective therapies for these patients is therefore an urgent, unmet clinical need. To address this challenge, we developed a nanoscale combination drug formulation that exploits ectopic expression of MERTK tyrosine kinase and dependency on BCL-2 family proteins for leukemia cell survival in pediatric AML and rearranged precursor B-cell ALL (infant ALL).

TMPRSS2 and SARS-CoV-2 SPIKE interaction assay for uHTS.

SARS-CoV-2, the coronavirus that causes the disease COVID-19, has claimed millions of lives over the past 2 years. This demands rapid development of effective therapeutic agents that target various phases of the viral replication cycle. The interaction between host transmembrane serine protease 2 (TMPRSS2) and viral SPIKE protein is an important initial step in SARS-CoV-2 infection, offering an opportunity for therapeutic development of viral entry inhibitors.

Hypomorph mutation-directed small-molecule protein-protein interaction inducers to restore mutant SMAD4-suppressed TGF-β signaling.

Tumor suppressor genes represent a major class of oncogenic drivers. However, direct targeting of loss-of-function tumor suppressors remains challenging. To address this gap, we explored a variant-directed chemical biology approach to reverse the lost function of tumor suppressors using SMAD4 as an example.

Development of a miniaturized 3D organoid culture platform for ultra-high-throughput screening.

The recent advent of robust methods to grow human tissues as 3D organoids allows us to recapitulate the 3D architecture of tumors in an in vitro setting and offers a new orthogonal approach for drug discovery. However, organoid culturing with extracellular matrix to support 3D architecture has been challenging for high-throughput screening (HTS)-based drug discovery due to technical difficulties. Using genetically engineered human colon organoids as a model system, here we report our effort to miniaturize such 3D organoid culture with extracellular matrix support in high-density plates to enable HTS.

Cyclosporine A as a Cardioprotective Agent During Donor Heart Retrieval, Storage, or Transportation: Benefits and Limitations.

Background: Storage of donor hearts in cardioplegic solutions supplemented with conditioning agents activating endogenous mitochondrial protective signaling enhanced their postreperfusion recovery. The present study investigates the role of timing and duration of cardiac exposure to cyclosporine A (CsA), another putative mitochondrial protectant, on cardiac functional recovery and potential mechanisms of CsA action in an isolated working rat heart model of donor heart retrieval and storage.

Methods: After measurement of baseline function, hearts were arrested and stored for 6 hours at 4°C in either Celsior alone or Celsior + CsA (0.

Functional recovery after dantrolene-supplementation of cold stored hearts using an ex vivo isolated working rat heart model.

Unlabelled: The ryanodine receptor antagonist dantrolene inhibits calcium release from the sarcoplasmic reticulum and reduces cardiac ischaemia-reperfusion injury (IRI) in global warm ischaemia models however the cardioprotective potential of dantrolene under hypothermic conditions is unknown. This study addresses whether the addition of dantrolene during cardioplegia and hypothermic storage of the donor heart can improve functional recovery and reduce IRI. Using an ex vivo isolated working heart model, Wistar rat (3 month and 12 month) hearts were perfused to acquire baseline haemodynamic measurements of aortic flow, coronary flow, cardiac output, pulse pressure and heart rate.

Development of a Time-Resolved Fluorescence Resonance Energy Transfer Ultrahigh-Throughput Screening Assay for Targeting the NSD3 and MYC Interaction.

Epigenetic modulators play critical roles in reprogramming of cellular functions, emerging as a new class of promising therapeutic targets. Nuclear receptor binding SET domain protein 3 (NSD3) is a member of the lysine methyltransferase family. Interestingly, the short isoform of NSD3 without the methyltransferase fragment, NSD3S, exhibits oncogenic activity in a wide range of cancers.

Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.

HIV-1 entry and fusion with target cells is an important target for antiviral therapy. However, a few currently approved treatments are not effective as monotherapy due to the emergence of drug resistance. This consideration has fueled efforts to develop new bioavailable inhibitors targeting different steps of the HIV-1 entry process.

Combination of heat shock protein 90 and focal adhesion kinase inhibitors synergistically inhibits the growth of non-small cell lung cancer cells.

Discovery of effective drug combinations is a promising strategy to improve patient survival. This study explores the impact of heat shock protein 90 (Hsp90) inhibition in combination with focal adhesion kinase (FAK) inhibitor on the growth of non-small cell lung cancer cells (NSCLC cells). Our data show that 17-N-Allylamino-17-demethoxygeldanamycin (17-AAG), a well-studied Hsp90 inhibitor, synergized with FAK inhibitor, PF-573228, on the growth inhibition of NSCLC cells.

High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors.

HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors.

A time-resolved fluorescence resonance energy transfer assay for high-throughput screening of 14-3-3 protein-protein interaction inhibitors.

Protein-protein interaction networks mediate diverse biological processes by regulating various signaling hubs and clusters. 14-3-3 proteins, a family of phosphoserine/threonine-binding molecules, serve as major interaction hubs in eukaryotic cells and have emerged as promising therapeutic targets for various human diseases. In order to identify chemical probes for mechanistic studies and for potential therapeutic development, we have developed highly sensitive bioassays to monitor the interaction of 14-3-3 with a client protein.

Ophiopogonin B-induced autophagy in non-small cell lung cancer cells via inhibition of the PI3K/Akt signaling pathway.

Ophiopogonin B (OP-B) is a bioactive component of Radix Ophiopogon Japonicus, which is often used in Chinese traditional medicine to treat pulmonary disease. However, whether or not OP-B has any potential antitumor activity has not been reported. Here, we show that the non-small cell lung cancer (NSCLC) cell lines NCI-H157 and NCI-H460 treated with OP-B grow more slowly and accumulate vacuoles in their cytoplasm compared to untreated control cells.

Small molecule antagonist reveals seizure-induced mediation of neuronal injury by prostaglandin E2 receptor subtype EP2.

With interest waning in the use of cyclooxygenase-2 (COX-2) inhibitors for inflammatory disease, prostaglandin receptors provide alternative targets for the treatment of COX-2-mediated pathological conditions in both the periphery and the central nervous system. Activation of prostaglandin E2 receptor (PGE(2)) subtype EP2 promotes inflammation and is just beginning to be explored as a therapeutic target. To better understand physiological and pathological functions of the prostaglandin EP2 receptor, we developed a suite of small molecules with a 3-aryl-acrylamide scaffold as selective EP2 antagonists.

A dual-readout F2 assay that combines fluorescence resonance energy transfer and fluorescence polarization for monitoring bimolecular interactions.

Förster (fluorescence) resonance energy transfer (FRET) and fluorescence polarization (FP) are widely used technologies for monitoring bimolecular interactions and have been extensively used in high-throughput screening (HTS) for probe and drug discovery. Despite their popularity in HTS, it has been recognized that different assay technologies may generate different hit lists for the same biochemical interaction. Due to the high cost of large-scale HTS campaigns, one has to make a critical choice to employee one assay platform for a particular HTS.

Sodium pyruvate modulates cell death pathways in HaCaT keratinocytes exposed to half-mustard gas.

2-Chloroethyl ethyl sulfide (CEES) or half-mustard gas, a sulfur mustard (HD) analog, is a genotoxic agent that causes oxidative stress and induces both apoptotic and necrotic cell death. Sodium pyruvate induced a necrosis-to-apoptosis shift in HaCaT cells exposed to CEES levels ≤ 1.5 mmol/L and lowered markers of DNA damage, oxidative stress, and inflammation.

Reversing chemoresistance by small molecule inhibition of the translation initiation complex eIF4F.

Deregulation of cap-dependent translation is associated with cancer initiation and progression. The rate-limiting step of protein synthesis is the loading of ribosomes onto mRNA templates stimulated by the heterotrimeric complex, eukaryotic initiation factor (eIF)4F. This step represents an attractive target for anticancer drug discovery because it resides at the nexus of the TOR signaling pathway.