Small Molecule Screening Research Articles

Development of Mitophagy-inducing Small Molecules as Therapeutics for Aging and Alzheimer's Disease

Alzheimer's disease (AD) is the leading cause of dementia in those over the age of65. The underlying molecular basis for the disease remains elusive. Rather than simply viewing individual diseases in isolation, researchers have turned to studying the contribution of common age-related processes. One such process is loss in mitochondrial function, normally maintained by defective mitochondrial clearance via mitophagy. Mitophagy slows with age and with various age-related conditions including AD. An agent which has received considerable attention is urolithin A (UA) which has recently been reported to elicit neuroprotection in a variety of AD models. As part of an independent small molecule screen, we identified a second structurally-related compound we named ‘mitophagy inducing compound’ (MIC). Using C. elegans as an initial validation tool, we showed that both compounds significantly improve mitochondrial function, protect against neuronal cell loss, and extend lifespan. These effects were translatable to both in vitro and in vivo mammalian models. To identify potential drug targets, we performed Cellular Thermal Shift Assay (CETSA). Preliminary data from knockdown of identified targets suggest the involvement of a mito-hormetic mechanism which acts to restore overall mitochondrial health. These studies may have important implications for how these compounds elicit their effects.

AKT2 inhibition accelerates the acquisition of phagocytic ability in induced pluripotent stem cell–derived neutrophils

Neutrophils are key components of the immune system that inhibit bacterial infections. Systemic bacterial infections can cause lethal conditions, especially in patients with neutropenia associated with chemotherapy or other systemic illnesses; hence, early detection of the symptoms and prompt management are crucial in such cases. Previously, we established expandable engineered neutrophil-primed progenitors (NeuPs-XL) using human-induced pluripotent stem cells (iPSCs), which can produce neutrophil-like cells at a clinically suitable scale within 4 days of inducing myeloid differentiation. In this study, using small-molecule compound-based screening, we detected that MK-2206, a selective pan-AKT inhibitor, can accelerate this differentiation process, promote phagocytic ability in neutrophils, and enhance cytokine and chemokine expression in response to lipopolysaccharides. The inhibition of AKT2 has been identified as the key mechanism underlying this acceleration. These results can make a substantial contribution to the development of strategies for the prompt production of clinically applicable iPSC-derived neutrophils, which can potentially lead to the management of severe infections associated with life-threatening neutropenia and the effective treatment of related health conditions in the future.

Screening and Characterization of Allosteric Small Molecules Targeting Bruton's Tyrosine Kinase.

Bruton's Tyrosine Kinase (BTK) is a nonreceptor tyrosine kinase that belongs to the TEC family. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) leading to an arrest in B-cell development. BTK is also a drug target for B-cell lymphomas that rely on an intact B-cell receptor signaling cascade for survival. All FDA approved drugs for BTK target the ATP binding site of the catalytic kinase domain, leading to potential adverse events due to off-target inhibition. In addition, acquired resistance mutations occur in a subset of patients, rendering available BTK inhibitors ineffective. Therefore, allosteric sites on BTK should be explored for drug development to target BTK more specifically and in combination with active site inhibitors. Virtual screening against nonactive site pockets and in vitro experiments resulted in a series of small molecules that bind to BTK outside of the active site. We characterized these compounds using biochemical and biophysical techniques and narrowed our focus to compound "C2". C2 activates full-length BTK and smaller multidomain BTK fragments but not the isolated kinase domain, consistent with an allosteric mode of action. Kinetic experiments reveal a C2-mediated decrease in Km and an increase in kcat leading to an overall increase in the catalytic efficiency of BTK. C2 is also capable of activating the BTK XLA mutants. These proof-of-principle data reveal that BTK can be targeted allosterically with small molecules, providing an alternative to active site BTK inhibitors.

Encoding Molecular Docking for Quantum Computers.

Molecular docking is important in drug discovery but is burdensome for classical computers. Here, we introduce Grid Point Matching (GPM) and Feature Atom Matching (FAM) to accelerate pose sampling in molecular docking by encoding the problem into quadratic unconstrained binary optimization (QUBO) models so that it could be solved by quantum computers like the coherent Ising machine (CIM). As a result, GPM shows a sampling power close to that of Glide SP, a method performing an extensive search. Moreover, it is estimated to be 1000 times faster on the CIM than on classical computers. Our methods could boost virtual drug screening of small molecules and peptides in future.

Design and structural validation of peptide–drug conjugate ligands of the kappa-opioid receptor

Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA–KOR–Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.

Selective and brain-penetrant HCN1 inhibitors reveal links between synaptic integration, cortical function, and working memory

Hyperpolarization-activated and cyclic-nucleotide-gated 1 (HCN1) ion channels are proposed to be critical for cognitive function through regulation of synaptic integration. However, resolving the precise role of HCN1 in neurophysiology and exploiting its therapeutic potential has been hampered by minimally selective antagonists with poor potency and limited invivo efficiency. Using automated electrophysiology in a small-molecule library screen and chemical optimization, we identified a primary carboxamide series of potent and selective HCN1 inhibitors with a distinct mode of action. In cognition-relevant brain circuits, selective inhibition of native HCN1 produced on-target effects, including enhanced excitatory postsynaptic potential summation, while administration of a selective HCN1 inhibitor to rats recovered decrement working memory. Unlike prior non-selective HCN antagonists, selective HCN1 inhibition did not alter cardiac physiology in human atrial cardiomyocytes or in rats. Collectively, selective HCN1 inhibitors described herein unmask HCN1 as a potential target for the treatment of cognitive dysfunction in brain disorders.

A Biomimetic Multiparametric Assay to Characterise Anti-Amyloid Drugs

Alzheimer’s disease (AD) is the most widespread form of senile dementia worldwide and represents a leading socioeconomic problem in healthcare. Although it is widely debated, the aggregation of the amyloid β peptide (Aβ) is linked to the onset and progression of this neurodegenerative disease. Molecules capable of interfering with specific steps in the fibrillation process remain of pharmacological interest. To identify such compounds, we have set up a small molecule screening process combining multiple experimental methods (UV and florescence spectrometry, ITC, and ATR-FTIR) to identify and characterise potential modulators of Aβ1-42 fibrillation through the description of the biochemical interactions (molecule–membrane Aβ peptide). Three known modulators, namely bexarotene, Chicago sky blue and indomethacin, have been evaluated through this process, and their modulation mechanism in the presence of a biomembrane has been described. Such a well-adapted physico-chemical approach to drug discovery proves to be an undeniable asset for the rapid characterisation of compounds of therapeutic interest for Alzheimer’s disease. This strategy could be adapted and transposed to search for modulators of other amyloids such as tau protein.

Chaetocin-mediated SUV39H1 inhibition targets stemness and oncogenic networks of diffuse midline gliomas and synergizes with ONC201.

Diffuse intrinsic pontine gliomas (DIPG/DMG) are devastating pediatric brain tumors with extraordinarily limited treatment options and uniformly fatal prognosis. Histone H3K27M mutation is a common recurrent alteration in DIPG and disrupts epigenetic regulation. We hypothesize that genome-wide H3K27M-induced epigenetic dysregulation makes tumors vulnerable to epigenetic targeting. We performed a screen of compounds targeting epigenetic enzymes to identify potential inhibitors for the growth of patient-derived DIPG cells. We further carried out transcriptomic and genomic landscape profiling including RNA-seq and CUT&RUN-seq as well as shRNA-mediated knockdown to assess the effects of chaetocin and SUV39H1, a target of chaetocin, on DIPG growth. High-throughput small-molecule screening identified an epigenetic compound chaetocin as a potent blocker of DIPG cell growth. Chaetocin treatment selectively decreased proliferation and increased apoptosis of DIPG cells and significantly extended survival in DIPG xenograft models, while restoring H3K27me3 levels. Moreover, the loss of H3K9 methyltransferase SUV39H1 inhibited DIPG cell growth. Transcriptomic and epigenomic profiling indicated that SUV39H1 loss or inhibition led to the downregulation of stemness and oncogenic networks including growth factor receptor signaling and stemness-related programs; however, D2 dopamine receptor (DRD2) signaling adaptively underwent compensatory upregulation conferring resistance. Consistently, a combination of chaetocin treatment with a DRD2 antagonist ONC201 synergistically increased the antitumor efficacy. Our studies reveal a therapeutic vulnerability of DIPG cells through targeting the SUV39H1-H3K9me3 pathway and compensatory signaling loops for treating this devastating disease. Combining SUV39H1-targeting chaetocin with other agents such as ONC201 may offer a new strategy for effective DIPG treatment.

High-Throughput, Quantitative Screening of Quorum-Sensing Inhibitors Based on a Bacterial Biosensor.

Quorum sensing (QS) is a cell-cell communication mechanism by which bacteria synchronize social behaviors such as biofilm formation and virulence factor secretion by producing and sensing small molecular signals. Quorum quenching (QQ) by degrading signals or blocking signal transmissions has become a promising strategy for disrupting QS and preventing bacterial infection and biofilm formation. However, studies of high-throughput screening and identification approaches for quorum-sensing inhibitors (QSIs) are still inadequate. In this work, we developed a sensitive, high-throughput approach for screening QSIs based on the bacterial biosensor strain Agrobacterium tumefaciens N5 (pBA7P), which contains a traG gene promoter induced by QS signals fused with a promoterless β-lactamase gene reporter. Using this approach, we identified 31 QQ bacteria from ∼2000 soil bacterial isolates, some belonging to the genera Bosea, Cupriavidus, and Flavobacterium that have not been reported previously as QQ bacteria. We also identified four QS inhibitory compounds and one QS signal analogue from ∼5000 small-molecule compounds, which profoundly affected the expression of QS-regulated genes and phenotypes of the pathogenic bacteria. This high-throughput screening system is effective and sensitive for screening of both QQ microbes and small molecules, enabling the discovery of a wide variety of biocompatible compounds.

Modeling cardiac injury using human cardiac slices to test therapeutic approaches for cardiac regeneration and repair

Abstract Background Due to limited regenerative capacity of the adult human heart, myocardial infarction causes irreversible damage by inducing the death of cardiomyocytes and the formation of a fibrotic scar. These remodeling mechanisms cause loss of contractility and eventually heart failure ensues in a significant proportion of patients. Purpose There is a need for a reliable model that can replicate the adult cardiac pathophysiology in order to test treatments against cardiac remodeling. Here, we developed a cardiac tissue culture model that can simulate in vitro the molecular changes occurring after cardiac injury using human cultured cardiac slices. Methods We obtained cardiac slices by sectioning ventricular myocardium from the surgical waste material of adult patients undergoing valve surgery or Morrow myectomy. Cardiac slices were cultured at a liquid-air interface and were viable up to 7 days of culture, even in the absence of stimulation. During this time cardiac slices underwent mechanical and chemical treatments to induce ischemia and/or local injury. Results We found that following these treatments we were able to simulate cardiac remodeling in terms of cardiomyocyte death, activation of cardiac fibroblasts and induction of inflammation and oxidative stress. Following small molecule screening, we discovered that addition of compounds with anti-inflammatory properties (e.g. dexamethasone) or targeting the TGF-β pathway (e.g. SB-431542) into our culture medium helped in preserving the microscopic structure of the slices. Additionally, we found that treatment with these compounds protected cardiac slices against remodeling following ischemic injury. Conclusions In conclusion, our model can emulate cardiac pathophysiology in culture, providing us a reliable platform to test therapies for cardiac regeneration and repair in an adult, multicellular, 3-D environment.

Abstract 17407: A Small-molecule Screening Strategy to Promote Cardiomyocyte Proliferation That Protects the Heart From Ischemic Injury

Restoring the ability of adult cardiac myocytes (ACMs) to proliferate and promote heart regeneration have been proposed as a promising therapeutic strategy. We have demonstrated that selective depletion of H3K9me3 by lysine demethylase 4D (KDM4D) overexpression increases ACM cell cycle activity, but proliferation was limited. To improve this, we performed a small-molecule screening in combination with KDM4D on neonatal rat cardiomyocytes (NRVMs), using a library of kinase inhibitors targeting different signaling pathways. Our study revealed that compounds targeting the PI3K and JAK signaling pathways significantly potentiated the effects of KDM4D and promote NRVM proliferation in vitro. Specifically, a JAK inhibitor (JAKi2) in combination with Ad-KDM4D treatment resulted in approximately 100%, 34%, and 62% increase in EdU+, phospho-H3+, and total cell number compared to control, respectively. To validate these findings in vivo, we tested JAKi2 in an inducible KDM4D transgenic mouse model (iKDM4D). The animals underwent ischemia/reperfusion (I/R) to induce infarction, and JAKi2 was administrated orally for up to 8 weeks. We found both early and late effects. 48 hours after I/R, the infarct size in iKDM4D+JAKi2 hearts was approximately 4-fold smaller than that in vehicle-treated or JAKi2 alone groups, and the combination increased EdU+, Ki67+, and phospho-H3+ CM number separately by 3.2-fold, 2.9-fold, and 6.5-fold. At 2 weeks post-I/R, the scar size was smaller in iKDM4D+JAKi2 hearts compared to vehicle-treated mice (9.0% vs 1.8%, P <0.05), and the combination resulted in a 3-fold, 5.5-fold, and 8.5-fold increases in EdU+, Ki67+, and phospho-H3+ CMs, respectively. Moreover, the iKDM4D+JAKi2 hearts demonstrated progressive improvement in left ventricular ejection fraction (26.8%, 27.3%, and 25.5%) and fraction shorten (37.9%, 38.1%, and 35.0%) at 2 weeks, 4 weeks, and 8 weeks post-I/R. The increased estimated ACM number and mononucleated CMs at 8 weeks post-I/R support that CM division was increased in the iKDM4D+JAKi2 hearts. These findings suggest that a combination therapy involving JAKi2, along with KDM4D, promotes robust CM proliferation and protects the heart after ischemic injury.

Results of a Phase 1 Trial Testing the Novel Combination Therapy of Venetoclax and Ruxolitinib in Relapsed/Refractory Acute Myeloid Leukemia Patients

Introduction : A functional small molecule screen on a large cohort of primary AML patient samples revealed that the combination of Ruxolitinib (Rux) and Venetoclax (Ven) exhibited ex vivo efficacy and synergy in both newly diagnosed and R/R AML. This observation motivated a Phase I multi-center trial to evaluate Rux+Ven in R/R AML, for which we now report complete toxicity and efficacy data for 30 subjects. Methods: This study was designed to identify a maximum tolerated or recommended Phase II dose (RP2D) and evaluate the overall safety and preliminary efficacy of Rux+Ven therapy. Eligible subjects were ≥18 years old with R/R AML. There was no restriction on the number of previous therapies or prior exposure to Ven or Rux. We included subjects with prior allogeneic transplant if GVHD was controlled and prior MDS (≥75 years old or having poor cardiac or kidney function) who progressed on a hypomethylating agent (HMA) but had no primary AML therapy. In cohorts of 3 to 4, subjects were assigned to receive Rux+Ven at 1 of 6 dose levels according to the “keyboard” Bayesian toxicity probability interval design applied to a target dose-limiting toxicity (DLT) rate of 30%. DLTs were defined as non-disease-related, Grade ≥3 non-hematologic toxicities, with exceptions for nausea, vomiting, infections, febrile neutropenia episodes, and electrolyte abnormalities that resolved within 48 hours. Hematologic DLT was specified as Grade 4 neutropenia after 42 days in the absence of disease. The DLT evaluation period covered the first cycle. Subjects could continue study therapy after two 28-day cycles if they had a response (ie, CR/CRi or MLFS) or were otherwise deriving clinical benefit. Results : This Phase 1 study has completed enrollment (n=30), with all but one patient off-treatment as of June 2023. Baseline patient characteristics are summarized in Table 1. Median age was 69 (range 29-84), 40% of subjects had ≥3 prior therapies, and 43% had prior Ven. At enrollment, 30% had prior MDS, 24% had mutated TP53 (of 25 patients with available data), and 47% had CD56+ blasts. The numbers of patients treated by dose level (DL) were: 3 on DL0 (200 mg qd Ven; 10 mg bid Rux), 4 on DL1 (400 mg qd Ven; 10 mg bid Rux), 3 on DL2 (400 mg qd Ven; 20 mg bid Rux), and 20 on DL3 (400 mg qd Ven; 30 mg bid Rux), the highest dose level of the study. Per prescribing guidelines, the cycle 1 Rux doses of 15 subjects and Ven doses of 20 subjects were reduced because of a concomitant CYP3A inhibitor. The regimen was well-tolerated. As no DLTs were observed, DL3 is the RP2D. The median (interquartile range) duration of therapy was 55 (36-93) days. The most common grade ≥3 hematologic AEs, regardless of attribution, were thrombocytopenia (50%; which was 30% at study enrollment), anemia (40%; 40% at enrollment), neutropenia (37%; 10% at enrollment), febrile neutropenia (33%), and leukopenia (33%; 7% at enrollment). Six subjects had a Grade 5 AE: 4 were unrelated to study drug (2 death NOS, 1 lung infection, 1 respiratory failure) and 2 were potentially related to Ven (1 sepsis, 1 neutropenic pneumonia). Over the first 2 cycles of study therapy, the Clinical Benefit Rate (CBR; defined as the proportion of subjects obtaining at least stable disease) was 63% (95% CI: 44%-80%). Morphologic leukemia-free state (MLFS) was achieved in 20% (95% CI: 8%-39%) and Composite Complete Remission in 10% (95% CI: 2%-27%). Median overall survival (OS) was 3.7 months (95% CI: 2.3-6.5), with 23% (95% CI: 10%-39%) alive 1 year after commencing Rux+Ven. Two participants were exceptional responders: the first achieving MLFS and receiving 16 cycles of study therapy and the second having a complete remission and remaining on-treatment after 31 cycles. Pre-Rux+Ven CD56 expression on blasts correlated with worse response (all 6 ≥MLFS patients were CD56-; Fig 1) and overall survival (HR=2.37, p=0.044; Table 1). Correlative studies such as ex vivo drug assessment, genomic analysis, CyTOF, and single-cell imaging will be presented to enhance this finding. Conclusion: The novel, all-oral combination of Rux+Ven in R/R AML patients was well-tolerated with no DLTs and an encouraging 63% CBR over cycles 1-2. Negative CD56 was a biomarker of response, although additional work is required to determine if this is a broad association with Ven-based therapies or unique to the Rux+Ven combination. The tolerable toxicity profile of Rux+Ven makes it a promising combination to test with HMAs in high-risk AML patients.

Broad spectrum post-entry inhibitors of coronavirus replication: Cardiotonic steroids and monensin

A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.

High-throughput screening identifies small molecule inhibitors of thioesterase superfamily member 1: Implications for the management of non-alcoholic fatty liver disease

ObjectiveThioesterase superfamily member 1 (Them1) is a long chain acyl-CoA thioesterase comprising two N-terminal HotDog fold enzymatic domains linked to a C-terminal lipid-sensing steroidogenic acute regulatory transfer-related (START) domain, which allosterically modulates enzymatic activity. Them1 is highly expressed in thermogenic adipose tissue, where it functions to suppress energy expenditure by limiting rates of fatty acid oxidation, and is induced markedly in liver in response to high fat feeding, where it suppresses fatty acid oxidation and promotes glucose production. Them1−/− mice are protected against non-alcoholic fatty liver disease (NAFLD), suggesting Them1 as a therapeutic target. MethodsA high-throughput small molecule screen was performed to identify promising inhibitors targeting the fatty acyl-CoA thioesterase activity of purified recombinant Them1.Counter screening was used to determine specificity for Them1 relative to other acyl-CoA thioesterase isoforms. Inhibitor binding and enzyme inhibition were quantified by biophysical and biochemical approaches, respectively. Following structure-based optimization, lead compounds were tested in cell culture. ResultsTwo lead allosteric inhibitors were identified that selectively inhibited Them1 by binding the START domain. In mouse brown adipocytes, these inhibitors promoted fatty acid oxidation, as evidenced by increased oxygen consumption rates. In mouse hepatocytes, they promoted fatty acid oxidation, but also reduced glucose production. ConclusionThem1 inhibitors could prove attractive for the pharmacologic management of NAFLD.

Fused Tetrahydroquinolines Are Interfering with Your Assay.

Tricyclic tetrahydroquinolines (THQs) have been repeatedly reported as hits across a diverse range of high-throughput screening (HTS) campaigns. The activities of these compounds, however, are likely due to reactive byproducts that interfere with the assay. As a lesser studied class of pan-assay interference compounds, the mechanism by which fused THQs react with protein targets remains largely unknown. During HTS follow-up, we characterized the behavior and stability of several fused tricyclic THQs. We synthesized key analogues to pinpoint the cyclopentene ring double bond as a source of reactivity of fused THQs. We found that these compounds degrade in solution under standard laboratory conditions in days. Importantly, these observations make it likely that fused THQs, which are ubiquitously found within small molecule screening libraries, are unlikely the intact parent compounds. We urge deprioritization of tricylic THQ hits in HTS follow-up and caution against the investment of resources to follow-up on these problematic compounds.

Graph neural networks for the identification of novel inhibitors of a small RNA

MicroRNAs (miRNAs) play a crucial role in post-transcriptional gene regulation and have been implicated in various diseases, including cancers and lung disease. In recent years, Graph Neural Networks (GNNs) have emerged as powerful tools for analyzing graph-structured data, making them well-suited for the analysis of molecular structures. In this work, we explore the application of GNNs in ligand-based drug screening for small molecules targeting miR-21. By representing a known dataset of small molecules targeting miR-21 as graphs, GNNs can learn complex relationships between their structures and activities, enabling the prediction of potential miRNA-targeting small molecules by capturing the structural features and similarity between known miRNA-targeting compounds. The use of GNNs in miRNA-targeting drug screening holds promise for the discovery of novel therapeutic agents and provides a computational framework for efficient screening of large chemical libraries.

Brazilin from Caesalpinia sappan L. as a Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitor: Pharmacophore-Based Virtual Screening, In Silico Molecular Docking, and In Vitro Studies.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a crucial regulator of low-density lipoprotein cholesterol (LDL-c) levels, as it binds to and degrades the LDL receptor (LDLR) in the lysosome of hepatocytes. Elevated levels of PCSK9 have been linked to an increased LDL-c plasma levels, thereby increasing the risk of cardiovascular disease (CVD), making it an attractive target for therapeutic interventions. As a way to inhibit PCSK9 action, we searched for naturally derived small molecules which can block the binding of PCSK9 to the LDLR. In this study, we carried out in silico studies which consist of virtual screening using an optimized pharmacophore model and molecular docking studies using Pyrx 0.98. Effects of the candidate compounds were evaluated using in vitro PCSK9-LDLR binding assays kit. Eleven natural compounds that bind to PCSK9 were virtually screened form HerbalDB database, including brazilin. Next, molecular docking studies using Pyrx 0.98 showed that brazilin had the highest binding affinity with PCSK9 at -9.0 (Kcal/mol), which was higher than that of the other ten compounds. Subsequent in vitro PCSK9-LDLR binding assays established that brazilin decreased the binding of PCSK9 to the EGF-A fragment of the LDLR in a dose-dependent manner, with an IC50 value of 2.19 μM. We have identified brazilin, which is derived from the Caesalpinia sappan herb, which can act as a small molecule inhibitor of PCSK9. Our findings suggest that screening for small molecules that can block the interaction between PCSK9 and the LDLR in silico and in vitro may be a promising approach for developing novel lipid-lowering therapy.

Precursor-Directed Biosynthesis and Biological Testing of omega-Alicyclic- and neo-Branched Tunicamycin N-Acyl Variants.

Tunicamycins (TUNs) are Streptomyces-derived natural products, widely used to block protein N-glycosylation in eukaryotes or cell wall biosynthesis in bacteria. Modified or synthetic TUN analogues that uncouple these activities have considerable potential as novel mode-of-action antibacterial agents. Chemically modified TUNs reported previously with attenuated activity on yeast have pinpointed eukaryotic-specific chemophores in the uridyl group and the N-acyl chain length and terminal branching pattern. A small molecule screen of fatty acid biosynthetic primers identified several novel alicyclic- and neo-branched TUN N-acyl variants, with primer incorporation at the terminal omega-acyl position. TUNs with unique 5- and 6-carbon ω-cycloalkane and ω-cycloalkene acyl chains are produced under fermentation and in yields comparable with the native TUN. The purification, structural assignments, and the comparable antimicrobial properties of 15 of these compounds are reported, greatly extending the structural diversity of this class of compounds for potential medicinal and agricultural applications.

Small molecule screen identifies pyrimethamine as an inhibitor of NRF2-driven esophageal hyperplasia

ObjectiveNRF2 is a master transcription factor that regulates the stress response. NRF2 is frequently mutated and activated in human esophageal squamous cell carcinoma (ESCC), which drives resistance to chemotherapy and radiation therapy. Therefore, a great need exists for NRF2 inhibitors for targeted therapy of NRF2high ESCC. DesignWe performed high-throughput screening of two compound libraries from which hit compounds were further validated in human ESCC cells and a genetically modified mouse model. The mechanism of action of one compound was explored by biochemical assays. ResultsUsing high-throughput screening of two small molecule compound libraries, we identified 11 hit compounds as potential NRF2 inhibitors with minimal cytotoxicity at specified concentrations. We then validated two of these compounds, pyrimethamine and mitoxantrone, by demonstrating their dose- and time-dependent inhibitory effects on the expression of NRF2 and its target genes in two NRF2Mut human ESCC cells (KYSE70 and KYSE180). RNAseq and qPCR confirmed the suppression of global NRF2 signaling by these two compounds. Mechanistically, pyrimethamine reduced NRF2 half-life by promoting NRF2 ubiquitination and degradation in KYSE70 and KYSE180 cells. Expression of an Nrf2E79Q allele in mouse esophageal epithelium (Sox2CreER;LSL-Nrf2E79Q/+) resulted in an NRF2high phenotype, which included squamous hyperplasia, hyperkeratinization, and hyperactive glycolysis. Treatment with pyrimethamine (30 mg/kg/day, p.o.) suppressed the NRF2high esophageal phenotype with no observed toxicity. ConclusionWe have identified and validated pyrimethamine as an NRF2 inhibitor that may be rapidly tested in the clinic for NRF2high ESCC.

Abstract PO-077: High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC

Abstract Cancer metastasis is a complex cascade that involves activation of cancer cell invasion and migration. This activation is greatly attributed by multiple factors, including the tumor-microenvironment (TME). Cancer-associated fibroblasts (CAF), a prominent cell type within the TME, have been shown to promote cancer cell invasion and migration, causing metastasis in various cancers including head and neck squamous cell carcinoma (HNSCC). The molecular mechanisms of how CAFs promote HNSCC invasion remain elusive. Our research goal is to understand how CAFs influence HNSCC cells to become invasive and potentially metastatic. Using a top-down research approach, our aim is to identify novel therapeutic chemical probe/drug regimens that potentially target CAF-dependent HNSCC cancer cell-invasion. In-order to perform high-throughput small molecule screens, we have established a 384-well format, three-dimensional (3D) spheroid invasion assay, as a powerful tool to study CAF-dependent HNSCC cancer cell invasion. This platform is currently being used to screen small molecule libraries and identify putative molecular targets, providing insights into underlying mechanisms of CAF-induced cancer cell invasion and candidate therapeutic strategies. Citation Format: Kunal Karve, Stephanie Poon, Panagiotis Prinos, Laurie Ailles. High-throughput 3D-spheroid invasion assay: A powerful tool to identify novel drugs targeting tumor micro-environment in HNSCC [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-077.