Library Of Molecules Research Articles

Discovery and preclinical development of LY3473329, a potent small molecule inhibitor of lipoprotein(a) formation

Abstract Background Lipoprotein(a) [Lp(a)] is a lipoprotein particle consisting of a low-density lipoprotein (LDL) particle and apolipoprotein(a) [apo(a)]. Lp(a) is a causal risk factor for cardiovascular disease. The formation of Lp(a) requires an initial interaction of apo(a) Kringle IV Domains 7 and 8 (KIV7, KIV8) with apoB-100 on LDL, followed by formation of a disulfide bond between apo(a) and apoB-100. Blocking the initial apo(a)-apoB interaction is a viable therapeutic approach to reduce circulating levels of Lp(a). The apo(a) protein is encoded by the LPA gene that arose from the plasminogen gene through an evolutionary gene duplication event. Because of sequence conservation between apo(a) and plasminogen, compound selectivity for apo(a) is imperative for clinical development. Purpose We report the discovery and preclinical characterization of LY3473329, a novel selective small molecule inhibitor of Lp(a) formation. Methods Computational and synthetic chemistry approaches coupled with multiple biochemical and biophysical assays were used to create apo(a) KIV7,8 binders. Chemical optimization led to molecules with potent inhibition of in vitro Lp(a) formation. Both apo(a) and plasminogen binding assays were conducted to evaluate compound selectivity. The potential for LY3473329 to lower steady state Lp(a) levels was assessed in human Lp(a) transgenic mice and in cynomolgus monkeys, and its effect on plasminogen activity was assessed in rat and monkey. Results Computational modeling, synthetic chemistry and small molecule library screens identified compounds that bind apo(a) KIV7 and KIV8 domains, and subsequent chemical optimization yielded compounds with sub-micromolar Lp(a) inhibition potency. Exploration of compound multivalency created inhibitors with sub-nanomolar potency, including LY3473329. LY3473329 engages multiple apo(a) KIV motifs, allowing it to potently inhibit Lp(a) formation in vitro. Oral doses of LY3473329 given to human Lp(a) transgenic mice and cynomolgus monkeys caused dose-dependent decreases in plasma Lp(a). The key binding motif for LY3473329 is shared by two KIV domains in rat and monkey plasminogen, but only one KIV domain in human plasminogen. LY3473329 was non-selective for rat and monkey plasminogen, but showed ∼ 50-fold selectivity for human plasminogen in in vitro binding assays. In cynomolgus monkeys and rats, LY3473329 significantly reduced plasminogen activity levels without adverse effects. Given its selectivity against human plasminogen, LY3473329 is expected to lower Lp(a) in human without affecting plasminogen. LY3473329 has been evaluated in a human Phase 1 study and is currently in Phase 2 clinical development. Conclusions LY3473329 is a potent and selective small molecule inhibitor of Lp(a) formation and is the only orally administered Lp(a) investigational therapeutic in clinical development.

Generation of focused drug molecule library using recurrent neural network.

With the wide application of deep learning in drug research and development, de novo molecular design methods based on recurrent neural network (RNN) have strong advantages in drug molecule generation. The RNN model can be used to learn the internal chemical structure of molecules, which is similar to a natural language processing task. Although techniques for generating target-specific molecular libraries based on RNN models are mature, research related to drug design and screening continues around the clock. Research based on de novo drug design methods to generate larger quantities of valid compounds is necessary. In this study, a molecular generation model based on RNN was designed, which abandoned the traditional way of stacked RNN and introduced the Nested long short-term memory network structure. To enrich the library of focused molecules for specific targets, we fine-tuned the model using active molecules from novel coronavirus pneumonia and screened the molecules using machine learning models. Following rigorous screening, the selected molecules underwent molecular docking with the SARS-CoV-2 M-pro receptor using AutoDock2.4 to identify the top 3 potential inhibitors. Subsequently, 100-ns molecular dynamics simulations were conducted using Amber22. Molecule parameterization involved the GAFF2 force field, while the proteins were modeled using the ff19SB force field, with solvation facilitated by a truncated octahedral TIP3P solvent environment. Upon completion of molecular dynamics simulations, stability of ligand-protein complexes was assessed by analysis of RMSD, H-bonds, and MM-GBSA. Reasonable results prove that the model can complete the task of de novo drug design and has the potential to be ideal drug molecules.

Design of a multi-target focused library for antidiabetic targets using a comprehensive set of chemical transformation rules.

Virtual small molecule libraries are valuable resources for identifying bioactive compounds in virtual screening campaigns and improving the quality of libraries in terms of physicochemical properties, complexity, and structural diversity. In this context, the computational-aided design of libraries focused against antidiabetic targets can provide novel alternatives for treating type II diabetes mellitus (T2DM). In this work, we integrated the information generated to date on compounds with antidiabetic activity, advances in computational methods, and knowledge of chemical transformations available in the literature to design multi-target compound libraries focused on T2DM. We evaluated the novelty and diversity of the newly generated library by comparing it with antidiabetic compounds approved for clinical use, natural products, and multi-target compounds tested in vivo in experimental antidiabetic models. The designed libraries are freely available and are a valuable starting point for drug design, chemical synthesis, and biological evaluation or further computational filtering. Also, the compendium of 280 transformation rules identified in a medicinal chemistry context is made available in the linear notation SMIRKS for use in other chemical library enumeration or hit optimization approaches.

Automated high-throughput cell culture scratch assay identifies wound healing promoting and inhibiting compounds from a small compound library of redox active molecules

Automated high-throughput cell culture scratch assay identifies wound healing promoting and inhibiting compounds from a small compound library of redox active molecules

Discovery of inhibitors of protein tyrosine phosphatase 1B contained in a natural products library from Mexican medicinal plants and fungi using a combination of enzymatic and in silico methods*.

This work aimed to discover protein tyrosine phosphatase 1B (PTP1B) inhibitors from a small molecule library of natural products (NPs) derived from selected Mexican medicinal plants and fungi to find new hits for developing antidiabetic drugs. The products showing similar IC50 values to ursolic acid (UA) (positive control, IC50 = 26.5) were considered hits. These compounds were canophyllol (1), 5-O-(β-D-glucopyranosyl)-7-methoxy-3',4'-dihydroxy-4-phenylcoumarin (2), 3,4-dimethoxy-2,5-phenanthrenediol (3), masticadienonic acid (4), 4',5,6-trihydroxy-3',7-dimethoxyflavone (5), E/Z vermelhotin (6), tajixanthone hydrate (7), quercetin-3-O-(6″-benzoyl)-β-D-galactoside (8), lichexanthone (9), melianodiol (10), and confusarin (11). According to the double-reciprocal plots, 1 was a non-competitive inhibitor, 3 a mixed-type, and 6 competitive. The chemical space analysis of the hits (IC50 < 100μM) and compounds possessing activity (IC50 in the range of 100-1,000μM) with the BIOFACQUIM library indicated that the active molecules are chemically diverse, covering most of the known Mexican NPs' chemical space. Finally, a structure-activity similarity (SAS) map was built using the Tanimoto similarity index and PTP1B absolute inhibitory activity, which allows the identification of seven scaffold hops, namely, compounds 3, 5, 6, 7, 8, 9, and 11. Canophyllol (1), on the other hand, is a true analog of UA since it is an SAR continuous zone of the SAS map.

DNA Chimeras as Electrochemical Biosensors for Host-Guest Measurements in Blood.

Few sensing platforms have become ubiquitous to enable rapid and convenient measurements at the point-of-care. Those, however, are "one-off" technologies, meaning that they can only detect a single target and are hardly adaptable. In response, we plan to develop a sensing platform that can be extended to detect other classes of molecules and that affords rapid, convenient, continuous measurements directly in undiluted complex matrices. For this, we decided to rely on a host molecule that presents reversible interactions toward specific guest molecules to develop a new class of sensors that we coined "Electrochemical DNA-host chimeras". As a proof-of-concept for our sensor, we decided to use cyclobis(paraquat-p-phenylene) ("blue box") that we attached on an electrode-bound DNA to allow measurements of electron-rich guests such as dopamine and aspirin. Doing so allows to promote host-guest complex that could be quantified using blue box's electrochemistry. Because of this unique sensor architecture, we achieve, to our knowledge, the first reagentless, continuous and rapid (< 5 min) host-guest measurements in undiluted whole blood. We envision that given the library of electroactive host molecules that this will allow the development of a sensing platform for measurements of several classes of molecules in complex matrices at the point-of-care.

Exploring the potential of machine learning to design antidiabetic molecules: a comprehensive study with experimental validation

Recent advances in hardware and software algorithms have led to the rise of data-driven approaches for designing therapeutic modalities. One of the major causes of human mortality is diabetes. Thus, there is a tremendous opportunity for research into effective antidiabetic designs. Therefore, in this study, we used machine learning-based small molecule design. We used various chemoinformatic and binary fingerprint techniques on small molecules to construct multiple models for alpha-amylase inhibitors. Among these models, the top models were used for ensemble-based machine learning predictions on libraries of organic molecules supplemented with synthetic scaffolds that could be used as antidiabetic agents. Further, involved identifying 10 promising molecules from computational studies and determining their inhibitory effects on alpha-amylase. These molecules were synthesised and thoroughly analysed to assess their biological inhibitory properties. Then, thermodynamic simulations were conducted to determine the stability and affinity of experimentally active molecules. The research results showcased the top 10 ML models recorded impressive statistics with an average model score of 0.8216, Pearson-r value of 0.827 and external validation yielding a Q 2 value of 0.835, proving their reliability and accuracy. Ten derivatives of benzothiophene dioxolane was prime research focus due to computational predictions. The biological inhibitory assay of synthesised molecules showed that small molecules with ID ALC5 and ALC6 exhibited inhibitory efficiencies (IC50) of 2.1 ± 0.14 µM and 5.71 ± 0.02 µM against alpha-amylase enzyme, whereas other molecules showed moderate inhibition. In conclusion, the positive results of the experiment indicate that researchers should explore machine learning-driven design. Communicated by Ramaswamy H. Sarma

Identification and Characterization of a Small Molecule that Activates Thiosulfate Sulfurtransferase and Stimulates Mitochondrial Respiration.

The enzyme Thiosulfate sulfurtransferase (TST, EC 2.8.1.1), is a positive genetic predictor of diabetes type 2 and obesity. As increased TST activity protects against the development of diabetic symptoms in mice, an activating compound for TST may provide therapeutic benefits in diabetes and obesity. We identified a small molecule activator of human TST through screening of an inhouse small molecule library. Kinetic studies in vitro suggest that two distinct isomers of the compound are required for full activation as well as an allosteric mode of activation. Additionally, we studied the effect of TST protein and the activator on TST activity through mitochondrial respiration. Molecular docking and molecular dynamics (MD) approaches supports an allosteric site for the binding of the activator, which is supported by the lack of activation in the E. coli. mercaptopyruvate sulfurtransferase. Finally, we show that increasing TST activity in isolated mitochondria increases mitochondrial oxygen consumption. This article is protected by copyright. All rights reserved.

Stereocontrolled access to thioisosteres of nucleoside di- and triphosphates.

Nucleoside diphosphates and triphosphates impact nearly every aspect of biochemistry; however, the use of such compounds as tools or medicinal leads for nucleotide-dependent enzymes and receptors is hampered by their rapid in vivo metabolism. Although a successful strategy to address the instability of the monophosphate moiety in oligonucleotide therapeutics has been accomplished by their isosteric replacement with phosphorothioates, no practical methods exist to rapidly and controllably access stereopure di- and triphosphate thioisosteres of both natural and unnatural nucleosides. Here we show how a modular, reagent-based platform can enable the stereocontrolled and scalable synthesis of a library of such molecules. This operationally simple approach provides access to pure stereoisomers of nucleoside α-thiodiphosphates and α-thiotriphosphates, as well as symmetrical or unsymmetrical dinucleoside thiodiphosphates and thiotriphosphates (including RNA cap reagents). We demonstrate that ligand-receptor interactions can be dramatically influenced by P-stereochemistry, showing that such thioisosteric replacements can have profound effects on the potency and stability of lead candidates.

Probing RNA-Small Molecule Interactions Using Biophysical and Computational Approaches.

Interest in small molecules that target RNA is flourishing, and the expectation set on them to treat diseases with unmet medical needs is high. However, several challenges remain, including difficulties in selecting suitable tools and establishing workflows for their discovery. In this context, we optimized experimental and computational approaches that were previously employed for the protein targets. Here, we demonstrate that a fluorescence-based assay can be effectively used to screen small molecule libraries for their ability to bind and stabilize an RNA stem-loop. Our screen identified several fluoroquinolones that bind to the target stem-loop. We further probed their interactions with the target using biolayer interferometry, isothermal titration calorimetry (ITC), and nuclear magnetic resonance spectroscopy. The results of these biophysical assays suggest that the fluoroquinolones bind the target in a similar manner. Armed with this knowledge, we built models for the complexes of the fluoroquinolones and the RNA target. Then, we performed fragment molecular orbital (FMO) calculations to dissect the interactions between the fluoroquinolones and the RNA. We found that the binding free energies obtained from the ITC experiments correlated strongly with the interaction energies calculated by FMO. Finally, we designed fluoroquinolone analogues and performed FMO calculations to predict their binding free energies. Taken together, the results of this study support the importance of conducting orthogonal assays in binding confirmation and compound selection and demonstrate the usefulness of FMO calculations in the rational design of RNA-targeted small molecules.

A CRISPR-based system to investigate HBV cccDNA biology.

Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.

Paper-Based Coculture Platform to Evaluate the Effects of Fibroblasts on Estrogen Signaling in ER+ Breast Cancers.

Cell-based assays enable molecular-level studies of cellular responses to drug candidates or potential toxins. Transactivation assays quantify the activation or inhibition of nuclear receptors, key transcriptional regulators of gene targets in mamalian cells. One such assay couples the expression of luciferase to the transcriptional activity of estrogen receptor-alpha (ERα). While this assay is regularly used to screen for agonists and antagonists of the estrogen signaling pathway, the setup relies on monolayer cultures in which cells are plated directly onto the surface of cell-compatible plasticware. The tumor microenvironment is more than a collection of cancerous cells and is profoundly influenced by tissue architecture, the presence of extracellular matrices, and intercellular signaling molecules produced by non-cancerous neighboring cells (e.g., fibroblasts). There exists a need for three-dimensional culture platforms that can be rapidly prototyped to assess new configurations and readily produced in the large numbers needed for translational studies and screening applications. Here, we demonstrate the utility of the paper-based culture platform to probe the effects of intercellular signaling between two cell types. We used paper scaffolds to generate tumor-like environments, forming a defined volume of breast cancer cells suspended in collagen. By placing the paper scaffolds in commercial 96-well plates, we compared monocultures of only breast cancer cells with coculture configurations containing fibroblasts in different locations that mimicked the stages of breast cancer progression. We show that ERα transactivation in the T47D-KBluc cell line is affected by the presence, number, and proximity of fibroblasts, and is a consequence of intercellular signaling molecules. After screening a small library of fibroblast-secreted signaling molecules, we showed that interleukin-6 (IL-6) was the primary driver of reduced estradiol sensitivity. These effects were mitigated in the coculture configurations by the addition of an IL-6 neutralizing antibody. We also assessed estrogen receptor expression and transcriptional regulation, further demonstrating the utility of the paper-based platform for detailed mechanistic studies.

Identification of small molecules that suppress cell invasion and metastasis promoted by WASF3 activation

The WASF3 gene promotes cancer cell invasion and metastasis, and genetic inactivation leads to suppression of metastasis. To identify small molecules that might interfere with WASF3 function, we performed an in silico docking study to the regulatory pocket of WASF3 using the National Cancer Institute (NCI) diversity set VI small molecule library. Compounds that showed the maximum likelihood of interaction with WASF3 were screened for their effect on cell movement in breast and prostate cancer cells, a well-established predictor of invasion and metastasis. Three hit compounds were identified that affected cell movement, and the same compounds also suppressed cell migration and invasion in vitro in both MDA-MB-231 breast cancer cells and Du145 prostate cancer cells. Using a zebrafish metastasis assay, one of these compounds, NSC670283, showed significant suppression of metastasis in vivo while not affecting cell proliferation. NSC670283 showed a consistent effect on suppression of invasion and metastasis, and cellular temperature shift assays provided support for physical interaction with WASF3. In addition, suppression of cell movement and invasion was accompanied by a decrease in actin filament polymerization. The data in this study suggest that these small molecules inhibit cancer cell invasion and metastasis, and to our knowledge, it is the first identification of a small molecule that can potentially inhibit WASF3-directed metastasis, laying the foundation for medicinal chemistry approaches to enhance the potency of the identified compounds.

In Silico Discovery and Investigation of Novel Small Molecules Targeting PSMA7 for the Treatment of GBM

The five-year survival rate for glioblastoma (GBM; WHO grade 4) remains only 5%, even with the current standard treatment of maximal safe surgical tumor resection followed by radiation treatment (RT) and concomitant and adjuvant temozolomide (TMZ). Therefore, alternative treatment therapies, such as blood-brain barrier (BBB)-permeable small molecule drugs, are needed to significantly improve patient outcomes by addressing other critical pathways in GBM progression and overcome GBM resistance to the standard treatment. Previously, we identified and validated proteasome subunit alpha type-7 (PSMA7), a subunit of the 20S proteasome, as a druggable, therapeutically vulnerable target in GBM by in vitro and in vivo methods. In this study, we performed in silico molecular docking screenings using a small molecule library to identify BBB-permeable small molecules predicted to bind to PSMA7 and investigated their potential as novel therapeutics for the treatment of GBM. In silico molecular docking screenings were performed using the National Cancer Institute Diversity Set VI small molecule library and the crystal structure of PSMA7 obtained from the native human 20S proteasome (RCSB PDB: 5LE5). Molecules were scored based on their predicted likelihood to bind to PSMA7. Top ranked molecules were screened using the SwissADME webtool, to select molecules predicted to be BBB-permeable and not a substrate for the permeability glycoprotein (P-gp), for testing with in vitro studies. Then, in vitro cell viability assays were used to determine IC50 values for molecules in human GBM PDX and normal human cell lines. The potential for molecules to sensitize GBM cells towards TMZ and/or RT was evaluated in vitro. In vitro proteasome activity assays were used to investigate whether molecules altered the three major protease activities performed by the proteasome. Computational virtual screenings using two docking algorithms (i.e., AutoDock Vina and Glide XP) identified and ranked the top small molecules most likely to bind to PSMA7. In silico screening using the SwissADME webtool predicted 99 of these molecules to be BBB-permeable and not a substrate for the P-gp. Subsequent in vitro cell viability studies determined eight of these molecules have lower IC50 values in human GBM PDX cells compared to normal human cells. Furthermore, we examined the potential of these eight molecules to sensitize GBM cells to TMZ and/or RT in vitro. Moreover, we found that three of these molecules inhibited all three major protease activities performed by the proteasome in GBM cells. Our study identified BBB-permeable small molecules predicted to target PSMA7 that demonstrated greater toxicity to GBM cells than normal cells and inhibited proteasome activities, suggesting a potential mechanism of action. Taken together, these small molecules are potential candidates for preclinical studies and may serve as novel targeted therapies for the treatment of GBM.

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.

Screening in serum-derived medium reveals differential response to compounds targeting metabolism

A challenge for screening new anticancer drugs is that efficacy in cell culture models is not always predictive of efficacy in patients. One limitation of standard cell culture is a reliance on non-physiological nutrient levels, which can influence cell metabolism and drug sensitivity. A general assessment of how physiological nutrients affect cancer cell response to small molecule therapies is lacking. To address this, we developed a serum-derived culture medium that supports the proliferation of diverse cancer cell lines and is amenable to high-throughput screening. We screened several small molecule libraries and found that compounds targeting metabolic enzymes were differentially effective in standard compared to serum-derived medium. We exploited the differences in nutrient levels between each medium to understand why medium conditions affected the response of cells to some compounds, illustrating how this approach can be used to screen potential therapeutics and understand how their efficacy is modified by available nutrients.

Deconstructive and Divergent Synthesis of Bioactive Natural Products.

Natural products play a key role in innovative drug discovery. To explore the potential application of natural products and their analogues in pharmacology, total synthesis is a key tool that provides natural product candidates and synthetic analogues for drug development and potential clinical trials. Deconstructive synthesis, namely building new, challenging structures through bond cleavage of easily accessible moieties, has emerged as a useful design principle in synthesizing bioactive natural products. Divergent synthesis, namely synthesizing many natural products from a common intermediate, can improve the efficiency of chemical synthesis and generate libraries of molecules with unprecedented structural diversity. In this review, we will firstly introduce five recent and excellent examples of deconstructive and divergent syntheses of natural products (2021-2023). Then, we will summarize our previous work on the deconstructive and divergent synthesis of natural products to demonstrate the high efficiency and simplicity of these two strategies in the field of total synthesis.

Abstract B020: A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma

Abstract TFE3 is a member of basic helix-loop-helix leucine zipper MiT transcription factor family and its chimeric proteins are associated with translocation renal cell carcinoma (tRCC). Despite the variety of genes fusions, most of TFE3 fusions partner genes are linked to spliceosome machinery. Dissecting the function of TFE3 fused to spliceosome machinery factors (TFE3-SF) could direct the development of effective therapies for this lethal disease, which is refractory to standard treatments for kidney cancer. Here, by using a combination of in silico structure prediction, molecular cloning, next generation transcriptomics, FRET technology, mutagenesis, proteomics, and high-throughput high-content screening (HTHCS) we interrogated a series of oncogenic mechanisms of TFE3-SF-containing fusions, including spontaneous nuclear translocation, nuclear paraspeckle occupancy, enhanced in vitro proliferation and in vivo growth, transcriptome remodeling, alternative splicing reprogramming, and novel dimer partner recruitment. Molecular inhibition of TFE3-SF dimerization reverses its oncogenic activity and represents a potential new target for therapeutic intervention. Using HTHCS combined with FRET technology we screened FDA approved drugs library (LOPAC) and small molecule library (Microsource) and identified hits compounds which inhibit TFE3-SF dimerization. Hits compounds were validated in 2D and 3D models utilizing patient derived xenolines expressing TFE3-SF. Ouabain and terfenadine demonstrated decreased cell proliferation reduced spheroid growth and in vivo tumor growth. Overall, our results unmask synthetic vulnerabilities of TFE3-SF dimerization for novel therapeutic strategies in patients with this aggressive type of kidney cancer. Citation Format: Ilaria Delle Fontane, Nuri Damayanti, Christopher Rupert, David E. Heppner, Roberto Pili. A mechanistic study of the TFE3-splicing machinery gene fusions reveals a new druggable target for translocation renal cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Advances in Kidney Cancer Research; 2023 Jun 24-27; Austin, Texas. Philadelphia (PA): AACR; Cancer Res 2023;83(16 Suppl):Abstract nr B020.

Rh-Catalyzed C–H Functionalization of the (Pyrazol-5-yl)pyridine Core of GBT-440

AbstractThe Rh-catalyzed cross dehydrogenative coupling (CDC)/alkylation of the pyrazolylpyridine unit of GBT-440 proceeded smoothly under ambient conditions and selectively on the pyrazole unit while directed by the pyridine. The scope of these reactions was established by employing simple as well as conjugated olefins for CDC and various diazo esters and the TIPS-EBX reagent for alkylation. At the outset, a focused small molecule library around the bis-heterocyclic core of GBT-440 was developed via C–H functionalization.

Abstract P2154: A Novel Allosteric Modulator Of The β 1 AR Identified By DNA-encoded Small Molecule Library Screening Demonstrates Unique Pharmacology And Function

While traditional β-blockers (i.e. competitive orthosteric antagonists of β-adrenergic receptors; βARs) are widely used as cardiovascular therapeutics, adverse effects such as fatigue and the nonselective inhibition of multiple receptor subtypes often limit maximal effectiveness. An emerging approach to enhance therapeutic targeting is to identify allosteric modulators that act cooperatively with orthosteric ligands. In contrast to orthosteric ligands which bind the endogenous ligand binding site, allosteric modulators bind to regions that are topographically distinct from the orthosteric pocket and can enhance (positive allosteric modulator; PAM) or reduce (negative allosteric modulator; NAM) the activities of orthosteric agonists/antagonists. Since allosteric regions exhibit greater sequence and structural diversity among receptor subtypes relative to the more highly conserved orthosteric pocket, allosteric modulators are more likely to be subtype specific and/or generate less adverse effects. We therefore embarked on a DNA-encoded small molecule library screen to identify novel allosteric modulators of the β 1 AR. Following multiple rounds of affinity selection using purified, functional, β 1 ARs reconstituted in lipid nanodiscs and HitGen’s OpenDEL® small molecule library containing more than 1 billion unique compounds, we identified Compound 11 (C11) as an allosteric modulator with unique pharmacological properties. Notably, C11 binds to the β 1 AR with micromolar affinity and enhances the binding affinity of orthosteric agonists and certain antagonists to the β 1 AR. In contrast to its positive cooperative effect on ligand binding, cell signaling assays showed C11 potently inhibits G protein and β-arrestin signaling downstream of the β 1 AR. Importantly, C11 showed high β 1 AR specificity with no effect on β 2 AR or AT 1 R signaling. These results suggest that C11 is a β 1 AR-specific PAM in terms of ligand binding but a NAM in terms of agonist efficacy, belonging to a largely under-characterized class of allosteric modulators termed PAM-antagonists. With an extremely unique pharmacological profile, C11 is a promising potential therapeutic and experiments evaluating its ability to modulate β 1 AR signaling in vivo are ongoing.