Drug Discovery Screening Research Articles

Studying the Collective Functional Response of a Receptor in Alchemical Ligand Binding Free Energy Simulations with Accelerated Solvation Layer Dynamics.

Ligand binding free energy simulations (LB-FES) that involve sampling of protein functional conformations have been longstanding challenges in research on molecular recognition. Particularly, modeling of the conformational transition pathway and design of the heuristic biasing mechanism are severe bottlenecks for the existing enhanced configurational sampling (ECS) methods. Inspired by the key role of hydration in regulating conformational dynamics of macromolecules, this report proposes a novel ECS approach that facilitates binding-associated structural dynamics by accelerated hydration transitions in combination with the λ-exchange of free energy perturbation (FEP). Two challenging protein-ligand binding processes involving large configurational transitions of the receptor are studied, with hydration transitions at binding sites accelerated by Hamiltonian-simulated annealing of the hydration layer. Without the need for pathway analysis or ad hoc barrier flattening potential, LB-FES were performed with FEP/λ-exchange molecular dynamics simulation at a minor overhead for annealing of the hydration layer. The LB-FES studies showed that the accelerated rehydration significantly enhances the collective conformational transitions of the receptor, and convergence of binding affinity calculations is obtained at a sweet-spot simulation time scale. Alchemical LB-FES with the proposed ECS strategy is free from the effort of trial and error for the setup and realizes efficient on-the-fly sampling for the collective functional response of the receptor and bound water and therefore presents a practical approach to high-throughput screening in drug discovery.

Abstract 6049: Cell-based PROTAC screening for cancer drug discovery

Abstract Proteolysis-Targeting Chimeras (PROTACs) have emerged as a promising strategy for selectively degrading specific target proteins, thereby offering a novel approach to precision cancer therapy. Among the most notable oncogenes, KRAS with the G12C mutation is responsible for 45%-50% of KRAS mutations in non-small cell lung carcinomas. Additionally, BRD4, a well-studied member of the Bromo- and Extra-Terminal (BET) protein family, is implicated in various hematological and solid tumors. The implementation of cell-based screening for PROTACs targeting either KRAS(G12C) or BRD4 represents a highly valuable platform for advancing cancer drug discovery. We utilized the NanoBRETTM Ternary Complex Assay, NanoBiTTM Assay, and Western Blot assay to evaluate the efficacy of PROTACs. The NanoBRETTM Ternary Complex Assay revealed that KRAS G12C degrader-1 and LC-2 effectively degrade KRAS(G12C), while dBET6 and MZ-1 demonstrated the ability to degrade BRD4. Notably, the NanoBiTTM assay showed that LC-2 dose-dependently degrades KRAS(G12C) in HEK293 cells co-transfected with KRAS(G12C)-LgBiT and KRAS(G12C)-SmBiT. Moreover, BET6 exhibited a dose-dependent degradation of endogenous BRD4 in HepG2 cells, and LC-2 demonstrated the degradation of endogenous KRAS(G12C) in MiaPaCa2 cells. In summary, the development of a robust cell-based assay platform for high-throughput screening of PROTACs marks a substantial advancement in cancer drug discovery. This approach not only enables the identification of promising PROTACs but also holds the potential for more effective and targeted interventions in the ongoing battle against cancer. Citation Format: Yong Wan, Haiqing Ma, Jianghong Wu. Cell-based PROTAC screening for cancer drug discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6049.

Abstract 6789: Development of an in vitro gastric cancer 3D co-culture platform using a microphysiological system (MPS) to improve outcomes in oncology clinical trials

Abstract Introduction: Despite significant advancements in scientific and diagnostic technologies, clinical trials for cancer often fail due to a lack of efficacy. Access to a platform that accurately predicts patient response is crucial to increase the success rate of clinical studies, and to minimize unnecessary exposure to ineffective and potentially harmful drug testing for patients. Because of the complexity of the disease, it would be beneficial to deploy patient samples for drug testing in a side-by-side comparison during the clinical trial. An accurate prediction of patient response earlier in the clinical study would aid decision making to determine the appropriate duration of treatment for each patient. Methods: We developed a 3D human cell co-culture model using our proprietary MPS technology, the Curio-spheroid Curiochip. Using patient samples isolated and expanded from either ascites fluids or biopsies to recapitulate the clinical phenotypes of advanced gastric cancer, we quantified the migratory properties of the patient cancer cells (i.e., the unique metastatic potential), and measured the level of secretion of VEGF. Somatic mutational analyses using NGS and RNAseq were performed for subtyping and clustering the samples. The patient cancer cells were expanded within 2 weeks post-collection, and used to form a spheroid that was loaded into a hydrogel embedded within the Curio-Spheroid, enabling the patient spheroids to interact through the hydrogel with neighboring (healthy) human endothelial cells. The patient spheroids induced aggressive vasculogenesis in the co-culture model, which was subdued by the anti-VEGFR2 antibody Ramucirumab. The platform was retrospectively validated with the Standard-of-Care (SOC) treatments that the patients received. Results: The patient cell testing took 3-4 weeks from sample collection to imaging. The patient cells preserved their migratory capacity, pro-angiogenic potential and drug responses in the vascularized model. The dose responses to SOC in the platform were consistent with the actual patient response assessments based on RECIST criteria, supporting the potential to use this platform to accurately predict patient clinical responses. Conclusions: The Curio-spheroid 3D co-culture model of vascularized gastric cancer provides a dynamic microenvironment to preserve the phenotypes of the patient cells and cell-cell interactions that are known to influence drug responses. This platform is compatible with high-throughput screening (HTS) for target or drug discovery with a good assay window. The ability to accurately recapitulate patient responses to SOC in the clinic make the Curio-Spheroid platform a promising method to test patient samples and measure drug responses of patients in real time during ongoing clinical trials. Citation Format: Yeongmin Choi, Sumin Kim, Youngsook Song, Yangji Kim, Jin Hawn Jang, Suryong Kim, Jihye Baek, Delaney Donnelly, Bushra Rajiput, Byungjun Lee, Woo Sun Kwon, Tsung-Li Liu, Kyusuk Baek, Sanghee Yoo, Sun Young Rha. Development of an in vitro gastric cancer 3D co-culture platform using a microphysiological system (MPS) to improve outcomes in oncology clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6789.

Abstract 4724: Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics

Abstract PRISM (Profiling Relative Inhibition Simultaneously in Mixtures), using DNA-barcoded cell lines cultured as ~25 cell line pools, has been an effective drug discovery screening platform to rapidly test large numbers of potential therapeutics on over 900 different cancer cell lines. However, the conventional 5-day assay may be too short to offer the resolution needed to identify the full cellular response for slower-acting therapeutics such as those directed against epigenetic pathways. Here, we describe development of the PRISM 10-day extended assay with ~500 adherent solid tumor cancer cell lines to overcome this challenge. We found that the ranges of cell line growth rates within the standard PRISM pools were too large and not amenable for a long-term assay beyond 6 days—after 10 days of growth, slower cell lines dropped below the limit of barcode detection as faster cell lines overtook the pools. We therefore regenerated pools to reduce the range of growth rates within a pool. We show that this re-pooling, coupled with seeding density optimization, enables recovery of > 98% of cell lines at day 10 in vehicle-treated wells, with acceptable data variability. The average estimated number of cell doublings across our cell lines, 7, is significantly greater than the number of doublings in the PRISM standard 5-day assay (~3 doublings). Importantly, we observe greater drug effects on day 10 versus day 6 when cells are treated with slow-acting DNA methyltransferase inhibitors such as decitabine; this also results in increased sensitivity to identify cellular biomarkers underlying differential response. In summary, we have successfully engineered a PRISM 10-day assay in ~500 adherent cell lines, where the extended duration can be beneficial in elucidating the effects and mechanism of action for epigenetic drug candidates. Citation Format: Melissa Ronan, Blanche C. Ip, Ellen Nguyen, Ursula Widocki, Tenzin Sangpo, Alvin Kalathungal, Nashielli Diaz-Gallegos, Anthony Fazio, Aydin Golabi, Maha Naim, Ashish Bino George, YuhJong Liu, Mustafa Kocak, Rishi Puram, Matthew G. Rees, Jennifer Roth. Optimizing 500 pooled PRISM cell lines for a 10-day assay to elucidate viability effects and mechanism of action for slower-acting therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4724.

Machine Learning Assisted Hit Prioritization for High Throughput Screening in Drug Discovery

Machine Learning Assisted Hit Prioritization for High Throughput Screening in Drug Discovery

Clustering of small molecules: new perspectives and their impact on natural product lead discovery

The clustering of small molecules is of considerable importance for computer-aided drug discovery and virtual screening applications. The structure of chemical data in appropriate subspaces of the chemical space is relevant to sample datasets in a representative manner, to generate small libraries with wide or narrow chemical coverage (depending on the specific goals), and to guide the selection of subsets of in silico hits that are submitted for experimental confirmation. In the field of natural products, identifying regions of the chemical space where bioactive compounds congregate and understanding the relationship between biosynthetic gene clusters and the molecular structure of secondary metabolites may have a direct impact on natural product discovery and engineering. Here, we briefly discuss general approximations and available resources for the clustering of small molecules, and how the clustering of small molecules can be boosted by the application of novel clustering approximations, namely subspace clustering and multi-view clustering, which represent opposite philosophies of the clustering paradigm. We present some specific applications of small molecule clustering in the field of natural products, and analyze how a chemogenomic perspective may be particularly embodied in the field of natural products.

Reporter cell lines to screen for inhibitors or regulators of the KRAS-RAF-MEK1/2-ERK1/2 pathway.

The RAS-regulated RAF-MEK1/2-ERK1/2 signalling pathway is activated in cancer due to mutations in RAS proteins (especially KRAS), BRAF, CRAF, MEK1 and MEK2. Whilst inhibitors of KRASG12C (lung adenocarcinoma) and BRAF and MEK1/2 (melanoma and colorectal cancer) are clinically approved, acquired resistance remains a problem. Consequently, the search for new inhibitors (especially of RAS proteins), new inhibitor modalities and regulators of this pathway, which may be new drug targets, continues and increasingly involves cell-based screens with small molecules or genetic screens such as RNAi, CRISPR or protein interference. Here we describe cell lines that exhibit doxycycline-dependent expression KRASG12V or BRAFV600E and harbour a stably integrated EGR1:EmGFP reporter gene that can be detected by flow cytometry, high-content microscopy or immunoblotting. KRASG12V or BRAFV600E-driven EmGFP expression is inhibited by MEK1/2 or ERK1/2 inhibitors (MEKi and ERKi). BRAFi inhibit BRAFV600E-driven EmGFP expression but enhance the response to KRASG12V, recapitulating paradoxical activation of wild type RAF proteins. In addition to small molecules, expression of iDab6, encoding a RAS-specific antibody fragment inhibited KRASG12V- but not BRAFV600E-driven EmGFP expression. Finally, substitution of EmGFP for a bacterial nitroreductase gene allowed KRASG12V or BRAFV600E to drive cell death in the presence of a pro-drug, which may allow selection of pathway inhibitors that promote survival. These cell lines should prove useful for cell-based screens to identify new regulators of KRAS- or BRAF-dependent ERK1/2 signalling (drug target discovery) as well as screening or triaging 'hits' from drug discovery screens.

An Enzyme-Cleavable Solubilizing-Tag Facilitates the Chemical Synthesis of Mirror-Image Proteins.

Mirror-image proteins (D-proteins) are useful in biomedical research for purposes such as mirror-image screening for D-peptide drug discovery, but the chemical synthesis of many D-proteins is often low yielding due to the poor solubility or aggregation of their constituent peptide segments. Here, we report a Lys-C protease-cleavable solubilizing tag and its use to synthesize difficult-to-obtain D-proteins. Our tag is easily installed onto multiple amino acids such as DLys, DSer, DThr, and/or the N-terminal amino acid of hydrophobic D-peptides, is impervious to various reaction conditions, such as peptide synthesis, ligation, desulfurization, and transition metal-mediated deprotection, and yet can be completely removed by Lys-C protease under denaturing conditions to give the desired D-protein. The efficacy and practicality of the new method were exemplified in the synthesis of two challenging D-proteins: D-enantiomers of programmed cell death protein 1 IgV domain and SARS-CoV-2 envelope protein, in high yield. This work demonstrates that the enzymatic cleavage of solubilizing tags under denaturing conditions is feasible, thus paving the way for the production of more D-proteins.

HIGH THROUGHPUT SCREENING IN DRUG DISCOVERY: PROBLEMS AND SOLUTIONS

World-wide introduction of high throughput screening (HTS) methods in drug discovery research did not result in the increased number of novel medications on the market. We discuss novel trends in drug discovery that came from the understanding that majority of diseases are multifactorial and that one enzyme has many protein substrates. Hence, new approaches are focused on development of drugs, which (1) trigger survival pathways to return the organism to homeostatic balance, and (2) inhibit enzymes modifying histones or transcription factors not at the active site, but by displacement of protein substrates from the enzyme complexes. A good example for both approaches comes from the development of activators of antioxidant defense. We analyze and illustrate problems of commonly used in vitro HTS assays, and briefl y discuss advantages and limitations of small animal models. The novel approaches are complementary to the standard HTS and do not substitute for testing in mammals. Development of transgenic reporter mice to monitor drug effects by means of in vivo imaging is extremely promising to select proper dosage and administration regimes for full-range PK studies.

A bioluminescent and homogeneous assay for monitoring GPCR-mediated cAMP modulation and PDE activity.

3',5'-Cyclic adenosine monophosphate (cAMP), the first identified second messenger, is implicated in diverse cellular processes involving cellular metabolism, cell proliferation and differentiation, apoptosis, and gene expression. cAMP is synthesized by adenylyl cyclase (AC), which converts ATP to cAMP upon activation of Gαs-protein coupled receptors (GPCRs) in most cases and hydrolyzed by cyclic nucleotide phosphodiesterases (PDEs) to 5'-AMP. Dysregulation of cAMP signaling is implicated in a wide range of pathophysiological conditions such as cardiovascular diseases, neurodegenerative and behavioral disorders, cancers, diabetes, obesity, cataracts, and others. Therefore, cAMP targeted therapies have been and are still undergoing intense investigation for the treatment of these and other diseases. This highlights the need for developing assays to detect and monitor cAMP levels. In this study, we show cAMP Lumit assay as a highly specific homogeneous bioluminescent assay suitable for high throughput screenings with a large assay window and a wide dynamic range for cAMP detection. We believe that this assay will aid and simplify drug discovery screening efforts for cAMP signaling targeted therapies.

Mycobacterial Targets for Thiourea Derivatives: Opportunities for Virtual Screening in Tuberculosis Drug Discovery.

Tuberculosis (TB) remains a primary global health concern, necessitating the discovery and development of new anti-TB drugs, mainly to combat drug-resistant strains. In this context, thiourea derivatives have emerged as promising candidates in TB drug discovery due to their diverse chemical structures and pharmacological properties. This review aimed to explore this potential, identifying and exploring molecular targets for thiourea derivatives in Mycobacterium tuberculosis (Mtb) and the potential application of virtual screening techniques in drug discovery. We have compiled a comprehensive list of possible molecular targets of thiourea derivatives in Mtb. The enzymes are primarily involved in the biosynthesis of various cell wall components, including mycolic acids, peptidoglycans, and arabinans, or targets in the branched-chain amino acid biosynthesis (BCAA) pathway and detoxification mechanisms. We discuss the potential of these targets as critical constituents for the design of novel anti-TB drugs. Besides, we highlight the opportunities that virtual screening methodologies present in identifying potential thiourea derivatives that can interact with these molecular targets. The presented findings contribute to the ongoing efforts in TB drug discovery and lay the foundation for further research in designing and developing more effective treatments against this devastating disease.

DMSO-Related Effects on Ligand-Binding Properties of Lysine Methyltransferases G9a and SETD8.

Being the standard solvent for preparing stock solutions of compounds for drug discovery, DMSO is always present in assay buffers in concentrations ranging from 0.1 % to 5 % (v/v). Even at the lowest concentrations, DMSO-containing solutions can have significant effects on individual proteins and possible pitfalls cannot be eliminated. Herein, we used two protein systems, the lysine methyltransferases G9a/KMT1 C and SETD8/KMT5 A, to study the effects of DMSO on protein stability and on the binding of the corresponding inhibitors, using different biophysical methods such as nano Differential Scanning Fluorimetry (nanoDSF), Differential Scanning Fluorimetry (DSF), microscale thermophoresis (MST), and surface plasmon resonance (SPR), all widely used in drug discovery screening campaigns. We demonstrated that the effects of DMSO are protein- and technique-dependent and cannot be predicted or extrapolated on the basis of previous studies using different proteins and/or different assays. Moreover, we showed that the application of orthogonal biophysical methods can lead to different binding affinity data, thus confirming the importance of using at least two different orthogonal assays in screening campaigns. This variability should be taken into account in the selection and characterization of hit compounds, in order to avoid data misinterpretation.

Molecular Characterization of the Recombinant Ig1 Axl Receptor Domain: An Intriguing Bait for Screening in Drug Discovery.

Axl receptor tyrosine kinase and its ligand Gas6 regulate several biological processes and are involved in both the onset and progression of tumor malignancies and autoimmune diseases. Based on its key role in these settings, Axl is considered a promising target for the development of molecules with therapeutic and diagnostic purposes. In this paper, we describe the molecular characterization of the recombinant Ig1 domain of Axl (Ig1 Axl) and its biochemical properties. For the first time, an exhaustive spectroscopic characterization of the recombinant protein through circular dichroism and fluorescence studies is also reported, as well as a binding analysis to its natural ligand Gas6, paving the way for the use of recombinant Ig1 Axl as a bait in drug discovery screening procedures aimed at the identification of novel and specific binders targeting the Axl receptor.

Photonic Crystal Surface Mode Real-Time Imaging of RAD51 DNA Repair Protein Interaction with the ssDNA Substrate.

Photonic crystals (PCs) are promising tools for label-free sensing in drug discovery screening, diagnostics, and analysis of ligand-receptor interactions. Imaging of PC surface modes has emerged as a novel approach to the detection of multiple binding events at the sensor surface. PC surface modification and decoration with recognition units yield an interface providing the highly sensitive detection of cancer biomarkers, antibodies, and oligonucleotides. The RAD51 protein plays a central role in DNA repair via the homologous recombination pathway. This recombinase is essential for the genome stability and its overexpression is often correlated with aggressive cancer. RAD51 is therefore a potential target in the therapeutic strategy for cancer. Here, we report the designing of a PC-based array sensor for real-time monitoring of oligonucleotide-RAD51 recruitment by means of surface mode imaging and validation of the concept of this approach. Our data demonstrate that the designed biosensor ensures the highly sensitive multiplexed analysis of association-dissociation events and detection of the biomarker of DNA damage using a microfluidic PC array. The obtained results highlight the potential of the developed technique for testing the functionality of candidate drugs, discovering new molecular targets and drug entities. This paves the way to further adaption and bioanalytical use of the biosensor for high-content screening to identify new DNA repair inhibitor drugs targeting the RAD51 nucleoprotein filament or to discover new molecular targets.

Advancements within Molecular Engineering for Regenerative Medicine and Biomedical Applications an Investigation Analysis towards A Computing Retrospective

The field of molecular engineering in medicine has witnessed remarkable progress in recent years, revolutionizing healthcare, diagnostics, and therapy development. However, the pandemic showcased there is still more requirement for progress along with further detailed investigation which is paramount and also a necessity moving forward. This research investigation delves into the interdisciplinary realm of molecular engineering, exploring its impact on regenerative medicine, biomaterials, tissue engineering, and the innovation from various advanced biotechnologies which has accelerated health science. The main objective for this research aims at providing an in depth investigative exploration of biomaterial applications with their respective roles within regenerative medicine and its associated advancements along with, tissue engineering, organ-on-a-chip device peripheral mechanics functionality and how bioprinting is paving the way for the creation of functional tissues and organs with a case study analysis on drug discovery, immune engineering, to the field of precision medicine, gene editing with the insight towards drug discovery processing, design and screening pipelined for biologics and the how therapeutics and drugs will play out in future healthcare. This exploration also provides many meaningful and remarkable conclusions on the advanced technologies which are explored and investigated throughout the step-by-step systematic technical computing methods approached for the research.

In Silico Screening of Leguminosae Phytochemicals as Potential Inhibitors of Lung Cancer: A Structure-Based Multi-Targeted Molecular Docking Analysis

About half of the chemotherapeutic drugs are naturally occurring anticancer compounds which are available in the market to date. In silico virtual screening for new anti-cancer drug discovery is a cost-effective method as compared to traditional drug synthesis. From Leguminosae family 144 phytochemicals which were screened from the Dictionary of Natural Products (DNP) undergo molecular docking in comparison to approved drugs Gemcitabine, osimertinib, and Nintedanib against targeted proteins EGFR, BRAF, and KRAS. Only five phytochemicals are 2-Pyrrolecarboxylate, O-(3, 5-Dihydroxy-4-methoxy benzoyl), ?-Aldotripiperideine, O8-(2-Pyrrolecarbonyl), and Benzoyl with high docking scores were selected for further studies based on ADMET lab. In this article, phytochemicals that can inhibit protein growth causing lung cancer were screened and selected by using molecular docking or ADME analysis. In silico ADME studies of these selected phytochemicals suggest that 2-Pyrrolecarboxylate, O-(3,5-Dihydroxy-4-methoxy benzoyl), and O8-(2-Pyrrolecarbonyl) are completely safe, non-toxic, non-carcinogenic, non GERG blocker, do not cause liver injury and have high excretion rate and can be taken for further in vivo and in vitro studies to discover new chemotherapy against lung cancer.  

GPU-optimized approaches to molecular docking-based virtual screening in drug discovery: A comparative analysis

Finding a novel drug is a very long and complex procedure. Using computer simulations, it is possible to accelerate the preliminary phases by performing a virtual screening that filters a large set of drug candidates to a manageable number. This paper presents the implementations and comparative analysis of two GPU-optimized implementations of a virtual screening algorithm targeting novel GPU architectures. This work focuses on the analysis of parallel computation patterns and their mapping onto the target architecture. The first method adopts a traditional approach that spreads the computation for a single molecule across the entire GPU. The second uses a novel batched approach that exploits the parallel architecture of the GPU to evaluate more molecules in parallel. Experimental results showed a different behavior depending on the size of the database to be screened, either reaching a performance plateau sooner or having a more extended initial transient period to achieve a higher throughput (up to 5x), which is more suitable for extreme-scale virtual screening campaigns.

T5S1607 identified as a antibacterial FtsZ inhibitor：Virtual screening combined with bioactivity evaluation for the drug discovery

Due to antibiotic overuse, many bacteria have developed resistance, creating an urgent need for novel antimicrobial agents. It has been established that the filamentous temperature-sensitive mutant Z (FtsZ) of the bacterial cell division protein is an effective and promising antibacterial target. In this study, the optimal proteins were assessed by early recognition ability and the processed compound libraries were virtually screened using Vina. This effort resulted in the identification of 14 potentially active antimicrobial compounds. Among them, the compound T5S1607 demonstrated remarkable antibacterial efficacy against Bacillus subtilis ATCC9732 (MIC = 1 μg/mL) and Staphylococcus aureus ATC5C6538 (MIC = 4 μg/mL). Furthermore, in vitro experiments demonstrated that the selected compound T5S1607 rapidly killed bacteria and induced FtsZ protein aggregation, preventing bacterial division and leading to bacterial death. Additionally, cell toxicity and hemolysis experiments indicate that compound T5S1607 exhibits minimal toxicity to LO2 cells and shows no significant hemolytic effects on mammalian cells in vitro at the MIC concentration range. All the results indicate that compound T5S1607 is a promising antibacterial agent and a potential FtsZ inhibitor. In conclusion, this work successfully discovered FtsZ inhibitors with good activity through the virtual screening drug discovery process.

Structure-based discovery of novel P-glycoprotein inhibitors targeting the nucleotide binding domains

P-glycoprotein (P-gp), a membrane transport protein overexpressed in certain drug-resistant cancer cells, has been the target of numerous drug discovery projects aimed at overcoming drug resistance in cancer. Most characterized P-gp inhibitors bind at the large hydrophobic drug binding domain (DBD), but none have yet attained regulatory approval. In this study, we explored the potential of designing inhibitors that target the nucleotide binding domains (NBDs), by computationally screening a large library of 2.6 billion synthesizable molecules, using a combination of machine learning-guided molecular docking and molecular dynamics (MD). 14 of the computationally best-scoring molecules were subsequently tested for their ability to inhibit P-gp mediated calcein-AM efflux. In total, five diverse compounds exhibited inhibitory effects in the calcein-AM assay without displaying toxicity. The activity of these compounds was confirmed by their ability to decrease the verapamil-stimulated ATPase activity of P-gp in a subsequent assay. The discovery of these five novel P-gp inhibitors demonstrates the potential of in-silico screening in drug discovery and provides a new stepping point towards future potent P-gp inhibitors.

ThermoBRET: A Ligand-Engagement Nanoscale Thermostability Assay Applied to GPCRs.

Measurements of membrane protein thermostability reflect ligand binding. Current thermostability assays often require protein purification or rely on pre-existing radiolabelled or fluorescent ligands, limiting their application to established targets. Alternative methods, such as fluorescence-detection size exclusion chromatography thermal shift, detect protein aggregation but are not amenable to high-throughput screening. Here, we present a ThermoBRET method to quantify the relative thermostability of G protein coupled receptors (GPCRs), using cannabinoid receptors (CB1 and CB2 ) and the β2 -adrenoceptor (β2 AR) as model systems. ThermoBRET reports receptor unfolding, does not need labelled ligands and can be used with non-purified proteins. It uses Bioluminescence Resonance Energy Transfer (BRET) between Nanoluciferase (Nluc) and a thiol-reactive fluorescent dye that binds cysteines exposed by unfolding. We demonstrate that the melting point (Tm ) of Nluc-fused GPCRs can be determined in non-purified detergent solubilised membrane preparations or solubilised whole cells, revealing differences in thermostability for different solubilising conditions and in the presence of stabilising ligands. We extended the range of the assay by developing the thermostable tsNLuc by incorporating mutations from the fragments of split-Nluc (Tm of 87 °C versus 59 °C). ThermoBRET allows the determination of GPCR thermostability, which is useful for protein purification optimisation and drug discovery screening.