Hit Validation Research Articles

Discovery and evaluation of novel SHIP-1 inhibitors

Src Homology 2-containing Inositol 5′-Phosphatase-1 (SHIP-1), encoded by INPP5D, has been identified as an Alzheimer’s disease (AD) risk-associated gene through recent genetic and epigenetic studies. SHIP-1 confers AD risk by inhibiting the TREM2 cascade and reducing beneficial microglial cellular processes, including phagocytosis. While several small molecules have been reported to modulate SHIP-1 activity, their limited selectivity and efficacy in advanced models restricted their potential as therapeutic agents or probes for biological studies. Herein, we validated and implemented a high-throughput screening platform to explore new chemotypes that can modulate the phosphatase activity of SHIP-1. We screened 49,260 central nervous system (CNS)-penetrate compounds sourced from commercial vendors using the malachite green-based assay for anti-SHIP-1 activity. Through analysis, prioritization, and validation of the screening hits, we identified three novel types of scaffolds that inhibit the SHIP-1 phosphatase activity with IC50s as low as 46.6 µM. To improve the inhibitory activity of these promising hits, we carried out structure–activity relationship (SAR) studies, resulting in a lead molecule SP3-12 that inhibits SHIP-1 with an IC50 value of 6.1 μM. Kinetic analyses of SP3-12 revealed that its inhibition mechanism is competitive, with a Ki value of 3.2 µM for SHIP-1 and a 7-fold selectivity over SHIP-2. Furthermore, results from testing in a microglial phagocytosis/cell health high content assay indicated that SP3-12 could effectively activate phagocytosis in human microglial clone 3 (HMC3) cells, with an EC50 of 2.0 µM, without cytotoxicity in the dose range. Given its potency, selectivity, and cellular activity, SP3-12 emerges as a promising small molecule inhibitor with potential for investigating the biological functions of SHIP-1.

Identification of Potent ADCK3 Inhibitors through Structure-Based Virtual Screening.

ADCK3 is a member of the UbiB family of atypical protein kinases in humans, with homologues in archaea, bacteria, and eukaryotes. In lieu of protein kinase activity, ADCK3 plays a role in the biosynthesis of coenzyme Q10 (CoQ10), and inactivating mutations can cause a CoQ10 deficiency and ataxia. However, the exact functions of ADCK3 are still unclear, and small-molecule inhibitors could be useful as chemical probes to elucidate its molecular mechanisms. In this study, we applied structure-based virtual screening (VS) to discover a novel chemical series of ADCK3 inhibitors. Through extensive structural analysis of the active-site residues, we developed a pharmacophore model and applied it to a large-scale VS. Out of ∼170,000 compounds virtually screened, 800 top-ranking candidate compounds were selected and tested in both ADCK3 and p38 biochemical assays for hit validation. In total, 129 compounds were confirmed as ADCK3 inhibitors, and among them, 114 compounds are selective against p38, which was used as a counter-target. Molecular dynamics (MD) simulations were then conducted to predict the binding modes of the most potent compounds within the ADCK3 active site. Through metadynamics analysis, we successfully detected the key amino acid residues that govern intermolecular interactions. The findings provided in this study can serve as a promising starting point for drug development.

Ligand-Based Virtual Screening for Discovery of Indole Derivatives as Potent DNA Gyrase ATPase Inhibitors Active against Mycobacterium tuberculosis and Hit Validation by Biological Assays.

Mycobacterium tuberculosis is the single most important global infectious disease killer and a World Health Organization critical priority pathogen for development of new antimicrobials. M. tuberculosis DNA gyrase is a validated target for anti-TB agents, but those in current use target DNA breakage-reunion, rather than the ATPase activity of the GyrB subunit. Here, virtual screening, subsequently validated by whole-cell and enzyme inhibition assays, was applied to identify candidate compounds that inhibit M. tuberculosis GyrB ATPase activity from the Specs compound library. This approach yielded six compounds: four carbazole derivatives (1, 2, 3, and 8), the benzoindole derivative 11, and the indole derivative 14. Carbazole derivatives can be considered a new scaffold for M. tuberculosis DNA gyrase ATPase inhibitors. IC50 values of compounds 8, 11, and 14 (0.26, 0.56, and 0.08 μM, respectively) for inhibition of M. tuberculosis DNA gyrase ATPase activity are 5-fold, 2-fold, and 16-fold better than the known DNA gyrase ATPase inhibitor novobiocin. MIC values of these compounds against growth of M. tuberculosis H37Ra are 25.0, 3.1, and 6.2 μg/mL, respectively, superior to novobiocin (MIC > 100.0 μg/mL). Molecular dynamics simulations of models of docked GyrB:inhibitor complexes suggest that hydrogen bond interactions with GyrB Asp79 are crucial for high-affinity binding of compounds 8, 11, and 14 to M. tuberculosis GyrB for inhibition of ATPase activity. These data demonstrate that virtual screening can identify known and new scaffolds that inhibit both M. tuberculosis DNA gyrase ATPase activity in vitro and growth of M. tuberculosis bacteria.

Permutational Encoding Strategy Accelerates HIT Validation from Single-Stranded DNA-Encoded Libraries.

DNA-Encoded Libraries (DELs) allow the parallel screening of millions of compounds for various applications, including de novo discovery or affinity maturation campaigns. However, library construction and HIT resynthesis can be cumbersome, especially when library members present an unknown stereochemistry. We introduce a permutational encoding strategy suitable for the construction of highly pure single-stranded single-pharmacophore DELs, designed to distinguish isomers at the sequencing level (e.g., stereoisomers, regio-isomers, and peptide sequences). This approach was validated by synthesizing a mock 921,600-member 4-amino-proline single-stranded DEL ("DEL1"). While screening DEL1 against different targets, high-throughput sequencing results showed selective enrichment of the most potent stereoisomers, with enrichment factors that outperform conventional encoding strategies. The versatility of our methodology was additionally validated by encoding 24 scaffolds derived from different permutations of the amino acid sequence of a previously described cyclic peptide targeting Fibroblast Activation Protein (FAP-2286). The resulting library ("DEL2") was interrogated against human FAP, showing selective enrichment of five cyclic peptides. We observed a direct correlation between enrichment factors and on-DNA binding affinities. The presented encoding methodology accelerates drug discovery by facilitating library synthesis and streamlining HIT resynthesis while enhancing enrichment factors at the DEL sequencing level. This facilitates the identification of HIT candidates prior to medicinal chemistry and affinity maturation campaigns.

Identification of Human TRIAC Transmembrane Transporters.

Background: 3,5,3'-Triiodothyroacetic acid (TRIAC) is a T3-receptor agonist pharmacologically used in patients to mitigate T3 resistance. It is additionally explored to treat some symptoms of patients with inactivating mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8, SLC16A2). MCT8 is expressed along the blood-brain barrier, on neurons, astrocytes, and oligodendrocytes. Hence, pathogenic variants in MCT8 limit the access of TH into and their functions within the brain. TRIAC was shown to enter the brain independently of MCT8 and to modulate expression of TH-dependent genes. The aim of the study was to identify transporters that facilitate TRIAC uptake into cells. Methods: We performed a whole-genome RNAi screen in HepG2 cells stably expressing a T3-receptor-dependent luciferase reporter gene. Validation of hits from the primary and confirmatory secondary screen involved a counter screen with siRNAs and compared the cellular response to TRIAC to the effect of T3, in order to exclude siRNAs targeting the gene expression machinery. MDCK1 cells were stably transfected with cDNA encoding C-terminally myc-tagged versions of the identified TRIAC-preferring transporters. Several individual clones were selected after immunocytochemical characterization for biochemical characterization of their 125I-TRIAC transport activities. Results: We identified SLC22A9 and SLC29A2 as transporters mediating cellular uptake of TRIAC. SLC22A9 encodes the organic anion transporter 7 (OAT7), a sodium-independent organic anion transporter expressed in the plasma membrane in brain, pituitary, liver, and other organs. Competition with the SLC22A9/OAT7 substrate estrone-3-sulfate reduced 125I-TRIAC uptake. SLC29A2 encodes the equilibrative nucleoside transporter 2 (ENT2), which is ubiquitously expressed, including pituitary and brain. Coincubation with the SLC29A2/ENT2 inhibitor nitrobenzyl-6-thioinosine reduced 125I-TRIAC uptake. Moreover, ABCD1, an ATP-dependent peroxisomal pump, was identified as a 125I-TRIAC exporter in transfected MDCK1 cells. Conclusions: Knowledge of TRIAC transporter expression patterns, also during brain development, may thus in the future help to interpret observations on TRIAC effects, as well as understand why TRIAC may not show a desirable effect on cells or organs not expressing appropriate transporters. The identification of ABCD1 highlights the sensitivity of our established screening assay, but it may not hold significant relevance for patients undergoing TRIAC treatment.

Phenotypic drug discovery: a case for thymosin alpha-1.

Phenotypic drug discovery (PDD) involves screening compounds for their effects on cells, tissues, or whole organisms without necessarily understanding the underlying molecular targets. PDD differs from target-based strategies as it does not require knowledge of a specific drug target or its role in the disease. This approach can lead to the discovery of drugs with unexpected therapeutic effects or applications and allows for the identification of drugs based on their functional effects, rather than through a predefined target-based approach. Ultimately, disease definitions are mostly symptom-based rather than mechanism-based, and the therapeutics should be likewise. In recent years, there has been a renewed interest in PDD due to its potential to address the complexity of human diseases, including the holistic picture of multiple metabolites engaging with multiple targets constituting the central hub of the metabolic host-microbe interactions. Although PDD presents challenges such as hit validation and target deconvolution, significant achievements have been reached in the era of big data. This article explores the experiences of researchers testing the effect of a thymic peptide hormone, thymosin alpha-1, in preclinical and clinical settings and discuss how its therapeutic utility in the precision medicine era can be accommodated within the PDD framework.

Abstract 3225: Profiling the DNA regulome to discover direct inhibitors of the brachyury transcription factor

Abstract Background: The human genome encodes more than 1,600 transcription factors (TFs), along with additional cofactors, chromatin regulators, and structural proteins that collectively execute the regulatory instructions encoded within the nuclear DNA. Dysfunctions of these proteins, collectively known as the Regulome, are known to drive multiple conditions such as cancer, autoimmune conditions, and fibrosis. Despite their importance, many of these proteins are considered undruggable due to challenges in modeling their activity in vitro. Methods: We have addressed these shortcomings by implementing a live-cell Regulome profiling approach that quantifies the global activity of TFs without the need for cell manipulation or genome engineering, called TF-Scan. The technology coupled rapid, automated purification of the DNA-bound proteome coupled to scalable Data Independent Acquisition (DIA) quantitative mass spectrometry proteomics and machine learning. Results: We completed a screen of covalent compounds across 8 tissue types to discover hotspot sites on TFs that control their DNA-binding activity. A chordoma cell line was included in this screen. The screen identified compounds capable of binding to and disrupting the activity of several previously undruggable transcription factors, including brachyury. Hit validation and hit-to-lead medicinal chemistry resulted in a covalent chemical probe for brachyury, with activity in cell line models of chordoma. Conclusion: Phenotypic drug discovery in live, unmodified human cells with systematic regulome profiling enables the discovery of compounds that disrupt previously undruggable targets by multiple mechanisms of action. Citation Format: Alexander Joel Federation, Yang Gao, Brian McEllin, Tonibelle Gatbonton-Schwager, Andrea Gutierrez, Julia Robbins, Bodhi Hueffmeier, Erin Broderick, Daniele Canzani, William Fondrie, Lindsay Pino. Profiling the DNA regulome to discover direct inhibitors of the brachyury transcription factor [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 3225.

Abstract 4662: High-throughput screening identifies potential combinatorial therapies to overcome lenvatinib resistance in liver cancer

Abstract Liver cancer has seen an increase in mortality rate over recent decades due to limited targeted therapies and growth in specific etiologies. Lenvatinib, a multi-TKI inhibitor, is used as a first-line therapy for liver cancer patients who cannot receive immunotherapy, which is a component of the current standard of care. Unfortunately, lenvatinib efficacy is short. Understanding why liver cancers become resistant and identifying interventions to restore sensitivity can help prolong the clinical efficacy of lenvatinib. A panel of 20 liver cancer lines was screened to identify four lenvatinib-sensitive lines. These lines were subsequently conditioned to grow under increasing lenvatinib concentrations to generate lenvatinib-resistant (LR) models. One such model, Hep3B LR, exhibited a stable resistance to lenvatinib for up to 12 weeks in culture. Hep3B LR showed cross-resistance to selective inhibitors of FGFR4 and FGFR1 but not of MAPK14 and PDGFR, suggesting that the resistance phenotype may result from FGFR dysfunction. Unlike parental Hep3B, Hep3B LR exhibited a 3.4X increase in migration. Western blot detected an increase in pAKT, a survival marker. Additionally, proteins associated with cancer drug resistance were higher in Hep3B LR cells: Notch receptors and ligands, pStat3, and Axl. To identify means of overcoming lenvatinib resistance in Hep3B LR, a Cell-Titer Glo high-throughput assay was developed to measure the growth inhibition of candidate drugs in the presence and absence of lenvatinib. Preliminary results from the first 24 drugs screened reproduced known drug sensitivities associated with lenvatinib resistance (i.e., MEK/ERK, EGFR, GPX4 inhibition) but also identified novel sensitivities to inhibition of PKMYT1 or γ-secretase, the latter a cleavage enzyme involved in Notch signaling activation. Validation of these candidate hits using a grid-based titration of both drugs showed additive growth inhibition of lenvatinib in combination with ERK, EGFR, or GPX4 inhibitors but not with PKMYT1 or γ-secretase inhibitors in resistant cells. Interestingly, EGFRi and lenvatinib were substantially synergistic in parental cells, suggesting that an upfront combination of these inhibitors could potentially be used before the emergence of lenvatinib resistance in liver cancer. We have established a lenvatinib-resistant liver cancer cell model and validated a drug screening assay to identify potential combinatorial therapies to overcome lenvatinib resistance in liver cancer. Citation Format: Kevin Chau, Martina S. McDermott, KeWei Gong, Dennis J. Slamon. High-throughput screening identifies potential combinatorial therapies to overcome lenvatinib resistance in liver cancer [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 4662.

Abstract 3107: SMASH: Single molecule antibody screening with high-throughput imaging system

Abstract Larger, more diverse antibody libraries are being screened in recent years. Despite enabling the high-throughput screening of extensive libraries, the process for identification and characterization of antibodies remains laborious and time-intensive. Here we report a platform that not only compiles hit finding and validation processes, but also provides high-resolution binding affinity as well as rank order among the library being screened. We streamlined the library construction and affinity screening via microscale transient expression in HEK293T cells with designed antibody sequence and direct quantification of antibody-antigen binding affinity which reduced the timeline from multiple weeks to a single week. We constructed the single-chain fragment antigen binding (scFab) libraries by replacing the duplex DNA fragment in CDR of interest while preserving the human framework of Fab. We have demonstrated that the SPID technique allows to detect antibody-antigen interactions from just dozens of pg of scFabs in total. This allowed us to bypass the conventional DNA cloning or even PCR amplification to achieve the minimum amount of DNA plasmids for expressing scFabs to be analyzed such as BLI and SPR. Therefore, we assembled the DNA plasmids through in vitro ligation with high efficiency and directly introduced it to HEK293T in 96-well microplate with microscale (200 ul) for transient transfection, which only takes three days for library generation. Next, we utilized SPID to quantify the number of single molecules between free and antigen-bound fractions of scFab, resulting in occupancy at the antibody-antigen binding equilibrium. This technique facilitated the determination of binding affinity (KD) for 200 antibody-antigen pairs within a 6-hour TAT encompassing both measurement and analysis. In total, we utilized 4,000 times less antibody for characterization while achieving enhanced sensitivity compared to both BLI and SPR methods. As a proof-of-concept, we generated a library consisting of 720 variants of Trastuzumab in scFab format to probe the landscape of CDRs affecting affinities against HER2 showing nM to low uM dynamic range of KD. Out of 720 analyzed variants, 5% were deleterious, 38% provided substantial changes, 40% showed little/no change, 17% enhanced affinity to HER2. To validate the robustness of the platform, we extensively expanded the library screening, assessing over 2,000 antibody variants for their affinity against more than three different therapeutic targets. To conclude, we demonstrated an efficient approach for monitoring changes in affinity resulting from single mutations in scFab. This rapid screening of site-saturated libraries would lead to a combinational variant library that our platform can screen. Ultimately, our platform aims to identify potential candidates for therapeutic antibody development throughput the phases of antibody discovery, maturation, and engineering. Citation Format: Jihye Jo, Changju Chun, Byeong-Kwon Sohn, Booyoung Yu, Jiyu Lee. SMASH: Single molecule antibody screening with high-throughput imaging system [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 3107.

Elder (Sambucus nigra), identified by high-content screening, counteracts foam cell formation without promoting hepatic lipogenesis

Cholesterol deposition in intimal macrophages leads to foam cell formation and atherosclerosis. Reverse cholesterol transport (RCT), initiated by efflux of excess cholesterol from foam cells, counteracts atherosclerosis. However, targeting RCT by enhancing cholesterol efflux was so far accompanied by adverse hepatic lipogenesis. Here, we aimed to identify novel natural enhancers of macrophage cholesterol efflux suitable for the prevention of atherosclerosis. Plant extracts of an open-access library were screened for their capacity to increase cholesterol efflux in RAW264.7 macrophages trace-labeled with fluorescent BODIPY-cholesterol. Incremental functional validation of hits yielded two final extracts, elder (Sambucus nigra) and bitter orange (Citrus aurantium L.) that induced ATP binding cassette transporter A1 (ABCA1) expression and reduced cholesteryl ester accumulation in aggregated LDL-induced foam cells. Aqueous elder extracts were subsequently prepared in-house and both, flower and leaf extracts increased ABCA1 mRNA and protein expression in human THP-1 macrophages, while lipogenic gene expression in hepatocyte-derived cells was not induced. Chlorogenic acid isomers and the quercetin glycoside rutin were identified as the main polyphenols in elder extracts with putative biological action. In summary, elder flower and leaf extracts increase macrophage ABCA1 expression and reduce foam cell formation without adversely affecting hepatic lipogenesis.

Asymmetry of Dual-Display DNA-Encoded Chemical Libraries.

While dual-display DNA-encoded chemical libraries (DELs) are increasingly employed for ligand discovery, some of their fundamental properties have not yet been studied in-depth. Aided with fluorescence polarization experiments, we demonstrate that dual-display DELs are intrinsically asymmetrical entities, and we deduce practical guidelines to perform better-informed on-DNA hit validation from these libraries.

An arrayed CRISPR knockout screen identifies genetic regulators of GLUT1 expression

Glucose, a primary fuel source under homeostatic conditions, is transported into cells by membrane transporters such as glucose transporter 1 (GLUT1). Due to its essential role in maintaining energy homeostasis, dysregulation of GLUT1 expression and function can adversely affect many physiological processes in the body. This has implications in a wide range of disorders such as Alzheimer’s disease (AD) and several types of cancers. However, the regulatory pathways that govern GLUT1 expression, which may be altered in these diseases, are poorly characterized. To gain insight into GLUT1 regulation, we performed an arrayed CRISPR knockout screen using Caco-2 cells as a model cell line. Using an automated high content immunostaining approach to quantify GLUT1 expression, we identified more than 300 genes whose removal led to GLUT1 downregulation. Many of these genes were enriched along signaling pathways associated with G-protein coupled receptors, particularly the rhodopsin-like family. Secondary hit validation confirmed that removal of select genes, or modulation of the activity of a corresponding protein, yielded changes in GLUT1 expression. Overall, this work provides a resource and framework for understanding GLUT1 regulation in health and disease.

In Vivo CRISPR Screening Platform for Studying Myeloid Inflammation and Clonal Dynamics

Previous experimental approaches using CRISPR to study factors governing hematopoiesis in vivo have been hampered by heterogeneity in cellular expansion during hematopoietic reconstitution. To control for such ‘cellular jackpotting’, we developed an in vivo hematopoietic stem and progenitor (HSPC)-based CRISPR screening system with unique molecular identifiers (UMIs) and applied these techniques to study myeloid inflammation and clonal expansion of Tet2-mutated HSPCs. Transplantation of Cas9-expressing Lin -Sca1 +c-Kit + (LSK) cells transduced with high-diversity UMI-labeled guide-RNAs (gRNAs) enabled sufficient gRNA representation at a genome-wide level after lentiviral integration of one gRNA per cell and controlled for 10 log-fold differences in cellular expansion during hematopoietic reconstitution. Simultaneous comparison of barcoded CRISPR screening with non-barcoded screening revealed that barcoding decreases false-positive biological processes related to cell division while improving sensitivity for detecting biological processes related to transcriptional events and cellular differentiation. To study inflammatory mediators, we applied this CRISPR screening tool towards studying the recruitment of CD101 +Ly6G +CD11b + mature neutrophils during an inflammatory insult, which revealed the majority of genes involved in human congenital neutropenia (Figure A). To demonstrate the reproducibility of this screening system, we performed a targeted transcription factor (TF)-based CRISPR screen, which revealed concordance between the genome-wide screen and the targeted screen. Next, we also applied this screening system to studying dynamics of Tet2-mutated clonal hematopoiesis. To study master regulators of clonal expansion in Tet2 KO, we performed a UMI-based CRISPR screen using chromatic and transcription factors, which revealed Tet3 and Bcor, genes previously identified to cooperate with Tet2 KO to promote myeloproliferation (Figure B). Validation of additional novel genetic hits is currently ongoing. We hypothesize that this screening platform will be useful for dissecting many aspects of hematopoiesis including inflammatory mediators and clonal dynamics of HSPCs.

Genome-Wide CRISPR/Cas9 Knock-out Screen Identifies Apoptosis-Relevant Genes in Multiple Myeloma

Background Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β family and have several biological functions in different cells and tissues. In multiple myeloma, BMPs enable growth arrest and apoptosis of the cancer cells. BMPs act as ligands and signal by binding a tetrameric complex of type I and type II receptors, leading to activation of SMAD1/5/8 transcription factors which allocate into the cell nucleus. The main type I receptor in myeloma cells is ALK2, encoded by the ACVR1 gene. Several BMP ligands can signal via ALK2, but the downstream signaling and mechanisms leading to apoptosis and growth arrest remain unknown. Mapping of this cascade could result in potential novel targets for treatment and therapies. Methods A genome-wide CRISPR/Cas9 knock-out screen with the GeCKO v2 library was performed in INA-6 myeloma cells. To induce apoptosis and growth arrest two treatments were performed in parallel: BMP9 and a combination of Activin B and FK506 (tacrolimus), compared with medium control. Both BMP9 and Activin B signal via the type I receptor ALK2 combined with one or more of the type II receptors ACVR2A, ACVR2B or BMPR2. Top hits were defined as genes over- or underrepresented compared with control in both treatment conditions with a p-value lower than 0.05. Reactome pathway analysis of the top hits from the screen was performed. Validation of the top ranking hits was performed by generating single-gene specific knockouts, silencing with siRNA or with inhibitors. CellTiter Glo viability assay and annexin V/propidium iodide staining analyzed by flow cytometry were used to determine apoptosis. RT-PCR and western blots were used to determine specific mRNA and protein abundance. Results The screen identified genes already known to be necessary in the BMP signaling pathway, such as ACVR1 and SMAD1 indicating technical robustness. Although the screen was designed to pick genes necessary for apoptosis and growth arrest, we were also able to identify genes that counteract BMP-induced apoptosis. Several novel genes/hits were found in the screen. Numerous well-established signaling cascades were identified from Reactome pathway analysis to be activated downstream of BMP mediated signaling. In particular, several genes belonging to the PI3K/AKT/mTOR cascade were significantly overrepresented in the screen, indicating they positively regulate ALK2-mediated apoptosis. The PI3K/AKT/mTOR pathway is known to be upregulated in several cancers, including multiple myeloma. Transcriptional regulators of TP53 were found to be significantly underrepresented, indicating negative regulation in ALK2-mediated apoptosis. The protein encoded by the TP53 gene is well known from several malignancies, including multiple myeloma. Additionally, 9 other genes with previously unknown apoptosis function were found to be relevant for its regulation. Conclusion In conclusion, BMP-induced apoptosis in multiple myeloma cells rely on the canonical BMP signaling pathway, but also on a number of previously unknown genes. The screen confirmed the importance of the PI3K/AKT/mTOR pathway for positive apoptosis regulation and transcriptional factors affecting TP53 for negative apoptosis regulation. Hits from this screen can be promising targets for future therapies and precision medicine targeting the apoptotic machinery in multiple myeloma cells.

A Multi-Modal Analysis of Acquired Resistance to Acalabrutinib and Pirtobrutinib Provides Potential Strategies to Augment Treatment Outcome with BTKi Drugs

Introduction: Acalabrutinib (acala), a covalent BTK inhibitor (BTKi), exhibits excellent efficacy and safety in CLL and other B cell malignancies. However, long term efficacy is often compromised by the acquisition of mutations at the acala-binding residue C481, the most prevalent being C481S. Pirtobrutinib (pirto), a reversible BTKi, is active clinically in patients who have progressed on a covalent BTKi with or without C481S mutations. Besides BTK mutations, several additional pathways are hypothesized to contribute to BTKi resistance and may be highly relevant to the sequencing and combining of CLL-directed therapies. To understand acquired resistance to both acala and pirto, we employed multiple approaches, including a genome-wide CRISPR knockout (KO) screen and the generation of BTK mutant drug resistant cell lines. Methods: Genome-wide CRISPR KO screen was performed in the BTKi-sensitive CD79B-MYD88 double mutant ABC-DLBCL cell line TMD8 with 3 guides/gene (best Vienna BioScore). Pooled library-transduced cells at 750x target coverage were cultured for 14 days with sublethal acala or pirto concentrations to achieve ~60% growth inhibition over DMSO. NGS/BAGAL/MAGeCK analyses identified enriched/depleted guides and corresponding resistance/sensitizing genes (FDR<0.1). Hits were validated by single gene KO (Cas9 RNP + 3-plex gRNA nucleofection) and 10-day growth assays with BTKi treatment. BTK mutant cell lines resistant to acala or pirto were derived in TMD8 and the BTKi-sensitive Karpas1718 MZL cell line by culturing through dose escalation. Exome/RNAseq was used to identify BTK mutations. TMD8 BTK C481R cell line was generated by CRISPR-HDR. BTK kinase and pathway activities were assayed by Western/Flow. Results: CRISPR screening results correlated highly between acala and pirto (Pearson's r=0.66), indicating shared pathways that impact response to both BTKi's. Collectively we identified 133 resistance and 57 sensitizing genes that span both known pathways (BCR/NFκB signaling) and novel modulators (autophagy, g-secretase, PBAF complex) of BTKi response. Systematic hit validation by single gene KO confirmed resistance phenotype for 6 genes: TNFAIP3 and TRAF3 negative regulators of canonical/non-canonical NFκB pathways; GRB2 adaptor protein for BCR signaling; PSEN1 in γ-secretase, PHF10 in PBAF complex; and SPOP substrate recognition protein for ubiquitin ligase. Interrogating the Project GENIE patient database identified loss-of-function mutations in these genes (8.6% CLL, 19.3% MCL, 25% MZL), supporting the hypothesis that they may be biomarkers for resistance to both BTKis. Sensitizing genes included CBL adaptor protein, PTPRCAP a CD45 tyrosine phosphatase, and RRAS2 small GTPase; KO of these genes did not alter TMD8 cell growth but enhanced the potency of both BTKis. Genomic analysis of BTKi-adaptive resistant cell lines identified causal BTK mutations, including nonclassical binding site mutation C481F for acala resistance and 2 novel mutations F442L and L460S for pirto resistance. C481F and C481R are kinase-inactive but cells still retain BCR signaling, pointing to BTK scaffolding activity. HCK is a proposed mediator of BTK scaffolding activity (Dhami et al., 2022; Yuan et al., 2022). However, while HCK activation was detected in C481R cells, HCK KO did not impair cell growth. In addition, no HCK activation was detected in C481F cells. Thus, scaffolding activity by C481F/R mutants does not depend on HCK. For the 2 pirto-resistant BTK mutations, F442L is kinase active but has reduced response to pirto, whereas L460S is kinase impaired. All our BTK mutant cell lines were still sensitive to the other BTKi, supporting a rationale for treatment sequencing between BTKi drugs to overcome resistance due to BTK mutations. Conclusions: We uncovered known and novel BTK resistance mutations and demonstrated BTK scaffolding activity independent of HCK, highlighting the need for other strategies to disrupt scaffolding-mediated BCR signaling. Beyond BTK mutations, our CRISPR KO screens illustrate a map of genetic modifiers of BTKi response and point to several potential resistance biomarkers for both acala and pirto. A better understanding of resistance mechanisms in the presence and absence of BTK mutations will help augment the use of BTKi treatments in patients.

Improved Antibacterial Activity of 1,3,4-Oxadiazole-Based Compounds That Restrict Staphylococcus aureus Growth Independent of LtaS Function.

The lipoteichoic acid (LTA) biosynthesis pathway has emerged as a promising antimicrobial therapeutic target. Previous studies identified the 1,3,4 oxadiazole compound 1771 as an LTA inhibitor with activity against Gram-positive pathogens. We have succeeded in making six 1771 derivatives and, through subsequent hit validation, identified the incorporation of a pentafluorosulfanyl substituent as central in enhancing activity. Our newly described derivative, compound 13, showed a 16- to 32-fold increase in activity compared to 1771 when tested against a cohort of multidrug-resistant Staphylococcus aureus strains while simultaneously exhibiting an improved toxicity profile against mammalian cells. Molecular techniques were employed in which the assumed target, lipoteichoic acid synthase (LtaS), was both deleted and overexpressed. Neither deletion nor overexpression of LtaS altered 1771 or compound 13 susceptibility; however, overexpression of LtaS increased the MIC of Congo red, a previously identified LtaS inhibitor. These data were further supported by comparing the docking poses of 1771 and derivatives in the LtaS active site, which indicated the possibility of an additional target(s). Finally, we show that both 1771 and compound 13 have activity that is independent of LtaS, extending to cover Gram-negative species if the outer membrane is first permeabilized, challenging the classification that these compounds are strict LtaS inhibitors.

Robust Strategy for Hit-to-Lead Discovery: NMR for SAR.

Establishing robust structure-activity relationships (SARs) is key to successful drug discovery campaigns, yet it often remains elusive due to screening and hit validation artifacts (false positives and false negatives), which frequently result in unproductive downstream expenditures of time and resources. To address this issue, we developed an integrative biophysics-driven strategy that expedites hit-to-lead discovery, mitigates false positives/negatives and common hit validation errors, and provides a robust approach to obtaining accurate binding and affinity measurements. The advantage of this method is that it vastly improves the clarity and reproducibility for affinity-driven SAR by monitoring and eliminating confounding factors. We demonstrate the ease at which high-quality micromolar binders can be generated from the initial millimolar fragment screening hits against an "undruggable" protein target, HRas.

Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery. However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

High-throughput screening of BAM inhibitors in native membrane environment

The outer membrane insertase of Gram-negative bacteria, BAM, is a key target for urgently needed novel antibiotics. Functional reconstitutions of BAM have so far been limited to synthetic membranes and with low throughput capacity for inhibitor screening. Here, we describe a BAM functional assay in native membrane environment capable of high-throughput screening. This is achieved by employing outer membrane vesicles (OMVs) to present BAM directly in native membranes. Refolding of the model substrate OmpT by BAM was possible from the chaperones SurA and Skp, with the required SurA concentration three times higher than Skp. In the OMVs, the antibiotic darobactin had a tenfold higher potency than in synthetic membranes, highlighting the need for native conditions in antibiotics development. The assay is successfully miniaturized for 1536-well plates and upscaled using large scale fermentation, resulting in high-throughput capacities to screen large commercial compound libraries. Our OMV-based assay thus lays the basis for discovery, hit validation and lead expansion of antibiotics targeting BAM.

Whole genome CRISPR screening strategy to identify genes contributing to SARS-CoV-2 spike and VSV-G mediated entry.

Understanding the cellular host factors that promote and inhibit viral entry is important for identifying viral countermeasures. CRISPR whole-genome screens can be used to rapidly discover host factors that contribute to or impair viral entry. However, when using live viruses and cellular lethality for selection, these screens can identify an overwhelming number of genes without specificity for the stage of the viral infection cycle. New screening methods are needed to identify host machinery contributing to specific steps of viral infection. Here, we developed a CRISPR whole-genome screen and counter-screen strategy based on a pseudoviral platform that allowed identification of genes specific to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike and vesicular stomatitis virus glycoprotein (VSV-G) mediated entry.Screening of SARS-CoV-2 spike and VSV-G on the same lentiviral pseudovirus allowed the identification of entry-specific genes relative to genes associated with retro-transcription, integration, and reporter expression from the lentiviral pseudovirus. Second, a Cre-Gag fusion protein packaged into the pseudovirus was used to bypass retro-transcription and integration by directly activating a floxed fluorescent protein reporter upon entry reduced the number of gene hits and increase specificity for viral entry.Our approach correctly identified SARS-CoV-2 and VSV-G receptors ACE2 and low-density lipoprotein receptors, respectively, and distinguished genes associated with retroviral reporter expression from envelope-mediated entry. Moreover, the CRE-Gag fusion/flox reporter increased the screen specificity for viral entry-associated genes. Validation of a few hits demonstrates that this approach distinguishes envelope-specific host factors from genes affecting reporter expression.Overall, this approach provides a new strategy for identifying host genes influencing viral entry without the confounding complexity of live-viral screens which produce long gene lists associated with all aspects of viral pathogenesis and replication.