To confirm target engagement of hits from our high-throughput screening efforts, we ran biophysical assays on several hundreds of hits from 15 different high-throughput screening campaigns. Analyzing the biophysical assay results from these screening campaigns led us to conclude that we could be more strategic in our biophysical analysis of hits by first confirming activity in a thermal shift assay (TSA) and then confirming activity in either a surface plasmon resonance (SPR) assay or a temperature-related intensity change (TRIC) assay. To understand how this new workflow shapes the quality of the final hits, we compared TSA/SPR or TSA/TRIC confirmed and unconfirmed hits to one another using four measures of compound quality: quantitative estimate of drug-likeness (QED), Pan-Assay Interference Compounds (PAINS), promiscuity, and aqueous solubility. In general, we found that the biophysically confirmed hits performed better in the compound quality metrics than the unconfirmed hits, demonstrating that our workflow not only confirmed target engagement of the hits but also enriched for higher quality hits.

Microphthalmia transcription factor (MITF) regulates melanocyte development and is the “lineage-specific survival” oncogene of melanoma. MITF is essential for melanoma initiation, progression, and relapse and has been considered an important therapeutic target; however, direct inhibition of MITF through small molecules is considered impossible, due to the absence of a ligand-binding pocket for drug design. Here, our structural analyses show that the structure of MITF is hyperdynamic because of its out-of-register leucine zipper with a 3-residue insertion. The dynamic MITF is highly vulnerable to dimer-disrupting mutations, as we observed that MITF loss-of-function mutations in human Waardenburg syndrome type 2 A are frequently located on the dimer interface and disrupt the dimer forming ability accordingly. These observations suggest a unique opportunity to inhibit MITF with small molecules capable of disrupting the MITF dimer. From a high throughput screening against 654,650 compounds, we discovered compound TT-012, which specifically binds to dynamic MITF and destroys the latter’s dimer formation and DNA-binding ability. Using chromatin immunoprecipitation assay and RNA sequencing, we showed that TT-012 inhibits the transcriptional activity of MITF in B16F10 melanoma cells. In addition, TT-012 inhibits the growth of high-MITF melanoma cells, and inhibits the tumor growth and metastasis with tolerable toxicity to liver and immune cells in animal models. Together, this study demonstrates a unique hyperdynamic dimer interface in melanoma oncoprotein MITF, and reveals a novel approach to therapeutically suppress MITF activity.

PDF file - 456K, Table S1: Growth inhibition of NCI-60 panel of cancer cell lines by L002 (NSC764414); Supplemental Figure 1: Distribution of hits in the primary potentiator screen ; Supplemental Figure 2: Hit distribution in the primary and counterscreen assays ; Supplemental Figure 3: Fluorescence-based in vitro p300 inhibition assay ; Supplemental Figure 4: Summary of the HTS campaign ; Supplemental Figure 5: L002 does not inhibit HDACs ; Supplemental Figure 6: L002 does not inhibit HMTs ; Supplemental Figure 7: Impacts of L002 and other hits on histone modifications

This special issue was conceived with the intent of highlighting the use of existing and progressive technologies in hit discovery and how these technologies may be combined to generate vital hits for drug discovery. These aspects now cross both small-molecule and large-molecule discovery in many organizations. In addition, the issue is intended to remind readers of the heritage of SLAS journals, and how they have supported the use and application of innovative hit identification approaches.Working with SLAS, we have compiled a list of the most downloaded articles from SLAS journals. All of the articles referenced below have been downloaded more than 4000 times. Many of these very nicely reflect the focus on new technology, its implementation, and the continued interest in how hit matter is identified, characterized, and progressed.It is very fitting that the article downloaded most frequently (more than 16,000 times) is one that describes the statistical definition of z-factor and how this can be used to define a high-throughput screening (HTS) window aiding the development of HTS assays.1Zhang J.H. Chung T.D. Oldenburg K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.J. Biomol. Screen. 1999; 4: 67-73Google Scholar Understanding your HTS assay is further highlighted by high-demand publications describing plate edge effects,2Lundholt B.K. Scudder K.M. Pagliaro L. A Simple Technique for Reducing Edge Effect in Cell-Based Assays.J. Biomol. Screen. 2003; 8: 566-570Google Scholar the relationship between % inhibition and IC50,3Gubler H. Schopfer U. Jacoby E. Theoretical and Experimental Relationships between Percent Inhibition and IC50 Data Observed in High-Throughput Screening.J. Biomol. Screen. 2013; 18: 1-13Google Scholar the time dependency of IC50,4Krippendorff B.F. Neuhaus R. Lienau P. et al.Mechanism-Based Inhibition: Deriving K(I) and k(inact) Directly from Time-Dependent IC(50) Values.J. Biomol. Screen. 2009; 14: 913-923Google Scholar and the effects of covalent and irreversible molecules on IC50 kinetics.5Strelow J.M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition.SLAS Discov. 2017; 22: 3-20Google Scholar In addition to achieving high-quality assays for hit identification, the data show how important it is to conserve the quality of hit matter. Two sample management publications describe the effects of freeze–thaw and managing compound storage in DMSO.6Kozikowski B.A. Burt T.M. Tirey D.A. et al.The Effect of Freeze/Thaw Cycles on the Stability of Compounds in DMSO.J. Biomol. Screen. 2003; 8: 210-215Google Scholar,7Waybright T.J. Britt J.R. McCloud T.G. Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives.J. Biomol. Screen. 2009; 14: 708-715Google ScholarThe list continues to illustrate many approaches that were new or had new implications at the time of the article coming to press. The impact of moving to more complex screening systems is highly represented, with six of the top 20 articles involving some form of screening approach or novel cellular assay system.8Forster J.I. Koglsberger S. Trefois C. et al.Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability.J. Biomol. Screen. 2016; 21: 496-509Google Scholar, 9Fang Y. Eglen R.M. Three-Dimensional Cell Cultures in Drug Discovery and Development.SLAS Discov. 2017; 22: 456-472Google Scholar, 10Ivascu A. Kubbies M. Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis.J. Biomol. Screen. 2006; 11: 922-932Google Scholar, 11Boehnke K. Iversen P.W. Schumacher D. et al.Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures.J. Biomol. Screen. 2016; 21: 931-941Google Scholar, 12Kunz-Schughart L.A. Freyer J.P. Hofstaedter F. et al.The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model.J. Biomol. Screen. 2004; 9: 273-285Google Scholar, 13Hou S. Tiriac H. Sridharan B.P. et al.Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening.SLAS Discov. 2018; 23: 574-584Google Scholar Finally, in this dataset, we can see how large molecules,14Razinkov V.I. Treuheit M.J. Becker G.W. Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities: Review of Methods and Tools.J. Biomol. Screen. 2015; 20: 468-483Google Scholar complex data analysis in combination screening,15Zhao W. Sachsenmeier K. Zhang L. et al.A New Bliss Independence Model to Analyze Drug Combination Data.J. Biomol. Screen. 2014; 19: 817-821Google Scholar as well as new technology16Pantoliano M.W. Petrella E.C. Kwasnoski J.D. et al.High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery.J. Biomol. Screen. 2001; 6: 429-440Google Scholar,17Shaw J. Dale I. Hemsley P. et al.Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1.SLAS Discov. 2019; 24: 121-132Google Scholar can advance hit identification approaches, and how the SLAS journals have constantly represented this section of the drug discovery industry.In this issue, we have tried to capture stories that exemplify how technologies have been applied successfully to find hits. These often require a combination of both new and existing technologies to identify the best-quality hits.Emery Smith et al.18Smith E. Dukovski D. Shumate J. et al.Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.SLAS Discov. 2021; 26: 204-214Google Scholar open up the special issue with a phenotypic assay approach; on the surface, the assay technology would not be out of place in any of the past 10 years of SLAS journals. This article, however, uses a well-established cellular approach in a membrane potential dye to rethink how this can be used to find new hits, generating a phenotypic assay with a novel approach looking for readthrough rescue of a response. It also nicely describes how, especially in cellular assays, a combination of assays in a hit triage is used and constructed to increase the confidence in hits.Similarly, Lorena Kallal et al.19Kallal L. Waszkiewicz A. Jaworski J.P. et al.High-Throughput Screening and Triage Assays Identify Small Molecules Targeting cMyc in Cancer Cells.SLAS Discov. 2021; 26: 215-228Google Scholar use several technologies that have been well used in HTS historically but combines them in a unique way to design both a successful screening assay and a powerful cascade. The article combines six different cellular assay systems to rule compounds in and out of project progression.Traditional HTS approaches remain extremely important for the mainstay of hit identification. The article from Saman Honarnejad et al.20Honarnejad S. van Boeckel S. van den Hurk H. et al.Hit Discovery for Public Target Programs in the European Lead Factory: Experiences and Output from Assay Development and Ultra-High-Throughput Screening.SLAS Discov. 2021; 26: 191-203Google Scholar reviewed European Lead Factory collaborative screening efforts that have developed significant experience in hit identification. In their article, they are able to describe a very wide array of screening technologies and successes.This brings us to newer approaches, as addressed in the technology review21McLaren D. Shah V. Wisniewski T. et al.High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives.SLAS Discov. 2021; 26: 167-190Google Scholar by David McLaren et al. An example article by Sahasrabuddhe et al.22Sahasrabuddhe A. Oakley D. Chen K. et al.Development of a High-Throughput Affinity Mass Spectrometry (AMS) Platform Using Laser Diode Thermal Desorption Ionization Coupled to Mass Spectrometry (LDTD-MS).SLAS Discov. 2021; 26: 229-240Google Scholar describes how mass spectrometry has established itself in recent years as a screening technology, capable of being applied as both a discrete screening assay and a multiplexed-affinity selection method.The next two articles reinforce the importance of compound quality and properties when developing small-molecule screening cascades. Wilson Shou et al.23Shou W.Z. Gerritz S.W. Harden D.H. et al.Rapid Compound Integrity Assessment for High-Throughput Screening Hit Triaging.SLAS Discov. 2021; 26: 241-246Google Scholar defined how proper high-throughput analysis ensured maintenance of compound integrity and determined the highest-quality molecules to be followed up for HTS. In their article, Gareth Davies et al.24Davies G. Semple H. McCandless M. et al.High-Throughput Mechanism of Inhibition.SLAS Discov. 2021; 26: 247-255Google Scholar shared a high-throughput approach to decipher the mechanism of action of compounds at enzyme targets.Alternative approaches to traditional HTS-based hit identification are covered in the last four article. Each challenges the way we see the future of hit identification. Gabriel Dreiman et al.25Dreiman G.H.S. Bictash M. Fish P.V. et al.Changing the HTS Paradigm: AI-Driven Iterative Screening for Hit Finding.SLAS Discov. 2021; 26: 256-261Google Scholar described how, with the increase in the capability of in silico approaches, iterative screening has gained favor, especially when reagents or resources are limited, such as with complex cell models. Timothy Foley et al.26Foley T. Burchett W. Chen Q. et al.Selecting Approaches for Hit Identification and Increasing Options by Building the Efficient Discovery of Actionable Chemical Matter from DNA-Encoded Libraries.SLAS Discov. 2021; 26: 262-279Google Scholar shared the history of DNA-encoded libraries within Pfizer, again indicating that often a combination of approaches to identify the best starting points is required. The authors also contrasted different hit identification paradigms. We are reminded that both complex cellular screening and large-molecule approaches now feature among the new paradigms of hit discovery. Hui Dou et al.27Dou H.H. Mallari R. Pipathsouk A. et al.An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics.SLAS Discov. 2021; 26: 280-290Google Scholar demonstrated this point with a nice example of a high-content imaging assay that combines branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by small interfering RNAs (siRNAs). And, finally, the possibility of not actually screening a compound collection but using knowledge-driven, phenotypic analysis is a very exciting proposition and a capability that is rapidly being established throughout the pharma and biotech industries. Johanna Nyffeler et al.28Nyffeler J. Haggard D.E. Willis C. et al.Comparison of Approaches for Determining Bioactivity Hits from High-Dimensional Profiling Data.SLAS Discov. 2021; 26: 291-307Google Scholar compared different approaches to determining bioactivity hits from high-dimensional profiling data and framed the concept that compound signatures can be identified and applied in drug discovery.Finally, we would like to thank everyone who has contributed to this special issue and hope that it serves as a useful, thought-provoking guide to the readers. This special issue was conceived with the intent of highlighting the use of existing and progressive technologies in hit discovery and how these technologies may be combined to generate vital hits for drug discovery. These aspects now cross both small-molecule and large-molecule discovery in many organizations. In addition, the issue is intended to remind readers of the heritage of SLAS journals, and how they have supported the use and application of innovative hit identification approaches. Working with SLAS, we have compiled a list of the most downloaded articles from SLAS journals. All of the articles referenced below have been downloaded more than 4000 times. Many of these very nicely reflect the focus on new technology, its implementation, and the continued interest in how hit matter is identified, characterized, and progressed. It is very fitting that the article downloaded most frequently (more than 16,000 times) is one that describes the statistical definition of z-factor and how this can be used to define a high-throughput screening (HTS) window aiding the development of HTS assays.1Zhang J.H. Chung T.D. Oldenburg K.R. A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.J. Biomol. Screen. 1999; 4: 67-73Google Scholar Understanding your HTS assay is further highlighted by high-demand publications describing plate edge effects,2Lundholt B.K. Scudder K.M. Pagliaro L. A Simple Technique for Reducing Edge Effect in Cell-Based Assays.J. Biomol. Screen. 2003; 8: 566-570Google Scholar the relationship between % inhibition and IC50,3Gubler H. Schopfer U. Jacoby E. Theoretical and Experimental Relationships between Percent Inhibition and IC50 Data Observed in High-Throughput Screening.J. Biomol. Screen. 2013; 18: 1-13Google Scholar the time dependency of IC50,4Krippendorff B.F. Neuhaus R. Lienau P. et al.Mechanism-Based Inhibition: Deriving K(I) and k(inact) Directly from Time-Dependent IC(50) Values.J. Biomol. Screen. 2009; 14: 913-923Google Scholar and the effects of covalent and irreversible molecules on IC50 kinetics.5Strelow J.M. A Perspective on the Kinetics of Covalent and Irreversible Inhibition.SLAS Discov. 2017; 22: 3-20Google Scholar In addition to achieving high-quality assays for hit identification, the data show how important it is to conserve the quality of hit matter. Two sample management publications describe the effects of freeze–thaw and managing compound storage in DMSO.6Kozikowski B.A. Burt T.M. Tirey D.A. et al.The Effect of Freeze/Thaw Cycles on the Stability of Compounds in DMSO.J. Biomol. Screen. 2003; 8: 210-215Google Scholar,7Waybright T.J. Britt J.R. McCloud T.G. Overcoming Problems of Compound Storage in DMSO: Solvent and Process Alternatives.J. Biomol. Screen. 2009; 14: 708-715Google Scholar The list continues to illustrate many approaches that were new or had new implications at the time of the article coming to press. The impact of moving to more complex screening systems is highly represented, with six of the top 20 articles involving some form of screening approach or novel cellular assay system.8Forster J.I. Koglsberger S. Trefois C. et al.Characterization of Differentiated SH-SY5Y as Neuronal Screening Model Reveals Increased Oxidative Vulnerability.J. Biomol. Screen. 2016; 21: 496-509Google Scholar, 9Fang Y. Eglen R.M. Three-Dimensional Cell Cultures in Drug Discovery and Development.SLAS Discov. 2017; 22: 456-472Google Scholar, 10Ivascu A. Kubbies M. Rapid Generation of Single-Tumor Spheroids for High-Throughput Cell Function and Toxicity Analysis.J. Biomol. Screen. 2006; 11: 922-932Google Scholar, 11Boehnke K. Iversen P.W. Schumacher D. et al.Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures.J. Biomol. Screen. 2016; 21: 931-941Google Scholar, 12Kunz-Schughart L.A. Freyer J.P. Hofstaedter F. et al.The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model.J. Biomol. Screen. 2004; 9: 273-285Google Scholar, 13Hou S. Tiriac H. Sridharan B.P. et al.Advanced Development of Primary Pancreatic Organoid Tumor Models for High-Throughput Phenotypic Drug Screening.SLAS Discov. 2018; 23: 574-584Google Scholar Finally, in this dataset, we can see how large molecules,14Razinkov V.I. Treuheit M.J. Becker G.W. Accelerated Formulation Development of Monoclonal Antibodies (mAbs) and mAb-Based Modalities: Review of Methods and Tools.J. Biomol. Screen. 2015; 20: 468-483Google Scholar complex data analysis in combination screening,15Zhao W. Sachsenmeier K. Zhang L. et al.A New Bliss Independence Model to Analyze Drug Combination Data.J. Biomol. Screen. 2014; 19: 817-821Google Scholar as well as new technology16Pantoliano M.W. Petrella E.C. Kwasnoski J.D. et al.High-Density Miniaturized Thermal Shift Assays as a General Strategy for Drug Discovery.J. Biomol. Screen. 2001; 6: 429-440Google Scholar,17Shaw J. Dale I. Hemsley P. et al.Positioning High-Throughput CETSA in Early Drug Discovery through Screening against B-Raf and PARP1.SLAS Discov. 2019; 24: 121-132Google Scholar can advance hit identification approaches, and how the SLAS journals have constantly represented this section of the drug discovery industry. In this issue, we have tried to capture stories that exemplify how technologies have been applied successfully to find hits. These often require a combination of both new and existing technologies to identify the best-quality hits. Emery Smith et al.18Smith E. Dukovski D. Shumate J. et al.Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.SLAS Discov. 2021; 26: 204-214Google Scholar open up the special issue with a phenotypic assay approach; on the surface, the assay technology would not be out of place in any of the past 10 years of SLAS journals. This article, however, uses a well-established cellular approach in a membrane potential dye to rethink how this can be used to find new hits, generating a phenotypic assay with a novel approach looking for readthrough rescue of a response. It also nicely describes how, especially in cellular assays, a combination of assays in a hit triage is used and constructed to increase the confidence in hits. Similarly, Lorena Kallal et al.19Kallal L. Waszkiewicz A. Jaworski J.P. et al.High-Throughput Screening and Triage Assays Identify Small Molecules Targeting cMyc in Cancer Cells.SLAS Discov. 2021; 26: 215-228Google Scholar use several technologies that have been well used in HTS historically but combines them in a unique way to design both a successful screening assay and a powerful cascade. The article combines six different cellular assay systems to rule compounds in and out of project progression. Traditional HTS approaches remain extremely important for the mainstay of hit identification. The article from Saman Honarnejad et al.20Honarnejad S. van Boeckel S. van den Hurk H. et al.Hit Discovery for Public Target Programs in the European Lead Factory: Experiences and Output from Assay Development and Ultra-High-Throughput Screening.SLAS Discov. 2021; 26: 191-203Google Scholar reviewed European Lead Factory collaborative screening efforts that have developed significant experience in hit identification. In their article, they are able to describe a very wide array of screening technologies and successes. This brings us to newer approaches, as addressed in the technology review21McLaren D. Shah V. Wisniewski T. et al.High-Throughput Mass Spectrometry for Hit Identification: Current Landscape and Future Perspectives.SLAS Discov. 2021; 26: 167-190Google Scholar by David McLaren et al. An example article by Sahasrabuddhe et al.22Sahasrabuddhe A. Oakley D. Chen K. et al.Development of a High-Throughput Affinity Mass Spectrometry (AMS) Platform Using Laser Diode Thermal Desorption Ionization Coupled to Mass Spectrometry (LDTD-MS).SLAS Discov. 2021; 26: 229-240Google Scholar describes how mass spectrometry has established itself in recent years as a screening technology, capable of being applied as both a discrete screening assay and a multiplexed-affinity selection method. The next two articles reinforce the importance of compound quality and properties when developing small-molecule screening cascades. Wilson Shou et al.23Shou W.Z. Gerritz S.W. Harden D.H. et al.Rapid Compound Integrity Assessment for High-Throughput Screening Hit Triaging.SLAS Discov. 2021; 26: 241-246Google Scholar defined how proper high-throughput analysis ensured maintenance of compound integrity and determined the highest-quality molecules to be followed up for HTS. In their article, Gareth Davies et al.24Davies G. Semple H. McCandless M. et al.High-Throughput Mechanism of Inhibition.SLAS Discov. 2021; 26: 247-255Google Scholar shared a high-throughput approach to decipher the mechanism of action of compounds at enzyme targets. Alternative approaches to traditional HTS-based hit identification are covered in the last four article. Each challenges the way we see the future of hit identification. Gabriel Dreiman et al.25Dreiman G.H.S. Bictash M. Fish P.V. et al.Changing the HTS Paradigm: AI-Driven Iterative Screening for Hit Finding.SLAS Discov. 2021; 26: 256-261Google Scholar described how, with the increase in the capability of in silico approaches, iterative screening has gained favor, especially when reagents or resources are limited, such as with complex cell models. Timothy Foley et al.26Foley T. Burchett W. Chen Q. et al.Selecting Approaches for Hit Identification and Increasing Options by Building the Efficient Discovery of Actionable Chemical Matter from DNA-Encoded Libraries.SLAS Discov. 2021; 26: 262-279Google Scholar shared the history of DNA-encoded libraries within Pfizer, again indicating that often a combination of approaches to identify the best starting points is required. The authors also contrasted different hit identification paradigms. We are reminded that both complex cellular screening and large-molecule approaches now feature among the new paradigms of hit discovery. Hui Dou et al.27Dou H.H. Mallari R. Pipathsouk A. et al.An Automated High-Throughput Fluorescence In Situ Hybridization (FISH) Assay Platform for Use in the Identification and Optimization of siRNA-Based Therapeutics.SLAS Discov. 2021; 26: 280-290Google Scholar demonstrated this point with a nice example of a high-content imaging assay that combines branched DNA (bDNA) technology with fluorescence in situ hybridization (FISH) to measure gene silencing by small interfering RNAs (siRNAs). And, finally, the possibility of not actually screening a compound collection but using knowledge-driven, phenotypic analysis is a very exciting proposition and a capability that is rapidly being established throughout the pharma and biotech industries. Johanna Nyffeler et al.28Nyffeler J. Haggard D.E. Willis C. et al.Comparison of Approaches for Determining Bioactivity Hits from High-Dimensional Profiling Data.SLAS Discov. 2021; 26: 291-307Google Scholar compared different approaches to determining bioactivity hits from high-dimensional profiling data and framed the concept that compound signatures can be identified and applied in drug discovery. Finally, we would like to thank everyone who has contributed to this special issue and hope that it serves as a useful, thought-provoking guide to the readers.

The orphan nuclear receptor COUP-TFII is expressed at a low level in adult tissues, but its expression is increased and shown to promote progression of multiple diseases, including prostate cancer, heart failure, and muscular dystrophy. Suppression of COUP-TFII slows disease progression, making it an intriguing therapeutic target. Here, we identified a potent and specific COUP-TFII inhibitor through high-throughput screening. The inhibitor specifically suppressed COUP-TFII activity to regulate its target genes. Mechanistically, the inhibitor directly bound to the COUP-TFII ligand-binding domain and disrupted COUP-TFII interaction with transcription regulators, including FOXA1, thus repressing COUP-TFII activity on target gene regulation. Through blocking COUP-TFII's oncogenic activity in prostate cancer, the inhibitor efficiently exerted a potent antitumor effect in xenograft mouse models and patient-derived xenograft models. Our study identified a potent and specific COUP-TFII inhibitor that may be useful for the treatment of prostate cancer and possibly other diseases.

GPR119 drug discovery efforts in the pharmaceutical industry for the treatment of type 2 diabetes mellitus (T2DM) and obesity, were initiated based on its restricted distribution in pancreas and GI tract, and its possible role in glucose homeostasis. While a number of lead series have emerged, the pharmacological endpoints they provide have not been clear. In particular, many lead series have demonstrated loss of efficacy and significant toxic side effects. Thus, we sought to identify novel, potent, positive modulators of GPR119. In this study, we have successfully developed and optimized a high-throughput screening strategy to identify GPR119 modulators using a live cell assay format that utilizes a cyclic nucleotide-gated channel as a biosensor for cAMP production. Our high-throughput screening (HTS) approach is unique to that of previous HTS approaches targeting this receptor, as changes in cAMP were measured both in the presence and absence of an EC10 of the endogenous ligand, oleoylethanolamide, enabling detection of both agonists and potential allosteric modulators in a single assay. From these efforts, we have identified positive modulators of GPR119 with similar as well as unique scaffolds compared to existing compounds and similar as well as unique signaling properties. Our compounds will not only serve as novel molecular probes to better understand GPR119 pleiotropic signaling and the underlying physiological consequences of receptor activation, but are also well-suited for translation as potential therapeutic agents.

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.

There is a pressing need to improve approaches for drug discovery related to neuropsychiatric disorders (NSDs). Therapeutic discovery in neuropsychiatric disorders would benefit from screening assays that can measure changes in complex phenotypes linked to disease mechanisms. However, traditional assays that track complex neuronal phenotypes, such as neuronal connectivity, exhibit poor scalability and are not compatible with high-throughput screening (HTS) procedures. Therefore, we created a neuronal phenotypic assay platform that focused on improving the scalability and affordability of neuron-based assays capable of tracking disease-relevant phenotypes. First, using inexpensive laboratory-level automation, we industrialized primary neuronal culture production, which enabled the creation of scalable assays within functioning neural networks. We then developed a panel of phenotypic assays based on culturing of primary neurons from genetically modified mice expressing HTS-compatible reporters that capture disease-relevant phenotypes. We demonstrated that a library of 1,280 compounds was quickly screened against both assays using only a few litters of mice in a typical academic laboratory setting. Finally, we implemented one assay in a fully automated high-throughput academic screening facility, illustrating the scalability of assays designed using this platform. These methodological improvements simplify the creation of highly scalable neuron-based phenotypic assays designed to improve drug discovery in CNS disorders.

This Letter describes the chemical optimization of a novel series of M4 positive allosteric modulators (PAMs) based on a 5,6-dimethyl-4-(piperidin-1-yl)thieno[2,3-d]pyrimidine core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent and selective, but not CNS penetrant. Potency was maintained, while CNS penetration was improved (rat brain:plasma Kp=0.74), within the original core after several rounds of optimization; however, the thieno[2,3-d]pyrimidine core was subject to extensive oxidative metabolism. Ultimately, we identified a 6-fluoroquinazoline core replacement that afforded good M4 PAM potency, muscarinic receptor subtype selectivity and CNS penetration (rat brain:plasma Kp>10). Moreover, this campaign provided fundamentally distinct M4 PAM chemotypes, greatly expanding the available structural diversity for this exciting CNS target.

There is interest in developing inhibitors of human group III secreted phospholipase A2 (hGIII-sPLA2) because this enzyme plays a role in mast cell maturation. There are no potent inhibitors for hGIII-sPLA2 reported to date, so we adapted a fluorescence-based enzyme activity monitoring method to a high-throughput screening format. We opted to use an assay based on phospholipid substrate present in phospholipid vesicles since this matrix more closely resembles the natural substrate of hGIII-sPLA2, as opposed to phospholipid/detergent mixed micelles. The substrate is a phospholipid analogue containing BODIPY fluorophores dispersed as a minor component in vesicles of nonfluorescent phospholipids. Action of hGIII-sPLA2 liberates a free fatty acid from the phospholipid, leading to a reduction in quenching of the fluorophore and hence an increase in fluorescence. The assay uses optical detection in a 1536-well plate format with an excitation wavelength far away from the UV range so as to minimize false-positive library hits that result from quenching of the fluorescence. The high-throughput screen was successfully carried out on a library of 370,276 small molecules. Several hits were discovered, and data have been uploaded to PubChem. This study describes the first high-throughput optical screening assay for secreted phospholipase A2 inhibitors based on a phospholipid vesicle substrate.

Microphthalmia transcription factor (MITF) is a master transcription factor expressed in melanocytes, essential for melanocyte survival, differentiation, and pigment formation, and is a key oncogenic factor in melanoma initiation, migration, and treatment resistance. Although identified as an important therapeutic target for melanoma, clinical inhibitors directly targeting the MITF protein are not available. Based on the functional state of MITF, we have designed an MITF dimerization-based AlphaScreen (MIDAS) assay that sensitively and specifically mirrors the dimerization of MITF in vitro. This assay is further exploited for identification of the MITF dimer disruptor for high-throughput screening. A pilot screen against a library of 1280 pharmacologically active compounds indicates that the MIDAS assay performance exhibits exceptional results with a Z' factor of 0.81 and a signal-to-background (S/B) ratio of 3.92 while identifying initial hit compounds that yield an ability to disrupt MITF-DNA interaction. The results presented demonstrate that the MIDAS assay is ready to screen large chemical libraries in order to discover novel modulators of MITF for potential melanoma treatment.

Although there has been substantial success in the development of specific inhibitors for protein kinases, little progress has been made in the identification of specific inhibitors for their protein phosphatase counterparts. Inhibitors of PP1 and PP5 are desired as probes for research and to test their potential for drug development. We developed and miniaturized (1536-well plate format) nearly identical homogeneous, fluorescence intensity (FLINT) enzymatic assays to detect inhibitors of PP1 or PP5. The assays were used in an ultra-high-throughput screening (uHTS) campaign, testing >315,000 small-molecule compounds. Both assays demonstrated robust performance, with a Z′ of 0.92 ± 0.03 and 0.95 ± 0.01 for the PP1 and PP5 assays, respectively. Screening the same library with both assays aided the identification of class inhibitors and assay artifacts. Confirmation screening and hit prioritization assays used [32P/33P]-radiolabel protein substrates, revealing excellent agreement between the FLINT and radiolabel assays. This screening campaign led to the discovery of four novel unrelated small-molecule inhibitors of PP1 and ~30 related small-molecule inhibitors of PP5. The results suggest that this uHTS approach is suitable for identifying selective chemical probes that inhibit PP1 or PP5 activity, and it is likely that similar assays can be developed for other PPP-family phosphatases.

This Letter describes the chemical optimization of a novel series of M4 PAMs based on a non-enolizable ketone core, identified from an MLPCN functional high-throughput screen. The HTS hit was potent, selective and CNS penetrant; however, the compound was highly cleared in vitro and in vivo. SAR provided analogs for which M4 PAM potency and CNS exposure were maintained; yet, clearance remained high. Metabolite identification studies demonstrated that this series was subject to rapid, and near quantitative, reductive metabolism to the corresponding secondary alcohol metabolite that was devoid of M4 PAM activity.

Imbalances in endoplasmic reticulum (ER) proteostasis are associated with etiologically-diverse degenerative diseases linked to excessive extracellular protein misfolding and aggregation. Reprogramming of the ER proteostasis environment through genetic activation of the Unfolded Protein Response (UPR)-associated transcription factor ATF6 attenuates secretion and extracellular aggregation of amyloidogenic proteins. Here, we employed a screening approach that included complementary arm-specific UPR reporters and medium-throughput transcriptional profiling to identify non-toxic small molecules that phenocopy the ATF6-mediated reprogramming of the ER proteostasis environment. The ER reprogramming afforded by our molecules requires activation of endogenous ATF6 and occurs independent of global ER stress. Furthermore, our molecules phenocopy the ability of genetic ATF6 activation to selectively reduce secretion and extracellular aggregation of amyloidogenic proteins. These results show that small molecule-dependent ER reprogramming, achieved through preferential activation of the ATF6 transcriptional program, is a promising strategy to ameliorate imbalances in ER function associated with degenerative protein aggregation diseases.

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

Improved therapies for the treatment of Trypanosoma brucei, the etiological agent of the neglected tropical disease human African trypanosomiasis, are urgently needed. We targeted T. brucei methionyl-tRNA synthetase (MetRS), an aminoacyl-tRNA synthase (aaRS), which is considered an important drug target due to its role in protein synthesis, cell survival, and its significant differences in structure from its mammalian ortholog. Previous work using RNA interference of MetRS demonstrated growth inhibition of T. brucei, further validating it as an attractive target. We report the development and implementation of two orthogonal high-throughput screening assays to identify inhibitors of T. brucei MetRS. First, a chemiluminescence assay was implemented in a 1536-well plate format and used to monitor adenosine triphosphate depletion during the aminoacylation reaction. Hit confirmation then used a counterscreen in which adenosine monophosphate production was assessed using fluorescence polarization technology. In addition, a miniaturized cell viability assay was used to triage cytotoxic compounds. Finally, lower throughput assays involving whole parasite growth inhibition of both human and parasite MetRS were used to analyze compound selectivity and efficacy. The outcome of this high-throughput screening campaign has led to the discovery of 19 potent and selective T. brucei MetRS inhibitors.

Muscarinic acetylcholine receptors (mAChRs) have long been viewed as viable targets for novel therapeutic agents for the treatment of Alzheimer’s disease and other disorders involving impaired cognitive function. In an attempt to identify orthosteric and allosteric modulators of the muscarinic acetylcholine receptor M4 (M4), we developed a homogenous, multiparametric, 1536-well assay to measure M4 receptor agonism, positive allosteric modulation (PAM), and antagonism in a single well. This assay yielded a Z′ of 0.85 ± 0.05 in the agonist, 0.72 ± 0.07 in PAM, and 0.80 ± 0.06 in the antagonist mode. Parallel screening of the M1 and M5 subtypes using the same multiparametric assay format revealed chemotypes that demonstrate selectivity and/or promiscuity between assays and modalities. This identified 503 M4 selective primary agonists, 1450 PAMs, and 2389 antagonist hits. Concentration-response analysis identified 25 selective agonists, 4 PAMs, and 41 antagonists. This demonstrates the advantages of this approach to rapidly identify selective receptor modulators while efficiently removing assay artifacts and undesirable compounds.

A recently completed functional, high throughput screen of the Molecular Libraries Probe Production Centers Network (MLPCN) screening deck of ∼360,000 compounds conducted by SRIMSC against three of the five muscarinic receptor subtypes (M1, M4 and M5) provided a number of interesting hits. As this was the first time a directed effort had been undertaken to indentify M5 selective leads, we were very pleased by the identification of nine M5 PAMs and nine M5 antagonists, despite the complete absence of M5 agonist hits. The most promising M5 PAM hit (CID 17516658), being particularly attractive due to the absence of the isatin core shared by all previous M5 PAMs, was quickly developed into a potent, selective and CNS penetrant probe molecule ML380 (hM5 PAM EC50 = 190 nM, hM5 ACh fold-shift = 9.3, rM5 PAM EC50 = 610 nM).