Jonathan A. Lee

Modern Phenotypic Drug Discovery Is a Viable, Neoclassic Pharma Strategy

Strategy Jonathan A. Lee,*,† Mark T. Uhlik,‡ Christopher M. Moxham, Dirk Tomandl, and Daniel J. Sall Departments of †Quantitative Biology, ‡Cancer-Angiogenesis, Discovery Informatics, and Discovery Chemistry Research and Technologies, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana ImClone Systems, a Wholly Owned Subsidiary of Eli Lilly and Company, New York, New York ■ INTRODUCTION The pharmaceutical industry (Pharma) is currently facing unprecedented challenges. In addition to strategic patent expirations, the rate of drug launches has been essentially constant for 60 years with overall productivity falling since the 1970s. Similarly, the appearance of novel targets to FDA approved drugs, a measure of industry innovation, has not dramatically improved since the 1980s. Although Pharma productivity is a multifaceted problem, detailed analyses of comprehensive, industry-wide data indicate that late stage clinical failures are a major contributor that has been attributed to poor target validation (TV) and the lack of predictive biomarkers that translate to the clinic. In the spirit of “reinventing innovation” Pharma needs to introspectively identify areas for improvement. This communication considers how choices in drug screening strategies may relate to target validation issues and influence the probability of identifying novel medicines. Target-directed drug discovery (TDD) and phenotypic drug discovery (PDD) are Pharma strategies that have roots in distinct but complementary philosophies. Advantages of genespecific, reductionist approaches include the formulation and testing of specific molecular hypotheses. TDD approaches utilize advances in automation, biochemistry, structural biology, and chemistry related technologies to provide efficient and high capacity testing of unprecedented numbers of compounds and molecular targets. In addition, deep mining of cDNA expressed sequence tags (ESTs) and subsequently entire genomes led to the discovery of thousands of unknown genes and the potential for deep insight into novel drug target biology. Taken together, these advances in science and technology, in conjunction with the innate human desire to seek new challenges, contributed to the rapid adoption of molecular-reductionist views of biology. In contrast, PDD tests compounds in complex biological systems and monitors physiological responses with minimal assumptions concerning the participation of specific molecular targets and/or signaling pathways. Analyses of new molecular entities (NMEs) approved by the FDA between 1999 and 2008 indicate that for first in class molecules, 37% resulted from projects that used phenotypic screening whereas target based screening identified 23%; moreover, the discovery rate of PDD NMEs was greater than TDD NMEs and was invariant over the 9-year study period. Since significantly more TDD efforts were conducted during this period, the overall launch rate for first in class drugs underestimates the intrinsic probability of technical success (pTS) of classic PDD. A hybrid of classic phenotypic and target-directed strategies, which blends the use of physiologically relevant biological systems with the high throughput and statistical robustness of modern assay technologies, may have a higher pTS for launching first in class drugs than either classic PDD or TDD. Academia has utilized modern phenotypic approaches to study cell cycle, stem cell renewal, cell migration, metastasis, and induction of pluripotent stem cells. However, such “neoclassic” PDD approaches are not widely used in Pharma because of concerns about assay performance, statistical robustness, perceived difficulties in establishing compound structure−activity relationships (SARs), anticipated limited applicability of chemoinformatics tools, and the difficulty/ requirement for elucidating a molecular drug target. The results outlined in this presentation address commonly perceived issues related to the use of complex biological systems for modern lead generation. Our data, using an angiogenesis assay incorporating a coculture of primary human endothelial and stromal progenitor cells, indicate that phenotypic assays can be statistically robust, can be used to identify novel compound scaffolds by chemoinformatics enabled hit expansion, and can provide evidence of compound structure−activity relationships. Identification of novel molecular targets important to angiogenesis, acetyl Co-A carboxylase (ACC) and a protein related to cellular β-secretase (β-sec) activity, demonstrates that PDD provides a means to test multiple biologically relevant pathways in a target agnostic fashion. These attributes of PDD enabled the discovery of chemical scaffolds that were readily differentiated, structurally and mechanistically, from antiangiogenic agents that constitute the current standard of care (SOC) and demonstrated in vivo activity. We conclude that PDD complements gene-specific target-directed strategies, may mitigate TV risks, and has the potential to enhance innovation in drug discovery.

Neoclassic Drug Discovery The Case for Lead Generation Using Phenotypic and Functional Approaches

Innovation and new molecular entity production by the pharmaceutical industry has been below expectations. Surprisingly, more first-in-class small-molecule drugs approved by the U.S. Food and Drug Administration (FDA) between 1999 and 2008 were identified by functional phenotypic lead generation strategies reminiscent of pre-genomics pharmacology than contemporary molecular targeted strategies that encompass the vast majority of lead generation efforts. This observation, in conjunction with the difficulty in validating molecular targets for drug discovery, has diminished the impact of the “genomics revolution” and has led to a growing grassroots movement and now broader trend in pharma to reconsider the use of modern physiology-based or phenotypic drug discovery (PDD) strategies. This “From the Guest Editors” column provides an introduction and overview of the two-part special issues of Journal of Biomolecular Screening on PDD. Terminology and the business case for use of PDD are defined. Key issues such as assay performance, chemical optimization, target identification, and challenges to the organization and implementation of PDD are discussed. Possible solutions for these challenges and a new neoclassic vision for PDD that combines phenotypic and functional approaches with technology innovations resulting from the genomics-driven era of target-based drug discovery (TDD) are also described. Finally, an overview of the manuscripts in this special edition is provided.

A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay.

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris™ Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris™ cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen™ Explorer laser microplate cytometer in combination with the Oris™ Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.

Open Innovation for Phenotypic Drug Discovery: The PD2 Assay Panel

Phenotypic lead generation strategies seek to identify compounds that modulate complex, physiologically relevant systems, an approach that is complementary to traditional, target-directed strategies. Unlike gene-specific assays, phenotypic assays interrogate multiple molecular targets and signaling pathways in a target “agnostic” fashion, which may reveal novel functions for well-studied proteins and discover new pathways of therapeutic value. Significantly, existing compound libraries may not have sufficient chemical diversity to fully leverage a phenotypic strategy. To address this issue, Eli Lilly and Company launched the Phenotypic Drug Discovery Initiative (PD2), a model of open innovation whereby external research groups can submit compounds for testing in a panel of Lilly phenotypic assays. This communication describes the statistical validation, operations, and initial screening results from the first PD2 assay panel. Analysis of PD2 submissions indicates that chemical diversity from open source collaborations complements internal sources. Screening results for the first 4691 compounds submitted to PD2 have confirmed hit rates from 1.6% to 10%, with the majority of active compounds exhibiting acceptable potency and selectivity. Phenotypic lead generation strategies, in conjunction with novel chemical diversity obtained via open-source initiatives such as PD2, may provide a means to identify compounds that modulate biology by novel mechanisms and expand the innovation potential of drug discovery.

Consideration of the cellular microenvironment: Physiologically relevant co-culture systems in drug discovery ☆

There is renewed interest in phenotypic approaches to drug discovery, using cell-based assays to select new drugs, with the goal of improving pharmaceutical success. Assays that are more predictive of human biology can help researchers achieve this goal. Primary cells are more physiologically relevant to human biology and advances are being made in methods to expand the available cell types and improve the potential clinical translation of these assays through the use of co-cultures or three-dimensional (3D) technologies. Of particular interest are assays that may be suitable for industrial scale drug discovery. Here we review the use of primary human cells and co-cultures in drug discovery and describe the characteristics of co-culture models for inflammation biology (BioMAP systems), neo-vascularization and tumor microenvironments. Finally we briefly describe technical trends that may enable and impact the development of physiologically relevant co-culture assays in the near future.

Novel Phenotypic Outcomes Identified for a Public Collection of Approved Drugs from a Publicly Accessible Panel of Assays

Phenotypic assays have a proven track record for generating leads that become first-in-class therapies. Whole cell assays that inform on a phenotype or mechanism also possess great potential in drug repositioning studies by illuminating new activities for the existing pharmacopeia. The National Center for Advancing Translational Sciences (NCATS) pharmaceutical collection (NPC) is the largest reported collection of approved small molecule therapeutics that is available for screening in a high-throughput setting. Via a wide-ranging collaborative effort, this library was analyzed in the Open Innovation Drug Discovery (OIDD) phenotypic assay modules publicly offered by Lilly. The results of these tests are publically available online at www.ncats.nih.gov/expertise/preclinical/pd2 and via the PubChem Database (https://pubchem.ncbi.nlm.nih.gov/) (AID 1117321). Phenotypic outcomes for numerous drugs were confirmed, including sulfonylureas as insulin secretagogues and the anti-angiogenesis actions of multikinase inhibitors sorafenib, axitinib and pazopanib. Several novel outcomes were also noted including the Wnt potentiating activities of rotenone and the antifolate class of drugs, and the anti-angiogenic activity of cetaben.

An In Vitro Cord Formation Assay Identifies Unique Vascular Phenotypes Associated with Angiogenic Growth Factors

Vascular endothelial growth factor (VEGF) plays a dominant role in angiogenesis. While inhibitors of the VEGF pathway are approved for the treatment of a number of tumor types, the effectiveness is limited and evasive resistance is common. One mechanism of evasive resistance to inhibition of the VEGF pathway is upregulation of other pro-angiogenic factors such as fibroblast growth factor (FGF) and epidermal growth factor (EGF). Numerous in vitro assays examine angiogenesis, but many of these assays are performed in media or matrix with multiple growth factors or are driven by VEGF. In order to study angiogenesis driven by other growth factors, we developed a basal medium to use on a co-culture cord formation system of adipose derived stem cells (ADSCs) and endothelial colony forming cells (ECFCs). We found that cord formation driven by different angiogenic factors led to unique phenotypes that could be differentiated and combination studies indicate dominant phenotypes elicited by some growth factors. VEGF-driven cords were highly covered by smooth muscle actin, and bFGF-driven cords had thicker nodes, while EGF-driven cords were highly branched. Multiparametric analysis indicated that when combined EGF has a dominant phenotype. In addition, because this assay system is run in minimal medium, potential proangiogenic molecules can be screened. Using this assay we identified an inhibitor that promoted cord formation, which was translated into in vivo tumor models. Together this study illustrates the unique roles of multiple anti-angiogenic agents, which may lead to improvements in therapeutic angiogenesis efforts and better rational for anti-angiogenic therapy.

Amphibian Melanophore Technology as a Functional Screen for Antagonists of G-Protein Coupled 7-Transmembrane Receptors

Xenopus laevis melanophores stably expressing 7-transmembrane G-protein-coupled receptors were established and evaluated, either as a primary screening utility for antagonists of the human calcium receptor, or as a screen to assign function to binding inhibitors of human cannabinoid receptors. Stably or transiently expressing melanophores responded selectively to respective effectors of the human calcium, cannabinoid, and neurokinin-1 receptors. Several selective cannabinoid receptor-binding inhibitors of known potency were characterized as agonists or antagonists of the human peripheral cannabinoid (CB2) receptor. The results were consistent with changes in cAMP content of hCB2-transfected human embryonic kidney (HEK) cells challenged with the same CB2-binding antagonists. A stable melanophore cell line expressing the human calcium receptor was used to screen a compound collection directly for functional antagonists, several of which were confirmed as antagonists in secondary screens by stimulating parathyroid hormone (PTH) secretion from bovine parathyroid cells. The percentage of hits in this cell-based screen was reasonably low (1.2%), indicating minimal interference due to toxic effects and validating melanophores as a primary screening modality. Also described is the development of a novel procedure for cryopreservation and reconstitution of cells retaining functional human receptors.

Empirical drug discovery: a view from the proteome

Jonathan Lee is a Senior Research Advisor in the department of Quantitative Biology at Eli Lilly and Company. Jonathan pharmaceutical experience includes portfolio strategy, research management and the evaluation, development, and deployment of new drug discovery technologies. Jonathan’s recent research emphasizes the use of complex, physiologically relevant cellular systems for target validation, biomarker discovery, lead generation, and SAR support. Jonathan was an early adopter of modern empirical drug discovery approaches and plays an active part in the emerging phenotypic drug discovery (PDD) community through publications, presentations, initiating the PDD Special Interest group, and initiating/coorganizing the Keystone Symposium ‘Modern Phenotypic Drug: Defining the Path Forward’.

Abstract 1612: Overexpression or inducible expression of Snail, Zeb1 or TGFβ recapitulates epithelial-mesenchymal transition (EMT) in colorectal cancer cell lines in vitro

Epithelial-mesenchymal transition (EMT) is one of the hallmarks of cancer and a critical process providing tumor cells with the ability to metastasize to distant sites. Several previous studies have revealed that EMT plays a crucial role in the progression and aggressiveness of colorectal cancer (CRC). Snail, Zeb1 and TGFβ are considered as master EMT genes and their overexpression has been linked to metastatic disease. Our study was conducted to establish an EMT platform in-house to determine the role of Snail, Zeb1 and TGF-β1 in mediating EMT in CRC cell lines in vitro and using them for studying drug sensitivity and target validation. Screening of six CRC cell lines for expression of epithelial (E) and mesenchymal (M) markers by western blot analysis suggested that DLD-1 and HT-29 cells are epithelial in nature and were used to generate stable overexpression and tet-inducible models. Overexpression of Snail or Zeb1 or TGF-β1 induced EMT in both DLD-1 and HT-29 colorectal cancer cells as indicated by downregulation of E-cadherin and upregulation of vimentin by western blot analysis and immunofluorescence staining. These cells which have undergone EMT also showed significant growth advantage in 3D growth on matrigel or soft agar. Stable ‘E’ and ‘M’ cells were also used to test the sensitivity of various inhibitors including erlotinib, salinomycin and chemotherapeutic agents such as paclitaxel, gemcitabine, cisplatin, oxaloplatin and 5-FU. Consistent with previous findings, erlotinib showed sensitivity only in ‘E’ cells and not ‘M’ cells. Chemotherapy agents have also shown higher sensitivity in ‘E’ cells than ‘M’ cells. In contrast, Salinomycin showed equipotent sensitivity in ‘E’ and ‘M’ cells. We have also generated tet-inducible models of Snail, Zeb1 and TGF-β1 and they behaved very similar to overexpression models. These inducible models were useful to determine whether any inhibitor treatment or knock down of specific gene (target) can block EMT. In our studies, testing of a Focal Adhesion Kinase (FAK) inhibitor or Dasatinib (SFK inhibitor) as examples showed blockade of EMT in these models. Overall, establishing several EMT models in-house enabled us to validate new targets involved in EMT, test new inhibitors whether it can block EMT or study drug sensitivity in ‘E’ vs. ‘M’ cells. Citation Format: Shripad V. Bhagwat, Nicole E. Hamm, Yu-Chih Hsu, Yuewei Qian, Robert Wild, Jonathan A. Lee, Sheng-Bin Peng. Overexpression or inducible expression of Snail, Zeb1 or TGFβ recapitulates epithelial-mesenchymal transition (EMT) in colorectal cancer cell lines in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1612.