In Focus pubs.acs.org/acschemicalbiology
Drug Discovery and Repurposing at Memorial Sloan Kettering Cancer Center: Chemical Biology Drives Translational Medicine Bhavneet Bhinder and Hakim Djaballah* HTS Core Facility, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, New York, New York 10065, United States
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rug discovery and pharmacological research have come a long way since their inception in the mid-19th century. The modern drug discovery pipeline is fairly well established, and an integral component of this streamlined workflow is high-throughput screening (HTS), playing a crucial role in driving the initial stages of “discovery” and laying the foundations for lead optimization. HTS is synonymous to a fishing expedition enabling parallel testing of millions of small molecules or RNA interference (RNAi) duplexes so as to identify novel drugs for therapy or novel molecular targets for diagnostics. Traditionally an expertise exclusive to the pharmaceutical sector, HTS has gradually found its way into academic institutions partly due to reduction in setup costs as well as global initiatives such as the National Institutes of Health (NIH) Roadmap in the USA, encouraging such ventures. Significant advancements in automation, miniaturization, and assay technologies have provoked the emergence of HTS as a prominent paradigm of drug discovery within the past two decades. Therefore, to fuel a rapid discovery of novel targets and drugs for cancer therapy, Memorial Sloan Kettering Cancer Center (MSKCC) opened doors to a High-Throughput Screening Core Facility (HTSCF) in 2003, under the Experimental Therapeutic Center. Since then, it has been instrumental in providing investigator-initiated drug discovery efforts and is equipped with amenities ranging from assay development and optimization to its miniaturization and automation and subsequent follow-up confirmatory studies.1 HTSCF has been motivated to foster successful collaborations with clinicians and basic researchers so as to provide them with cost-effective HTS solutions to understand complex disease states and to drive innovation. The state-of-the-art robotics, cutting edge instrumentation, innovative high-content assay technologies, and personnel expertise enabled optimal integration of chemical biology and functional genomics approaches conducive to adoption on a high-throughput platform. The probes discovered from large-scale screens would be further used to investigate key signaling pathways or interrogated for hit to lead transformation and eventually drug development. Translational medicine is arising as a contemporary discipline of drug discovery. With this foresight, the US Food and Drug Administration (FDA)-approved drugs made up an integral part of our diverse chemical collections for screening. This has presented an exciting opportunity to repurpose old drugs for newer treatments, opening avenues to rapidly transition drugs from laboratory to clinic, a critical achievement toward addressing unmet clinical needs, and with direct impact on patient care (Figure 1). Besides drug repurposing efforts and novel inhibitor identification for lead generation, a broadspectrum brute force approach is also undertaken to elucidate novel pathways for therapeutic intervention, to study genetic © 2014 American Chemical Society
Figure 1. Translational medicine as a means to rapid drug discovery. Triggers prompt transition of drugs from the scientist’s bench to the patient’s bedside. Illustration is courtesy of Wenjing Wu, from Medical Graphics at MSKCC.
abnormalities among nonresponder populations, for biomarker discovery, and to explore drugs for combination therapy. In this report, we discuss the fundamentals of academic screening as a means to discover drugs for clinical use through the lens of the HTSCF.
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THE NUMBERS GAME IS OVER: SCREENING OF LARGE CHEMICAL LIBRARIES? HTS has evolved over the years to enable rapid investigation of millions of compounds for activity in disease-relevant model systems. The success or failure of an HTS campaign has long been attributed to the quality of the chemical libraries screened. So much so that this belief triggered a rat race toward procuring the largest chemical library driven by numbers and designed primarily by man-made chemistry. Despite an astronomical Published: July 18, 2014 1394
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thereof. As expected, the fame for RNAi screening was rather short-lived; issues pertaining to data reproducibility and lack of confirmation started to gain notice, causing the pharma giants to shy away from taking this technology under their wing. A well-known case would be that of STK33, identified in an RNAi screen by a reputed academic institution but failed to confirm in the hands of Amgen.3 We have also expanded our capabilities to perform RNAi screens using the small interfering RNA (siRNA), short hairpin RNA (shRNA), endoribonuclease-prepared siRNA (esiRNA), and microRNA (miRNA) technologies using both focused as well as genome-scale libraries. However, we adopted a more strategic approach toward handling RNAi screens, focusing mainly on arrayed formats, which allow targeting one duplex per well as opposed to pools. We devised a systematic and unbiased methodology, the BDA method, to analyze RNAi screen data outputs.3 This method was used to nominate quality hits based on H score, OTE filtering, and biological relevance in context of cellular pathways. Amidst the RNAi screening turmoil, our experiences taught us three critical lessons: (1) Extent of data reproducibility is far more serious than we think; key essential genes such as PLK1 fail to be identified across the board among published lethality screens. (2) Data discordance persists even in the controlled environment of screen execution and analysis; performance is most likely technology biased. (3) There is a newly discovered novel route of cell-type-dependent nonspecific gene silencing due to differential cleavage of shRNA hairpins, a phenomenon we termed ATSG (alternate targeting sequence generator).4,5 These findings highlight some alarming issues pertaining to perhaps an early adoption of RNAi technologies to HTS, especially at a time when we are in deficit of novel therapeutic targets. As dust gathers on the RNAi screening technology, the focus has shifted to other exciting genome editing technologies such as CRISPR (clustered regularly interspaced short palindromic repeats), the motivation being their quick transformation to high-throughput usage. This begs the questions as to whether, blinded by enthusiasm, we have a tendency to embrace newer technology platforms prior to their optimization, and if so, are we headed down the same old road as with RNAi and is history set to repeat itself with CRISPR?
growth in the number of chemicals in today’s screening libraries, the approval rate of drugs emerging out of HTS has significantly declined over the years. This begs the question as to whether this numbers game holds any relevance. Perhaps the answer lies in the actual composition of these libraries. The number of chemicals synthesized to date constitute only a miniscule proportion relative to the unexplored chemical space, since the synthetic and combinatorial chemistry approaches are restrictive and the desired level of chemical diversity remains practically unattainable. More so, man-made chemistry remains unmatched when compared to the diversity represented in Mother Nature, which is perhaps the most promising yet under-tapped avenue for exploring novel therapeutic agents. Natural products (NP) have been used in traditional medicine for ages; World Health Organization (WHO) estimates 80% of world’s population to be dependent on traditional medicine for healthcare. Also, bear in mind that over 50% of the drugs in clinic are either analogues or derivatives of NPs; specifically, seven out of the ten blockbuster drugs in the U.S. have origins from NPs, e.g., Lipitor (Pfizer).2 We have realized early on that the key lay in compiling a collection of compounds that would exhibit a comprehensive structural and functional diversity, libraries built over time based on quality rather than sheer quantity. We currently possess approximately 400,000 compounds obtained from various commercial vendors designed following a loose exclusion criteria for compounds and encompassing diverse small molecules, which include FDA-approved drugs, known bioactives, and several pharmacophore classes. Among them, is a vast coverage of NPs to include deep sea extracts. Our latest addition has been approximately 2,000 unique plant, actinomycetes, and fungal extracts obtained from the Sarawak Biodiversity Center (Malaysia), one of the richest resources of natural biodiversity procured from the rainforests of Borneo. Taken together, our diversified library collection has helped position us better to drive fruitful screening endeavors for drug discovery.
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A NEW ERA OF TECHNOLOGY PLATFORMS: EXTINCT BEFORE THEY EVER EXISTED In recent years, drug discovery has been heavily reliant upon hypothesis-driven target-based approaches. Therefore, completion of the human genome sequencing was viewed as a treasured resource to identify and characterize novel molecular targets. The “omics” wave took over the entire scientific community by storm; one of the landmarks of the new era was discovery of RNAi, with a potential to revolutionize the field of functional genomics. The technology enthusiasts wasted no time in adopting RNAi as a tool to explore up to the entire human genome in high-throughput settings, so as to find disease-relevant targets. Consequently, RNAi screening was never given enough time to evolve and mature as an individual technology prior to its large-scale implementation. The peril lay in the fact that this technology lacked its own learning curve before adaptation to HTS; most of the concepts were borrowed directly from the classic chemical screening practices and therefore were never standardized. As an example, Z′ factor, a routinely used indicator of assay performance in chemical screens, holds no value proposition in assessing RNAi screens due to their heterogeneity and higher noise. Importantly, one key factor that set RNAi screens apart from chemical screens remained widely unaddressed, that of widespread off-target effects (OTEs) and lack of dedicated hit nomination strategies
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INFORMAL COLLABORATIONS ARE MORE SUCCESSFUL: LET US MEET IN THE CAR PARK? To expand the frontiers of drug discovery, it has become more crucial than ever to cultivate a multidisciplinary collaborative spirit. Therefore, we believe in initiating and building fruitful collaborations, more in terms of transforming corridor conversations into conceptualization of an experimental design. In our experience, such informal collaborations often share a common goal so as to have a positive impact on a patients’ well being. Such efforts provide an exceptional opportunity to combine the investigator’s knowledge base with our expertise, thereby enhancing productivity and achieving rewarding outcomes. Discussed below are three such inspirational stories from our collaborative screening campaigns for drug discovery at HTSCF.1 Drug Repurposing: Beginning an Era of Translational Medicine. This is an example of a collaboration undertaken with Dr. David Abramson, Chief of Ophthalmic Oncology Service at MSKCC. Dr. Abramson has a profound passion to develop therapy for retinoblastoma, a rare form of pediatric 1395
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to study this behavior in NIH-3T3 cells expressing KP and HRAS oncogenes. We achieved tremendous success in developing the first high content assay using automated whole well imaging to study 3D morphological features induced by oncogenic transformation under KP expression. Our robust image analysis algorithms exhibited sensitivity in differentiating among cells in monolayer versus clusters versus debris. We implemented this assay system to screen approximately 6,000 compounds, followed by a focused screen of 56 compounds.1 Among many promising candidates, we identified inhibitors of PDGFR such as imatinib, benchmark inhibitors that validate the merits of this approach. The hits identified in our screen for phenotype reversal are currently being pursued in Dr. Holland’s lab for mechanistic investigation.
cancer that often culminates into enucleation of the affected eye. What appears to be more unfortunate is the fact that one in three affected children will most likely develop retinoblastoma in both their eyes, calling for an urgent need to develop an effective therapy. Therefore, as a part of this joint project, we conducted a chemical screen in two retinoblastoma cell lines, Y79 and RB355, to test the activity of 2,640 FDA-approved drugs and bioactive compounds. The hits identified in the screen were enriched in cardenolide-like scaffolds, and one such cardenolide, digoxin, was selected for testing directly in Dr. Abramson’s clinic. The FDA had approved digoxin in 1975 for cardiac arrhythmia and heart failure, but its feasibility for cancer treatment has never been demonstrated before, perhaps due to the systemic toxicity of the required dosage. Fortunately, the development of intra-arterial delivery of drugs opened doors to retest such drugs. Dr. Abramson administered an intra-arterial dose of digoxin to a 4-year-old boy with stage Vb retinoblastoma, and to our astonishment the patient was responsive to this treatment. The effect observed by digoxin therapy was modest and the success marginal, as the boy developed secondary tumors seemingly resistant to the administered dose of digoxin.1 Further studies are ongoing to address the challenge of ensuring a sustained dosage of digoxin in the eye. Taken together, this highlights the value of screening FDA-approved drugs as a means to their quick transition from lab to clinic. Mining Genetic Hypervariability To Develop Comprehensive Therapy. Genetic variabilities among patients is the driving force behind resistance to conventional chemotherapy, increased instances of relapse, and poor prognosis. A prominent example in this category is that of refractory adult acute myeloid leukemia (AML), cancer of the bone marrow. In collaboration with Dr. Mark Frattini, Director of Research for the Hematologic Malignancies at the Columbia University Medical Center, we conducted a screen using approximately 250,000 compounds so as to identify promising candidates for clinical development. From our screen, we identified MSK-777, an inhibitor of CDC7 (cell division cycle 7) kinase.6 CDC7 phosphorylates the MCM proteins, plays a key role in regulating DNA replication in the S-phase, and has a proposed role in AML progression. MSK-777 was successful when tested in mouse tumor models, and an IND application was filed with the FDA; it is set to enter clinical trials in early 2015. While awaiting the clinical outcomes, we pre-empted the value of characterizing the landscape for MSK-777 efficacy. For the purpose, we conducted a genome-wide shRNA screen at two doses of this drug, IC20 and IC50, with an aim to identify genes that synergize or rescue cells from the action of MSK-777. While the synergizers would help enhance the efficacy of MSK777, the rescuers would constitute the biomarkers for refractory AML so as to enable prediction of responder population.7 Phenotype Reversal of Oncogenic Transformed Cells for Anticancer Drugs. The hallmark of cancer is genetic alterations that modulate the cell’s growth and morphology. Aberrant expression of oncogenes such as KP (a fusion protein of platelet derived growth factor receptor alpha (PDGFRα) and kinase insert domain receptor (KDR)) has been reported to transforms cell’s phenotype, causing them to form threedimensional (3D) clusters. Therefore, one of the approaches to develop anticancer treatments would be to identify small molecule inhibitors that would reverse the altered phenotype. For this purpose, we collaborated with Dr. Eric Holland, a world-renowned neurosurgeon working at MSKCC at the time,
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THE FUTURE OF DRUG DISCOVERY: SERENDIPITY REMAINS KEY! HTS has traditionally been the forte of the big pharma, but the current trends seem to reflect a change in this paradigm. High risk and high R&D investments coupled with diminished productivity are pushing the pharma to downsize. To add fuel to the fire, loss of patented drugs has presented competition from the generics while the worldwide pressure to minimize prices is at an all time high. Furthermore, in this era of translational medicine where screening FDA-approved drugs is opening excellent avenues to repurpose old drugs for novel treatments, pharma is still at bay from adopting this practice, perhaps to avoid conflicts pertaining to positioning of intellectual property (IP). On the other hand, academic screening centers have come of age and are now ready to successfully undertake large-scale ventures, while also being open to collaborations that drive innovation. It is now incumbent on academia and the CROs to carry the torch forward in terms of the research aspect of R&D while the pharma would most likely concentrate on the aspect of development. A rapid decline in the number of FDA-approved drugs reaching the clinic has also emphasized the need to overhaul and re-energize the drug discovery process. Target-based techniques have for long been the most accepted and widely used models to search for novel therapeutic agents. Assuming there are 22,000 genes in the human genome, only 20% of the genes have been identified as drug targets so far. This brings to bear the limitations of the target-based approach and, in part, explains the relative stagnation in the drugs entering market. Therefore, it might be lucrative to switch gears and re-adopt the classical function-based drug discovery practices, so as to test compounds primarily for their therapeutic activity in disease models. After all, these methodologies have shown success in finding first-in-class drugs in the past. More so, utilizing patientderived samples in such models would be received as a leap forward in translational medicine. Since the inception of high content screening, it has become possible to perform cell-based assays with phenotypic end point readouts, thus further enhancing the feasibility to efficiently implement functionbased models. Polypharmacology had been viewed as a persistent challenge in drug discovery; promiscuous compounds emerging from screening have often been neglected. However, polypharmacology is rather debatable with regards to its perils and promise; on one hand it compromises drug safety, resulting in withdrawals of approved drugs from market, while on the 1396
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tions in gene silencing and pooled RNAi screens. PLoS One, (In Press). (6) Frattini, M. G., Shum, D., O’Dwyer, K. M., Brentjens, R. J., Yeh, R., Maslak, P. G., Djaballah, H., and Kelly, T. J. (2009) Discovery and validation of a novel class of small molecule inhibitors of the CDC7 kinase: modulation of tumor growth in vitro and in vivo. AACR Abstr. 50, 1125. (7) Liu-Sullivan, N., Bhinder, B., Shum, D., Ramirez, C., Radu, C., Djaballah, H., and Frattini, M. G. (2011) Preclinical assessment of a novel CDC7 inhibitor: Genomewide RNAi screening identifies unique synergetic and resistance genes. Blood Abstr. 118, 1524.
other hand it has played an instrumental role in discovery of novel drugs (e.g., sorafenib) as well as repurposing of old drugs (imatinib, asprin). The technological advancements, different discovery models, and so on and so forth, have all been the fundamental determinants in finding successful novel treatments; however, accidental discoveries remain common-place in all scientific research. Such pleasant surprises, defined by the term serendipity, have been integral contributors to the world of clinical drugs. Right from the discovery of early psychotropic drugs in 1857 to the discovery of penicillin in 1928 to the more recent achievements in repurposing existing drugs, all signify the value of serendipitous drug discovery. Therefore, it would be reasonable to argue that, in the end, serendipity would continue to hold the key to the future of drug discovery.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Notes
The authors declare no competing financial interests.
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ACKNOWLEDGMENTS The HTS Core Facility is partially supported by Mr. William H. Goodwin and Mrs. Alice Goodwin and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of MSKCC, the William Randolph Hearst Fund in Experimental Therapeutics, the Lillian S. Wells Foundation, and NIH/NCI Cancer Center Support Grant 5 P30 CA008748-44.
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ABBREVIATIONS HTS, high-throughput screening; RNAi, RNA interference; NIH, National Institutes of Health; MSKCC, Memorial Sloan Kettering Cancer Center; HTSCF, High-Throughput Screening Core Facility; FDA, Food and Drug Administration; NP, natural products; WHO, World Health Organization; OTE, offtarget effects; siRNA, small interfering RNA; shRNA, short hairpin RNA; esiRNA, endoribonuclease-prepared RNA; miRNA, microRNA; BDA, Bhinder−Djaballah analysis; H score, hit rate per gene score; ATSG, alternate targeting sequence generator; CRISPR, clustered regularly interspaced short palindromic repeats; AML, adult acute myeloid leukemia; R&D, research and development; IP, intellectual property; CRO, Contract Research Organization
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REFERENCES
(1) Bhinder, B., Antczak, C., Shum, D., Radu, C., Mahida, J. P., LiuSullivan, N., Ibanez, G., Raja, B. S., Calder, P. A., and Djaballah, H. (2014) Chemical & RNAi screening at MSKCC: A collaborative platform to discover and repurpose drugs to fight disease. Comb. Chem. High Throughput Screening 17, 298−318. (2) Djaballah, H. (2013) Chemical space, high throughput screening and the world of blockbuster drugs. DDW, spring. (3) Bhinder, B., and Djaballah, H. (2012) A simple method for analyzing actives in random RNAi screens: introducing the “H Score” for hit nomination and gene prioritization. Comb. Chem. High Throughput Screening 15, 686−704. (4) Bhinder, B., and Djaballah, H. (2013) Systematic analysis of RNAi reports identifies dismal commonality at gene-level and reveals an unprecedented enrichment in pooled shRNA screens. Comb Chem. High Throughput Screening 16, 665−681. (5) Bhinder, B.; Shum, D.; Li, M.; Ibáñez, G.; Vlassov, A. V.; Magdaleno, S.; Djaballah, H. (2014) Discovery of a dicer-independent, cell-type dependent alternate targeting sequence generator: implica1397
dx.doi.org/10.1021/cb500479z | ACS Chem. Biol. 2014, 9, 1394−1397