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DRUG DISCOVERY
A new vision of drug discovery and development D. Lansing Taylor Director, University of Pittsburgh Drug Discovery Institute and Allegheny Foundation Professor of Computational and Systems Biology, University of Pittsburgh School of Medicine
The pharmaceutical industry has experienced a decade of turbulence driven by the ‘patent cliff’ as major revenue generators are lost to generic status, coupled to the absence of a sustainable pipeline of drug candidates in development that have 1,2 a good chance of being approved and launched . It is generally agreed that the lowest hanging drug discovery ‘fruit’ has been harvested and the industry is addressing diseases that are more complex. The current one target, one drug discovery and development paradigm continues to exhibit more than 90 per cent attrition mainly due to the lack of success in translating preclinical efficacy and 1 safety data into successful human trials . It has also become clear that efficient drug discovery and development requires a deeper understanding of the complexity 3 of human biology early in the process . The high attrition rates increase the costs and with the science indicating that precision therapeutics will replace the 4 blockbuster model , the challenge of drug discovery and development is even greater. The traditional business model of pharmaceutical companies working in silos is no longer sustainable.
focused efforts in order to gain the advantage of collaboration. All potential partners must be willing to negotiate terms of agreements that benefit all partners. In many cases, this will require modifications of institutional policies created in a very different era. In a new environment of collaboration, each potential partner must also identify and optimise the strengths of their organisation to make it a good complementary fit for potential partners. Academic medical centres are in a great position to take part in the collaborative business model of drug discovery and development, clinical trials and diagnostics, if they are willing to negotiate mutually beneficial terms, including ownership / licensing of intellectual property
A new vision of strategic collaborations
nologies and basic knowledge. Life sciences
However, there is a new vision where strategic
and diagnostic companies have recognised
The importance of adopting the collabor-
collaborations between pharmaceutical
the emergence of personalised or precision
ative business model to increase the potential
companies, government agencies, venture
medicine and have begun commercialising
for innovation is reflected in the fact that the
capital-backed biotechnology companies and
the ‘omics’ platforms required for realising
dominant paradigm in drug discovery for
academic medical centres will create a new
personalised (precision) medicine. There is also a
the last 25 years has been the target-centric
breadth of approaches that have a good chance
heightened focus on coupling diagnostic /
model or the one gene, one target, one New
of increasing the innovation that has been
prognostic tests with new therapeutics to
Chemical Entity (NCE) approach. Most
stagnant in recent years5-8. Government funding
identify the patient sub-populations that will
pharmaceutical companies embraced this
agencies are increasing the emphasis on
benefit from a treatment. This evolution of the
philosophy due to the expectation of a near-
translational research, even in a period of
pharmaceutical industry offers opportunities
term impact of the human genome project,
funding pressures. Academic medical centres
and challenges to the potential partners.
technological advances in screening auto -
arising from the collaboration.
are also increasing their efforts in translational
It is necessary to harness the expertise of the
mation, creation of large chemical libraries,
science, while continuing to create new tech-
collaborating partners and to be innovative in
increases in the number of solutions to protein
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DRUG DISCOVERY structures and the creation of in silico software tools. A major challenge has been the slow and incomplete determination of the relationship between the disease genotype and the phenotype. It is now clear that putting most of the intellectual and experimental ‘eggs’ in this one basket has not produced the predicted productivity1.
A road to higher innovation High Throughput Screening (HTS) had become the standard in target-centric discovery programs by the early 1990s, but in the late 1990s, High Content Screening (HCS) was introduced as an alternative or at least a complement to HTS9,10. The concept of HCS was to fully automate fluorescence microscopic analysis of cells and later small experimental organisms, allowing the creation of deeper
Blending, Granulation, Drying …
biological knowledge based on the physiological disease relevance to better guide decisions on the progression of compounds. HCS offered the opportunity to initiate discovery based on a disease phenotype and not a specific molecular target. However, most discovery programs in the early days used HCS only in secondary assays and to follow a specific molecular target activity, since the speed of reading primary assays using HTS was viewed as paramount. However, given the late-stage failure of drug candidates, especially in phase II clinical trials1, the speed of reading a plate in discovery is insignificant compared to the time to make good decisions on progressing candidates based on the necessary biological and chemical knowledge11. Importantly, it has been demonstrated that phenotypic discovery was the most successful approach for first-in-class small molecule drugs approved by the US Food and Drug Administration (FDA) between 1999 and 200812,13. Combining the application of multiplexed HCS to more disease relevant (and more complex) biological screening models such as mixed cell cultures, 3D human tissue engineered models of organs and small experimental organisms such as yeast, C. elegans, and zebrafish should have a major impact in the early discovery process
14,15
. Especially promising for the future is the potential of
human induced pluripotent stem cells (iPSCs) from patients with a range of genomic backgrounds to serve as the source for disease relevant human tissue models to be used in discovery and early
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phenotypic discovery
methods to reduce the data to knowledge.
A critical role for quantitative systems pharmacology Quantitative systems pharmacology (QSP) is an emerging field that integrates experimentation, including screening data, with computational tools to define the actions of drugs across multiple scales of biological complexity from molecules to cells, tissues, organs and organisms for a ‘systems’ level perspective20-22. QSP is the merger of pharmacology, especially pharmacokinetics (PK) and
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pharmacodynamics (PD) with the field of systems biology 23. You can see us at:
European Pharmaceutical Review Volume 17 | Issue 6 | 2012
IFPAC - January 2013, Baltimore
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DRUG DISCOVERY extended to define the complex processes of living systems 23,27. The Output from this approach is expected to yield advanced functional knowledge on the pathways, including potential feedback loops between pathways, as well as physiological processes interrogated experimentally and compu tationally. Additionally, the output will identify any ‘emergent properties’ or properties of the system not predicted from investigating the components, such as heterogeneity of system responses22. Another important output should be improved and predictive drug efficacy and safety as a ‘knowledgebase’ of information as QSP is applied. The major output should be novel drugs, including drug combinations working on connected pathways 28. There will be ‘feedback loops’ between input, Figure 1 QSP in drug discovery and development in the UPDDI begins with Inputs of ‘omics’ data, the development of inferred pathways involved in the disease and a phenotypic disease model for the screen. The application of QSP involves the phenotypic discovery approach using HCS and computational and systems biology, as well as mathematical modeling. The outputs of QSP include functional knowledge including any emergent properties, improved drug efficacy and safety and most importantly, novel drugs
QSP and output. For example, the QSP results will help build new hypotheses in regards to pathways and MOA that could feedback to input to perform new assays. Likewise, the outputs (e.g. drugs, combination therapies
24,25
The promise of QSP also includes the ability
knowledge, including polypharmacology
.
or polypharmacological strategies) would also
to understand the interaction of drugs in indi-
Furthermore, a number of machine learning
be tested against HCS assays and newly
vidual patients.
approaches have the potential to reduce the
collected omics data (e.g. from patients
We view QSP as involving Inputs, as well as
amount of screening measurements required26.
that have received treatment). QSP will also
the application of the elements of experi -
Mathematical models of refined pathways, as
help to understand side effects, or mechanisms
mentation and computational methods of QSP
well as PK and PD, are developed and then
of drug resistance.
and finally Outputs that result from the approach (Figure 1). The Input (diseaseassociated genotypes and phenotypes) of the QSP approach includes all of the available ‘omics’ generated on the disease model systems and patient samples including multiplexed fluorescence-based pathology. Disease relevant
‘‘It is necessary to harness the expertise of the collaborating partners and to be innovative in focused efforts in order to gain the advantage of collaboration’’ pathways are inferred from the ‘omics’ data to guide the development of the optimal phenotypic screen. The best possible phenotypic disease model is then validated as a High Content Screen (HCS) assay. The disease model/assay can range from multiplexed cells to engineered human tissue to experimental organisms. QSP integrates the data from the phenotypic discovery approach (see Figure 3, page 23) and applies a range of computational chemistry, computational biology and systems biology software tools to reduce the large screening/experimental data sets to functional
Figure 2 Starting discovery campaigns with computational chemistry and biology allows the integration of existing knowledge of the targeted pathway(s) and the identification of predicted binding of existing drugs to pathway components that could identify useful ‘probes’ of pathway modulations, as well as starting points for rational drug design. Some of the databases used in target prediction include: STITCH – chemical-protein interactions, http://stitch.embl.de/ . DrugBank – approved/experimental drugs with target information from literature, http://www.drugbank.ca/ ,SIDER – drug side effects database, http://sideeffects.embl.de/ , ChEMBL – chemicalprotein interactions from bioassays/literature, https://www.ebi.ac.uk/chembl/ , Target Hunter-identify possible targets of small molecules, http://www.cbligand.org/targethunter, and MDDR – chemical bioactivity database http://accelrys.com/products/databases/bioactivity/mddr.html
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DRUG DISCOVERY The University of Pittsburgh Drug Discovery Institute (UPDDI) as an example
and development process starting with the
campaign is performed identifying ‘hits’ and lead
application of phenotypic screening using
generation and lead optimisation is subse -
HCS11,14,15. Figure 3 shows the steps in performing
quently performed. An optimally designed HCS
The University of Pittsburgh Drug Discovery
a phenotypic screen. In phenotypic drug
assay can be used to guide the structure activity
Institute (UPDDI) has embraced a two-fold
discovery, the detailed, basic and clinical science
relationship (SAR) in lead optimisation15,32,33.
strategy in creating an environment for the new
is a critical component, as in target-centric
In fact, the FDA does not require molecular
vision of drug discovery and development
discovery. However, a functional, disease
target identification for approval13. Extensive
(www.upddi.pitt.edu): Collaboration – through integrated activities across internal departments, centres and institutes, along with collaborations with other academic centres, government agencies and commercial partners; and Innovation – by developing and practicing new approaches and technologies to complement the present paradigm of discovery. Technologically, the UPDDI has key strengths in HCS and phenotypic discovery, chemistry / medicinal chemistry and molecular biophysics applied to target-centric discovery and is investing heavily in the implementation of QSP. The focus of this discussion is on QSP coupled to phenotypic discovery, but the power of molecular biophysics in target-centric programs is evident when applied to the right target and can be performed subsequent to an initial phenotypic discovery step when a target is identified29. The University of Pittsburgh has the
Figure 3 Phenotypic drug discovery and development using HCS is a major commitment of the UPDDI. The implementation of QSP involves the best experimental methods, including HCS screening data, coupled with computational chemistry and biology, as well as systems biology. Although not required by the FDA, the identification of the molecular target(s) is investigated by chemical proteomics. The goal is to integrate and to analyse data across the complete discovery-development continuum from early discovery to clinical data
depth and breadth to support a major effort data mining on 890 approved drugs demon-
in drug discovery and development and in cooperation with the University of Pittsburgh Medical Center (UPMC), the ability to translate basic research to clinical trials and clinical practice. Ready access to clinician scientists, patients and patient samples is crucial to the success of this new paradigm.
‘‘The phenotypic drug discoverydevelopment approach allows the application of QSP from early discovery on a range of scales of biological complexity from molecules to mammalian models to human patient cohorts’’
strated that on average, each of the analysed drugs interact with six known molecular targets34. This latter fact gives promise to the drug repurposing efforts now underway35. However, when optimised leads are identified, it is important to apply chemical proteomics approaches to try to identify
A major advance in computational chemistry and computational biology in recent
relevant phenotype is selected, validated
potential molecular target(s)36,37. Identification
years has been the harnessing of genomics and
and profiled by HCS rather than selecting and
of molecular target(s) allows the application of
proteomics databases and sophisticated
validating a single molecular target. This
mechanism of action (MOA) studies that might
algorithms to infer pathways responsible for cell
broadens the potential molecular targets that
involve multiple molecular targets across
functions as a starting point for defining the
can be modulated by compounds and puts the
multiple pathways. Furthermore, one or a
actual pathways (Figure 2, page 22). It is also
initial focus on functions and pathways. This is
combination of identified molecular targets
now possible to harness drug and target protein
very different from applying hypothesis driven
could be useful as biomarkers. In vitro early
databases to predict drug interactions with
research to follow a single molecular target, but
safety assessment can also be performed using
specific target proteins. Positive ‘hits’ from the
allows an unbiased, physiological profile to
HCS at this stage to help guide the prioritisation
in silico profiling can be used to identify useful
define the cellular functions and pathways
of leads38,39. The pre-clinical and clinical stages
‘chemical probes’ of pathways and cellular
directly involved in the disease. Optimally, one
are the same functionally, as with the target-
functions, as well as a starting point to design
or more biomarkers can be identified in the
centric approach, but with the addition of
a phenotypic screening strategy for drug
process, quantified in the HCS assay and carried
computational and systems biology that
discovery and/or as a reference point for
through the whole discovery-development
permits quantification and modelling of
pursuing a molecular biophysics / target-centric
process. A critical step is the selection of
the data. The phenotypic drug discovery-
compound libraries based on both diversity and
development approach allows the application
novelty. In addition, exploring existing drugs for
of QSP from early discovery on a range of
Critical role of HCS in QSP
repurposing and natural products for added
scales of biological complexity from molecules
The UPDDI is applying QSP in the drug discovery
diversity has great promise. The screening
to mammalian models to human patient
30,31
approach to drug discovery
.
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DRUG DISCOVERY cohorts. The phenotypic approach can be
References
extended from early discovery through pre-
1.
clinical and clinical trials and represents a natural path to the development and use of biomarkers using multiscale imaging modalities40.
2.
Munos B. Lessons from 60 years of pharmaceutical innovation. Nature Reviews Drug discovery. 2009;8(12):959-68
3.
Butcher E. Can cell systems biology rescue drug discovery? Nature Reviews Drug Discovery 2005;4:461-7
4.
Eichler HG, Abadie E, Breckenridge A, Flamion B, Gustafsson LL, Leufkens H, et al. Bridging the efficacyeffectiveness gap: a regulator's perspective on addressing variability of drug response. Nature Reviews Drug Discovery. 2011;10(7):495-506
Conclusion A new vision of drug discovery and development is based on collaborations between pharmaceutical companies, government agencies, venture-backed biotechnology companies and academic medical centres to expand the potential for innovation, while
5.
sharing the complexity and technologies involved in discovering and developing novel therapeutics. Quantitative systems pharma-
Frye S, Crosby M, Edwards T, Juliano R. US academic drug discovery. Nature Reviews Drug Discovery. 2011;10(6):409-10
7.
Kaitin KI. Deconstructing the drug development process: the new face of innovation. Clin Pharmacol Ther. 2010;87(3):356-61. PMCID: 2953249
University of Pittsburgh Drug Discovery Institute. Personalised / precision medicine is
8.
Edmondson G, Valigra L, Kenward M, Hudson R, Belfield H. Making industry-university partnerships work: Science/Business Innovation Board, AISBL; 2012 Contract No.: Document Number|
9.
Taylor DL. A personal perspective on high-content screening (HCS): from the beginning. J Biomol Screen. 2010;15(7):720-5
dependent of the development of diagnostic tests to guide the optimal delivery of therapeutics to patient sub-populations.
Acknowledgements I would like to thank my colleagues for valuable discussions on how to harness the new vision of drug discovery and development including Mark Schurdak, Bert Gough, Andy Stern, Andreas Vogt, Dutch Boltz, Ivet Bahar, Jeremy Berg, Ahmet Bakan, Tim Lezon and Bob Murphy.
Vallance P, Williams P, Dollery C. The future is much closer collaboration between the pharmaceutical industry and academic medical centers. Clin Pharmacol Ther. 2010;87(5):525-7
6.
cology coupled with high content screening is being developed as one innovative path at the
Paul S, Mytelka D, Dunwiddie C, Persinger C, Munos B, Lindborg S, Schacht A. How to improve R&D productivity: the pharmaceutical industry's grand challenge. Nature Reviews Drug Discovery. 2010;9:203-14
10. Taylor D. Past, Present, and Future of High Content Screening and the Field of Cellomics. In: Taylor D, Haskins J, Giuliano K, editor. Methods in Molecular Biology, 2007; 356: 3-18. High Content Screening: A Powerful Approach to Systems Cell Biology and Drug Discovery. Totowa, NJ: Humana Press 11. Alcock P, Bath C, Blackett C, Simpson P. High content cell based primary screening for oncology targets--a perspective. European Pharmaceutical Review. 2010;3 12. Swinney D, Anthony J. How were new medicines discovered? Nature Reviews Drug Discovery. 2011;10
Biography Dr. D. Lansing Taylor began his academic career at Harvard University developing and applying novel fluorescence-based reagents and imaging technologies to investigate fundamental cellular processes. He then moved to Carnegie Mellon University and became the Director of the National Science Foundation, Center for Light Microscope Imaging and Biotechnology, and in 1995 was named Vice Dean of CMU’s Division of Molecular Sciences. Together with his students, post-docs and collaborators, Dr. Taylor developed reagent and imaging technologies including fluorescent analog cytochemistry, ratio imaging, fluorescence anisotropy imaging microscopy, the multimode light microscope and standing wave fluorescence microscopy. Dr. Taylor co-founded and led a series of biotechnology companies including Biological Detection Systems (cyanine dyes with Alan Waggoner), Cellomics (High Content Screening), Cellumen (predictive early safety assessment) and Cernostics (tissue systems pathology). Dr. Taylor is a co-inventor on more than 25 US patents, including six focused on cell-based imaging. He was the recipient of an NIH MERIT award and a Pioneer Award from the NSF. Dr. Taylor returned to academia at the end of 2010 to continue his academic interests which now link largescale cell, tissue and experimental organism profiling with computational and systems biology to optimise drug discovery and development. As the Director of the Drug Discovery Institute, he is focusing on the application of Quantitative Systems Pharmacology in order to change the paradigm in drug discovery and development.
13. Lee J, Uhlik M, Moxham C, Tomandl D, Sall D. Modern phenotypic drug discovery is a viable, neoclassic pharma strategy. Journal of Medicinal Chemistry. 2012;55:4527-38. 14. Gough A, Taylor D, et al. High Content Screening. The Molecular Basis of Cancer: Elsevier; In Press 15. Zanella F, Lorens JB, Link W. High content screening: seeing is believing. Trends Biotechnol. 2010;28(5): 237-45 16. Grskovic M, Javaherian A, Strulovici B, Daley GQ. Induced pluripotent stem cells--opportunities for disease modelling and drug discovery. Nature Reviews Drug Discovery. 2011;10(12):915-29 17. Perlman ZE, Slack MD, Feng Y, Mitchison TJ, Wu LF, Altschuler SJ. Multidimensional drug profiling by automated microscopy. Science. 2004;306(5699):1194-8 18. Giuliano KA, Chen YT, Taylor DL. High-content screening with siRNA optimizes a cell biological approach to drug discovery: defining the role of P53 activation in the cellular response to anticancer drugs. J Biomol Screen. 2004;9(7):557-68 19. Loo LH, Lin HJ, Steininger RJ, 3rd, Wang Y, Wu LF, Altschuler SJ. An approach for extensibly profiling the molecular states of cellular subpopulations. Nat Methods. 2009;6(10):759-65. PMCID: 2818727 20. Yang R, Niepel M, Mitchison TK, Sorger PK. Dissecting variability in responses to cancer chemotherapy through systems pharmacology. Clin Pharmacol Ther. 2010;88(1):34-8. PMCID: 2941986 21. Zhao S, Iyengar R. Systems pharmacology: network
European Pharmaceutical Review Volume 17 | Issue 6 | 2012
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analysis to identify multiscale mechanisms of drug action. Annu Rev Pharmacol Toxicol. 2012;52:505-21 22. Sorger P, Allerheiligen S. Quantitative and systems pharmacology in the post-genomic era: new approaches to discovering drugs and understanding therapeutic mechanisms. National Institutes of Health White Paper. 2011 23. van der Graaf PH, Benson N. Systems pharmacology: bridging systems biology and pharmacokineticspharmacodynamics (PKPD) in drug discovery and development. Pharm Res. 2011;28(7):1460-4 24. Pujol A, Mosca R, Farres J, Aloy P. Unveiling the role of network and systems biology in drug discovery. Trends Pharmacol Sci. 2010;31(3):115-23 25. Hopkins AL. Network pharmacology: the next paradigm in drug discovery. Nat Chem Biol. 2008;4(11):682-90 26. Murphy RF. An active role for machine learning in drug development. Nat Chem Biol. 2011;7(6):327-30 27. Faeden J, Blinov M, Hlavacek W. Rule-based modeling of biochemical systems with BioNetGen. In: Maly I, editor. Methods in Molecular Biology: Systems Biology. Totowa, NJ: Humana Press; 2009 28. Bernards R. A missing link in genotype-directed cancer therapy. Cell. 2012;151(3):465-8. 29. Molina G, Vogt A, Bakan A, Dai W, Queiroz de Oliveira P, Znosko W, et al. Zebrafish chemical screening reveals an inhibitor of Dusp6 that expands cardiac cell lineages. Nat Chem Biol. 2009;5(9):680-7. PMCID: 2771339 30. Liu Z, Fang H, Reagan K, Xu X, Mendrick DL, Slikker W, Jr., et al. In silico drug repositioning – what we need to know. Drug Discov Today. 2012;00(00):13-6 31. Keiser MJ, Setola V, Irwin JJ, Laggner C, Abbas AI, Hufeisen SJ, et al. Predicting new molecular targets for known drugs. Nature. 2009;462(7270):175-81. PMCID: 2784146 32. Link W, Oyarzabal J, Serelde BG, Albarran MI, Rabal O, Cebria A, et al. Chemical interrogation of FOXO3a nuclear translocation identifies potent and selective inhibitors of phosphoinositide 3-kinases. J Biol Chem. 2009;284(41):28392-400. PMCID: 2788888 33. Zanella F, Rosado A, Garcia B, Carnero A, Link W. Chemical genetic analysis of FOXO nuclearcytoplasmic shuttling by using image-based cell screening. Chembiochem. 2008;9(14):2229-37 34. Mestres J, Gregori-Puigjane E, Valverde S, Sole RV. The topology of drug-target interaction networks: implicit dependence on drug properties and target families. Mol Biosyst. 2009;5(9):1051-7 35. Mullard A. Drug repurposing programmes get lift off. Nature Reviews Drug Discovery. 2012;11(7):505-6 36. Cong F, Cheung AK, Huang SM. Chemical geneticsbased target identification in drug discovery. Annu Rev Pharmacol Toxicol. 2012;52:57-78 37. Laggner C, Kokel D, Setola V, Tolia A, Lin H, Irwin JJ, et al. Chemical informatics and target identification in a zebrafish phenotypic screen. Nat Chem Biol. 2012;8(2):144-6. PMCID: 3262069 38. Vernetti L, Irwin W, Giuliano K, Gough A, Johnston K, Taylor D. Cellular Systems Biology Applied to PreClinical Safety Testing: A Case Study of CellCiphr™ Cytotoxicity Profiling. In: Xu J, Ekins S, editor. Drug Efficacy, Safety, and Biologics Discovery: Emerging Technologies and Tools. New Jersey: John Wiley and Sons; 2008. p. 53-73 39. Giuliano K, Gough A, Taylor D, Vernetti L, Johnston K. Early Safety Assessment Using Cellular Systems Biology Yields Insights into Mechanisms of Action. J Biomol Screen. 2010;15:783-97 40. Prescher JA, Contag CH. Guided by the light: visualizing biomolecular processes in living animals with bioluminescence. Curr Opin Chem Biol. 2010;14(1):80-9