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Combinatorial Chemistry & High Throughput Screening, 2014, 17, 241-252 241 A Perspective on 10-Years HTS Experience at the Walter and Institute of Medical Research – Eighteen Million Assays and Counting

Kurt Lackovic1,2,3,4,5, Guillaume Lessene2,3,4,6, Hendrik Falk1,2,3,4,5, Karl-Johan Leuchowius1,2,4, Jonathan Baell7,§ and Ian Street*,1,2,3,4,5,§

1Division of Systems Biology and Personalized Medicine, Walter and Eliza Hall Institute of Medical Research, Parkville, , 3052, 2Division of Chemical Biology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia 3Division of Structural Biology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia 4Department of Medical Biology, University of , Parkville, Victoria, 3010, Australia 5Cancer Therapeutics CRC, Bundoora, Victoria, 3083, Australia 6Department of Pharmacology and Therapeutics, , Parkville, Victoria, 3010, Australia 7Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Campus, Victoria, 3052, Australia

Abstract: The Walter and Eliza Hall Institute of Medical Research (WEHI) is Australia’s longest serving medical research institute. WEHI’s High Throughput Screening (HTS) Facility was established in 2003 with $5 million of infrastructure funds invested by WEHI, and the Victorian State Government’s Strategic Technology Initiative through Bio21 Australia Ltd. The Facility was Australia’s first truly academic HTS facility and was one of only a handful operating in publicly funded institutions worldwide at that time. The objectives were to provide access to enabling HTS technologies, such as assay design, liquid handling automation, compound libraries and expertise to promote translation of basic research in a national setting that has a relatively young biotech sector and does not have a big Pharma research presence. Ten years on and the WEHI HTS Facility has participated in over 92 collaborative projects, generated over 18 million Ian Street data points, and most importantly, projects that began in the Facility have been commercialized successfully (due to strong ties with Business Development and emphasis on intellectual property management) and now have molecules progressing in clinical trials. Keywords: Academic, , chemical libraries, HTS, , therapeutics, trypanosomiasis.

INTRODUCTION The Walter and Eliza Hall Institute of Medical Research (WEHI) is Australia’s longest serving medical research High Throughput Screening (HTS) is a widely applied and institute, celebrating its centenary in 2015. WEHI currently extremely effective technology for bioactive compound employs close to 1000 staff across three sites in Melbourne, discovery. HTS is a technology platform that enables the large Australia, dedicated to discovery and translational research in scale screening of compound libraries in a rapid and cost the fields of cancer, and . WEHI’s HTS effective manner. HTS emerged in the late 1980s [1], and has Facility was established in 2003 with $5 million of matured into the principal hit identification method and a crucial infrastructure funds invested by WEHI, and the Victorian source of drug discovery leads within pharmaceutical and State Government’s Strategic Technology Initiative through biotechnology companies. In the late 1990’s two evolutionary Bio21 Australia Ltd. This Facility was Australia’s first truly changes occurred in the industry that allowed HTS to be academic HTS facility and one of only a handful operating in adopted by academia. First, liquid handling automation reached publicly funded institutions worldwide at that time. The a state of maturity and stability allowing it to be usefully objectives of the new Facility were clear, provide access to deployed in academic organizations. Second and most enabling HTS technologies, compound libraries and expertise important, a chemical vendor industry emerged, and for the first to (i) promote translation of basic research in a national setting time large screening libraries, which had previously been the that does not have a big Pharma research presence, and only a sole domain of Pharma and Biotech companies became relatively young biotech community, and (ii) promote the use available to the wider research community. of chemical libraries in basic research.

*Address correspondence to this author at the Division of Systems Biology BUILDING BLOCKS OF THE WEHI HTS FACILITY and Personalized Medicine, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, 3052, Australia; Tel: +61 3 9345 2100; Our main HTS laboratory is based at the WEHI E-mail: [email protected] Biotechnology Centre, which is situated some 16 km north §These authors contributed equally to this work from the Institute’s main campus. In late 2012, an Assay

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Development Laboratory was also opened on the Institute’s only for the resolution of multiple parameters from cellular main campus, designed to provide walk-up access to basic imaging in microtitre plates, but also imaging of HTS technologies for a wide section of the academic multicellular, three-dimensional aggregates (e.g. community. The Assay Development Lab is situated in the mammospheres), which more accurately reflect the cell-cell center of the Parkville precinct, home to over ~10,000 interactions of the body tissue compared to traditional two- research scientists, and houses a high-end multi-label plate dimensional cell culture. The ability to employ more reader, plate washer, multiple reagent dispensers, a liquid complex and relevant cellular models, in conjunction with handler (96- and 384-heads), pin tool dispensers and a Perkin the additional information gained in high-content imaging, Elmer Operetta high content imager. The availability of the more than offsets the additional cost and time associated labs in both the Biotechnology Centre and the Parkville with such screens. Our high-content screening platforms, campus allows us to run both large-scale screens for together with other technologies, have enabled the WEHI commercially focused collaborations and also provide a HTS Facility to execute phenotypic screening campaigns, mechanism to engage with the broader research community where compound effects are measured in terms of a whose primary interest is in running focused screens against biological response, with no bias towards a particular target. our smaller boutique libraries. Given 62% of approved first-in-class small molecule drugs originated from phenotypic rather than targeted approaches Major infrastructure housed within the WEHI HTS Facility includes multiple liquid handling platforms such as a Perkin throughout the period 1999-2008 [4], despite the pharmaceutical industry’s preference towards targeted Elmer Minitrak, Perkin Elmer Janus and Hamilton Star. The approaches, we believe phenotypic screening holds great Hamilton Star is integrated with a cell culture incubator and promise in advancing truly novel therapies in a range of multi-label plate reader allowing fully automated cell based disease areas. Target-based drug discovery has been one of screening. We also maintain a number of stand-alone multi- the central paradigms of the past decade. While that label plate readers that enable us to use a wide range of technologies in designing our assays, including Alphascreen, approach will remain important in delivering drugs with a defined mechanism of action, phenotypic screening has more luminescence, fluorescence, absorbance, TR-FRET, Mesoscale recently demonstrated its ability to identify new and the Roche xCelligence label-free platform. The flexible opportunities of previously unsuspected interactions [5]. nature of the facility allows us to develop and run fully Both approaches together have the potential to generate new automated screening campaigns or where desired deploy a insights into cellular function. While phenotypic screening semi-automated work station approach. In ten years of operation, the WEHI HTS Facility has executed many often produces unexpected insights into cellular pathways, target-based design of drugs or tool molecules remains the technically challenging projects, including miniaturized assays most effective way to optimize HTS leads. in 1536-well formats, high-content assays using cell lines and primary cells, and parallel screening campaigns testing over 1,000,000 compounds each at a rate of 70,000 assay points per DATA MANAGEMENT day. Over the last 10 years the Facility has executed In a HTS Facility capable of generating in excess of approximately 18 million assays, including 11,604,262 primary 70,000 assay points per day, as well as thousands of high- screening events, 305,139 dose response curves (3,356,529 data content images, good data management and analysis tools points) and 2,992,158 control assays. Infrastructure has never are an imperative. Since its inception, our Facility has used limited our screening capacity; the major rate limiting factor has IDBS ActivityBase as our main data capture, processing and always been project funding. storage repository. Based on positive experience in commercial collaborations, we also acquired Tibco’s SURFACE PLASMON RESONANCE Spotfire, predominantly for data visualization. Spotfire provided a vastly improved ability to view data trends, Funding from the ’s Super visualize results in real-time, and to identify and exclude Science Initiative recently allowed us to acquire a Biacore 4000 platform, replacing our aging Biacore S51. The new systematic errors as they occur, saving both time and reagents. In addition, the visualization export functions have instrument allows detailed characterization of binding enhanced our ability to prepare reports in a timely fashion. interactions for both protein-ligand (small molecules or The lead discovery option also enables us to link compound peptides) and for protein-protein interactions, and also structure with assay results, allowing our medicinal chemists fragment based drug discovery where we are in the process to more easily determine structure-activity-relationships. of building a fragment screening library with local collaborators [2]. Our Biacore instruments have been essential for validating screening hits across multiple HTS EXPERTISE projects, as well as the more detailed characterization of We currently maintain a core group of 12 analogues from lead generation and lead optimization. The scientists/technologists with broad expertise in assay Biacore 4000 is a component of the Therapeutic Innovation development and HTS (biochemical and cell based Australia national infrastructure platform [3], and access is technologies), HTS automation and IT systems. One of the also available to the wider research community. key challenges in establishing and growing the WEHI HTS Facility has been recruitment of experienced HTS personnel HIGH CONTENT SCREENING in a national setting without big Pharma research and only a Increasingly we are finding that our high-content imaging relatively young Biotech sector. It has been relatively rare to recruit staff with HTS experience from overseas and in most platforms are in high demand. These platforms allow not cases we have recruited scientists from the appropriate basic 10-Years HTS Experience at the WEHI Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 243 science field and provided “on the job” training in HTS. follows. Reactive, non-drug-like and known frequent hitter More recently we have also had several PhD students and scaffolds were first removed from the vendor collections by post-doctoral scientists placed within our Facility to elimination of compounds containing functional groups as complete assay development and perform HTS campaigns described in detail elsewhere by Baell [7, 8]. Remaining and associated follow up experiments under our guidance. compounds were selected on lead-likeness. Finally, This model has worked particularly well, reducing labor compounds were removed if more than 85% similar to each costs for collaborators as well as transferring HTS other and additionally, for libraries processed subsequent to knowledge to the broader scientific community. Stage 1, if more than 85% similar to previous stage libraries. Table 1 summarizes the processes and interrelationship of Over the last 10 years many of our trained personnel each library, from the earliest Stage 1 in 2003 to the most have moved to other appointments within the Australian Biotech sector, to establish another academic HTS group at recent Stage 6 in 2010. the Children’s Cancer Institute Australia, overseas academic The process to establish Stage 1, Stage 4, Stage 5 and organizations and Australian technology vendors. In this Stage 6 libraries was broadly the same as just described, and capacity the Facility has played a substantial role in growing we call these collections the Lead-Like Stages. Stage 2 and the Australian technology sector. Stage 3 libraries were obtained through collaboration. Selection criteria for each of the individual stages of the CHEMICAL LIBRARIES Lead-Like Stages were very similar to each other, but there were distinct differences (Table 2). For example, the number At the core of our success is the quality of our chemical of vendors varied. For Stage 1, vendors were Maybridge, libraries. The WEHI Facility offers a range of “boutique Specs, ChemDiv and LeadQuest; Stage 4, ChemDiv; Stage libraries”, in conjunction with lead-like diversity collections now 5, ChemDiv and ChemBridge; Stage 6, Asinex, comprising over 350,000 small molecules. Our boutique libraries ChemBridge, ChemDiv, Enamine, InterBioScreen, Life currently comprise kinase inhibitors (212), epigenetic modifiers Chemicals, Maybridge, Specs, and TimTec. Other (89), targeted agents (93), and a collection of almost 6,000 differences centered on definitions and quantification of known pharmacologically active agents, including the Lopac, lead-likeness. For example, Stage 1 selection was unique in Tocris and Prestwick compound sets. These boutique libraries that we developed a Muegge-like filter [9] which was are constantly expanded as new molecules become available or designed to select compounds based on degree of are published in the literature. Our boutique libraries are functionalization with polar groups. This appeared to particularly well suited to phenotypic screening and compound discriminate better than use of cLogP, minimum heavy atom repurposing projects [6], and are inexpensive to access. count, and rotatable bonds filters in establishing the Stage 1 library (Fig. 1). For the Stage 5 library on the other hand, DIVERSITY SETS around 30,000 compounds were selected from the compounds that had been excluded purely because they were In brief, since inception WEHI has developed three major in the molecular weight range of 400-450. These included high quality libraries of lead-like compounds: the combined compounds identified as containing kinase inhibitor cores Stage 1/3 Libraries (WEHI Library), the combined Stage 3/5 (similarity <85%), compounds that were highly diverse Libraries (CTX 153,000 compounds), and the Stage 6 HTS (<80% similarity to any other compound) and compounds Library (WECC). The lead-like libraries were developed with halogen or sulfonyl group (similarity <85%; we felt using extensive filtering, and the process involved has been such compounds are unfairly penalized by molecular weight published previously [7, 8]. – consider the CF3 group, for example). The Stage 5 library was also different in that compounds in the molecular weight SELECTION OF “LEAD LIKE” COMPOUNDS range 150-400 were allowed to be up to 90% similar to each other. The Stage 4 Library is essentially a subset of high Compound databases were obtained from selected similarity to the Stage 5 Library. Operationally, the Stage 1 vendors and lead-like subsets were compiled from each, as

Table 1. Different HTS Libraries Established Over Time and Selection Principles

Number of Number of Broad Selection PAINS Similarity Stage (Name, Year Established) Compounds Vendors Principlesa Filterb Filterc

Stage 1, (2003) (WEHI Set) 93,000 4 Lead-like N Y Stage 2 (2003) (ChemBridgeDiversity Set) 30,000 1 Diversity N Y Stage 3 (2007) (CTX-DUNDEE) 16,000 20 Clustering N Y Stage 4 (2007) (WEHI ChemDiv Set,) 15,000 1 Lead-like Y Y Stage 5 (2007) (CTX Set), 136,000 2 Lead-like Y Y Stage 6 (2010) (WECC Set) 114,000 10 Lead-like Y Y a Lead-like criteria generally as follows: mw 150-400(450); rings 1-4; cLogPmax 5; rot. bondsmax 10; chiralmax 3; HBDmax 3-5; HBAmax 6-8; inappropriate functional groups; bEarly versions were used for Stages 4 and 5. cGenerally analogs could not be more than 85% similar to each other (or 90% similar to each other for most of the Stage 4 and Stage 5 libraries) as defined by the Tanimoto coefficient, and not more than 85% similar to compounds in other Stages. The Stage 2 and Stage 3 libraries were sourced in collaboration with independent 3rd parties and contain some compounds more than 90% similar to those in Stages 4-6. 244 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 Lackovic et al.

Table 2. Selection Criteria for Lead-Like Stages, with Selected Average Values in Parentheses

Property Stage 1 Stage 4 Stage 5 Stage 6

Molecular Weight 150-400 150-450 150-450 (351) 150-400 (328) Maximum number of rings 4 4 4(3) 4 (2.9) Minimum number of rings 1 1 1 1 Maximum cLogP na 5.0 5.0 (2.9) 5.0 (3.0) Maximum number of freely rotatable bonds na 10 10 (5) 10 Maximum number of chiral centres 3 3 3 3 (0.3) Maximum number of Hydrogen Bond Donors 5 5 5 3 (1.4) Maximum number of Hydrogen Bond Acceptors 8 8 8 6 (3.3) Minimum number of Hydrogen Bond Acceptors 1 1 1 1 Average PSA 67 59 and Stage 4 Libraries are combined to form the WEHI HTS example, no compound has more than 3 hydrogen bond library, although in practice the Stage 1 Library is now donors or 6 hydrogen bond acceptors. The establishment of essentially exhausted, having been in operation for over 10 the Stage 6 library has been discussed in detail elsewhere [7, years and screened over 30 times. The Stage 6 library was 8]. developed based on more stringent criteria, so that, for

Br H Br N O ** HO ** ** Br N NH S 2 N F Br O O O CH2OH **

Br O ** ** Br Cl Br O COOH CO2H Cl O NH2 Cl N HO Cl O O Br N ** Cl I H **

Br Br Cl ** NH Cl N Cl 2 NH2 ** N Br O N O S I O O H O Br N N N Cl HN O O Cl N Br O ** ** O O NH H N N O S 2 O N N HN N

Fig. (1). Muegge-modified filters for the Stage 1 library led to retention of compounds that at the time in 2002 we judged to be more acceptable (left of the vertical line), with exclusion of compounds we thought less acceptable (right of the vertical line). This filter discriminated when typical values used for cLogP (pink), minimum heavy atom count (double asterisk), and maximum rotatable bonds (red), did not. 10-Years HTS Experience at the WEHI Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 245

Despite some differences in compound selection criteria, COLLABORATIVE LIBRARIES the profiles of each of the Lead-Like Stage libraries were The Stage 2 Library is simply the ChemBridge 30K broadly similar to each other. The Stage 5 CTx library is diverse set, which we share with a collaborator. The Stage 3 representative, generally falling within the criteria for lead- Library was established as follows: Brenk et al. [13] like compounds as described by Oprea [10, 11] and others [12]. developed a computational approach for selecting focused cluster sets around common medicinal chemistry scaffolds, We determined that approximately 94% of the rather than using a pairwise dissimilarity selection method compounds in this Stage 5 library have a molecular weight such as the Tanimoto coefficient. We collaborated with them less than 400 daltons. Similarly, 67% of the compounds to refine their compound selection methodology, and then in contain only 3 rings and 72% have no chiral centres collaboration used this technique to select compounds from indicating that the majority of the compounds in the the following vendors: ChemDiv, Asinex_gold, Asinex_ collection are generally simple structures that are likely to platinum, Biofocus, Bionet, Chembridge, Chemivate, provide a good starting point for medicinal chemistry. Enamine, IBS, Life_chemicals, Mdd_inc, Myriad, Otava, Likewise, the general physicochemical properties of the Peakdale, Pharmeks, Princeton, Sigma_Aldrich, Specs, compounds also suggest that the majority of the compounds Timtec, Toslab. Operationally, the Stage 3 Library is in the library are likely to be endowed with drug-like combined with the Stage 5 Library, and we collectively refer characteristics. For example, 73% of compounds have a total to these as the CTx library or the CTx 153K HTS Library. polar surface area (tPSA) of less than 70 Å2, while 97% have 2 a tPSA of less than 100 Å . This favorable range of tPSA TARGET FOCUSED SETS values coupled with the observation that ~78% of the library compounds have calculated cLogP values between 0 and 3 Our chosen vendors roll their focus sets into the general suggests that we have a good chance of identifying screening collections and so in our bulk purchase, we compounds with favorable permeability and absorption necessarily have also imported established focus sets of lead properties in many of our HTS campaigns. like compounds. This obviated the need to purchase focus sets separately and in addition is a much more economical Scrutiny of the recently published profile of the Stage 6 approach as compounds purchased as focus sets are library confirms the similarity of different Stages of the significantly more expensive. For the Kinase Focus Set, we Lead-Like Stages (6)., apart from the obvious point that the have additionally identified and labeled compounds that Stage 6 library has a molecular weight cutoff of 400 rather contain any of the 113 cores used by the Wyatt Dundee than 450. Compound molecular weight distribution in these group to select kinase focus set compounds. The focus sets libraries merits further comment: in Table 2, it can be seen identified in the CTx 153,000 HTS Library are listed in that even though the molecular weight range for compound Table 3. This target focus sets profile is broadly similar selection is 150-400 for Stage 6, the average molecular across all Stages. weight is surprisingly high, at 328. The histogram shown in Fig. (2) helps explain this, where rather than a bell-curve, a Table 3. Target Focus Sets in the 153,000 HTS Library continued increase in compound numbers is observed. This starkly suggests how lead-likes of vendor libraries could be Target Type Number of Compounds greatly improved through populating lower molecular weight space, in particular in the 300-350 range. This would GPCR 1,717 admittedly be a synthetically intensive task to create new diversity and much harder than simply increasing compound GPCR/Ion Channel 31 numbers by “adding things on”, leading to higher molecular GPCR/Kinase 234 weights. GPCR/Peptidomimetic 49 GPCR/Ro3 2 Ion Channels 1,088 Peptidomimetics 570 Fragments 1,646 Kinase Focus Set 16,727

COMPOUND STORAGE Each of our chemical libraries is stored in the Compound Library (QCL) in Brisbane, Australia [14]. QCL

was purpose built in 2007 to support the Australian translational research community, with funds from the Queensland Government’s “Smart State Research Facilities Fig. (2). Histogram of binned molecular weight for the Stage 6 Fund” and Australian Government funding provided under library, illustrating that vendor lead-likeness could be greatly the “Super Science Initiative” and financed from the improved by better populating the lower molecular weight space in Education Investment Fund. QCL operates as a national the range 250-350. facility, and now stores in excess of 400,000 compounds and 246 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 Lackovic et al. over 200,000 natural products in both microtube and molecules will follow suit, progressing into formal pre- microplate formats. Compounds are routinely dispensed in clinical development in the next 12 months. nanolitre volumes into 96, 384 or 1536-well microtitre plate formats using a Labcyte Echo acoustic liquid handling WEHI, GENENTECH ABBOT LABORATORIES system, and shipped on dry ice overnight to our HTS (ABBVIE) TRIPARTITE COLLABORATION (2006 – Facility. Such assay ready plates are screened within a week 2010) of receipt. For the past 25 years WEHI’s Molecular Genetics of Cancer Division has been at the forefront of apoptosis MEDICINAL CHEMISTRY research [15]. A number of Bcl-2 family proteins, which It is rare that hits from an HTS campaign are suitable for govern this essential biological process, have been immediate use as pharmacological tools. Normally extensive discovered and characterized at the Institute. The discovery structure modification is required to improve potency, of the oncogenic role of pro-survival proteins such as Bcl-2, selectivity and physicochemical properties of the active Bcl-XL or Mcl-1, by promoting cancer cell survival, led to molecules. One of our strengths has been to not only work in the notion that small molecule antagonists of these pro- close collaboration with WEHI’s biologists, but also with survival proteins would induce apoptosis in tumors, and thus our medicinal chemistry group where we often provide represent a novel strategy for treating cancer. Funding from ongoing assay support for the optimization of molecules the NHMRC Program and Development Grant Schemes emerging from our screening campaigns. The importance of (Australia) and the and Society (USA) close collaboration with medicinal chemists was recognized Specialized Center of Research have enabled WEHI to early on by WEHI and a medicinal chemistry team, which undertake a HTS campaign targeting the challenging protein- has grown to over 30 medicinal chemists, many with protein interactions (PPIs) underpinning the regulation of significant industry experience, was established in Bcl-2-driven apoptosis. partnership with the HTS Facility. In collaboration with Historically, PPIs have proven intractable drug discovery external groups that can provide deep expertise in the targets, and HTS has had a poor track record in identifying relevant area of biology, hits identified by our HTS inhibitors of PPIs (although these results are probably more a campaigns can therefore progress through medicinal reflection of the libraries used in these screens rather than the chemistry development to the point of delivering useful tool approach itself). We screened our initial WEHI library compounds or molecules suitable for in vivo proof of against the pro-survival protein Bcl-W, a close relative of concept. This gap between biological hypothesis and pre- Bcl-X , identified and structurally characterized at WEHI. clinical proof of concept has often been referred to as “the L Specifically, the HTS Facility developed a competition assay valley of death”: too transnationally focused for utilizing the bead based Alphascreen technology. Since the conventional academic grant funding, too early (too risky) Bcl-2 proteins mediate the apoptotic signal through contacts for commercial funding. Co-location of medicinal chemistry between large, shallow and largely hydrophobic surfaces, it and HTS has enabled us to bridge the “valley of death” for a was expected that a large number of false positives, number of projects, and has allowed the WEHI HTS Facility especially lipophilic acids, would appear in the primary hit to play an ongoing role through Lead Generation and Lead list. This was indeed a feature of many literature reports Optimization stages of compound development. using for example Fluorescence Polarization (FP).

COLLABORATION HIGHLIGHTS FROM THE PAST The Alphascreen assay performed extremely well in this TEN YEARS context and avoided large number of false positives. The screen identified one validated hit displaying good affinity Since its inception, the WEHI HTS Facility has been for Bcl-XL and very low interference with the assay involved in over 92 HTS collaborations, including technology [16]. This successful screening campaign was partnerships with many WEHI researchers, numerous followed by a highly productive hit-to-lead effort comprising external academic organizations, Australian biotechnology medicinal chemists, structural biologists and screening companies, and international Pharma companies. These biologists assembled from three WEHI Divisions. Access to collaborations have the potential to impact disease areas the HTS Facility’s then state-of-the-art Biacore S51 allowed spanning cancer, infection and immunity, inflammation and us to measure the direct binding constant to Bcl-XL, though neglected diseases. The Facility has collaborated with many the nature of the interactions we were attempting to target Australian biotechnology companies, including Progen required careful design of the SPR assay conditions. This Pharmaceuticals, Bionomics, Biopharmica, Murigen, Biota, academic endeavor yielded the first drug-like small molecule Alchemia and Cancer Therapeutics CRC. In addition, we selective ligand of Bcl-XL with IC50 and KD values ranging have collaborated with most major Australian national 15 to 20 nM [16, 17], which complemented the dual Bcl- universities and research institutes in addition to several XL/Bcl-2 inhibitor ABT-737 discovered by Abbott major universities from New Zealand, USA and Europe. To Laboratories (now AbbVie) [18, 19]. date the work of the HTS Facility has enabled filing of These efforts in combination with our deep knowledge of international patent applications, publications, foundation of the background biology attracted the attention of leading a spin-out company, and collaborations with international biotechnology company Genentech Inc., and in 2007, WEHI pharmaceutical companies. Importantly, two projects that entered into a formal collaboration aimed at developing started their journey in the WEHI HTS Facility now have compounds in clinical trials, and we anticipate a further three novel inhibitors of BCL-2 proteins. This team was joined a year later by Abbott Laboratories; a formidable team in the 10-Years HTS Experience at the WEHI Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 247 field of Bcl-2 drug discovery. This tripartite collaboration profiling malaria antigens in serum samples collected from produced: (i) a Bcl-2 selective compound currently in the malaria endemic regions of the south pacific. clinic (Phase 2) for the treatment of Chronic Lymphocytic Leukemia (ABT-199) [20], and (ii) a series of Bcl-XL CANCER THERAPEUTICS COOPERATIVE inhibitors currently in late stage pre-clinical development. RESEARCH CENTRE (2006-PRESENT) Finally, although the original HTS campaign which led to the development of the Bcl-XL selective inhibitors was run in Founded in 2007, The Cancer Therapeutics Collaborative 2003, the constrains on publication required to maintain Research Centre (CTx) [28] is an alliance comprised of two commercial value and protect the intellectual property biotechnology companies (Bionomics and Cancer Research generated by this project imposed considerable delay on Technology UK), six Australian research organizations publication of this research. However, the recently published (CSIRO, Griffith University, Monash University, The Peter accounts of this work describe the success of this close MacCallum Cancer Centre, St. Vincent’s Institute and The collaboration between academic screening, medicinal Walter and Eliza Hall Institute of Medical Research) and a chemistry, and biology with industry. cancer council (The Cancer Council of Victoria). CTx specializes in small molecule drug discovery based on innovative cancer biology and is supported through MALARIA AND NEGLECTED DISEASES (2004-2007) contributions from member institutes and a AU$37.6 million The WEHI HTS Facility has been an active participant in grant from the Australian Government Cooperative Research the search for new treatments of the neglected diseases Centre scheme. CTx provides the expertise, funding and including, trypanosomiasis, Chagas disease, leishmania [21] resources to translate new ideas to the stage of pre-clinical and malaria. An early collaboration was established with the proof of concept and once a project has reached this stage, UNICEF/UNDP/World Bank/WHO special program for typically licenses the project to a Biotechnology or Pharma research and training in tropical diseases (TDR), and later company to progress through clinical development. the continuing medicinal chemistry was supported by the Promising targets are derived from many different areas Drugs for Neglected Disease Initiative. The initial molecular of cancer biology and include novel pathways associated targets were selected by TDR, and HTS was conducted at the with the tumor microenvironment and tumor growth. CTx WEHI Facility in collaboration with a “biology champion” focuses on with a clear clinical need, including for each target. The underlying principle was to connect breast, prostate, melanoma, lung and brain. New projects are researchers with deep knowledge of the target biology with proposed by scientists from our consortium members and our expertise in HTS and drug discovery. Three projects external contributors. were selected: trypanothione reductase from T. cruzi [22] in collaboration with Prof Alan Fairlamb, (Division of Bio- CTx draws on the strengths of consortium members by logical Chemistry and Drug Discovery University of combining their capabilities, expertise and infrastructure to Dundee, UK), farnesyl pyrophosphate synthase in colla- develop new therapeutics through the early stages of drug boration with Prof Eric Oldfield, (Department of Chemistry, discovery (hit identification, lead generation and lead University of Illinois, U.S.A.) and dihydropterin pyrophos- optimization) to deliver pre-clinical proof of concept (Fig. phokinase domain of dihydropterin pyrophosphokinase 3). Routinely, the consortium is working on multiple parallel dihydropteroate synthase (DHPS) in collaboration with Prof projects, and collaboration is facilitated by centralized Worachart Sirawaraporn, (Mahidol University, Mahidol, project management and an information technology system Thailand). Hits were selected from each of the screens in whereby experimental data is deposited and analyzed in a consultation with TDR medicinal chemistry, and compounds central database linked to an industry-standard electronic lab were prioritized and tested against intact parasites at the notebook. This combination makes all data generated at all Swiss Tropical and Public Health Institute [23]. The active of the geographically dispersed sites available to all project molecules from the DHPS screen were also tested for personnel via a virtual private network. malarial antiparasitic activity under low folate growth CTx includes two established Australian HTS Facilities conditions by Prof. John Hyde (Faculty of Life Sciences, at WEHI and Griffith University. These groups provide both University of Manchester, UK). Compounds that were complementary and overlapping expertise and infrastructure, inhibitory against the molecular target and also possessed delivering CTx a robust capability to develop and execute a significant antiparasitic activity were progressed to wide range of molecular and phenotypic discovery medicinal chemistry. Although there was generally poor campaigns. CTx also has access to comprehensive correlation between activity against the molecular target and recombinant protein production facilities with scale-up the antiparasitic activity, there were notable exceptions that available to support HTS, structural biology and other progressed to medicinal chemistry. For example two novel applications. CTx HTS campaigns are often complemented classes of trypanothione reductase inhibitors were by fragment screening to identify high quality molecular developed, 2-imidobenzimdazoles [24] and dihydroquinazo- starting points for medicinal chemistry optimization. CTx’s lines [25]. In addition, a novel class of potent benzhydryl- medicinal chemistry leadership draws on many years of large tropanetrypanocides, highly selective for Trypanosoma Pharma experience and is supported by 22 full-time cruzi, were also identified [26]. medicinal chemists. Analyses of physicochemical, One other area where we have had beneficial impact is absorption, distribution, and metabolism characteristics of by enabling other studies though developing and validating candidate molecules are conducted at the Centre for Drug robust high capacity assays. For example in collaboration candidate Optimization (CDCO) at Monash University [29], with Fowkes et al. [27] we developed a robust assay for and the Translational Research Lab at the Peter MacCallum 248 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 Lackovic et al.

CTx

Medicinal Protein Structural Translational HTS DMPK Chemistry Production Biology Cancer Biology

Centre For Drug Peter St Vincent’s WEHI WEHI Candidate CSIRO MacCallum Institute Optimization Cancer Institute

Griffith Monash CSIRO Bionomics University University

Griffith University

Fig. (3). Combined capabilities, expertise and infrastructure across multiple partners enables CTx to develop new therapeutics through the early stages of drug discovery (hit identification, lead generation and lead optimization) to deliver pre-clinical proof of concept.

Cancer Institute develops mouse models of various human NATIONAL BREAST CANCER FOUNDATION tumors to provide proof of concept in pre-clinical models. EMPATHY BREAST CANCER NETWORK (2010- 2015) Over the past six years, CTx has conducted more than 25 projects ranging in scope from early hit discovery to lead The National Breast Cancer Foundation-funded EMPathy optimization. The biological targets included kinases Breast Cancer Network [42] is a consortium of Australian important for cancer cell proliferation, migration, invasion research groups collaborating to develop new therapeutics to [30, 31], and lymphangiogenesis. In recent years, CTx has prevent the recurrence of breast cancer. In addition to also pursued a number of non-kinase targets, such as identifying biological targets for the development of new isoprenyl carboxymethyl transferase, a novel anti-mitotic, therapeutics, the network is also developing improved and epigenetic modifiers such as arginine methyltransferases diagnostics to predict the likelihood of breast cancer (PRMT5) [32] and lysine acetyltransferases (MYST3/MOZ) recurrence in newly diagnosed patients and likely response 33], novel target classes that are at the frontiers of drug to targeted therapies (companion diagnostics). discovery [34, 35]. CTx has embarked on technically The approach taken is to target the cellular process of challenging targets including protein-protein and protein- epithelial mesenchymal plasticity (EMP), with which DNA interactions. Notable in this regard, CTx has developed epithelial cells can gain mesenchymal traits such as a versatile discovery platform targeting ubiquitin increased motility and invasiveness, and increased resistance transferases. This high-throughput 1536-well assay platform reliably quantifies the ubiquitination of physiologically to cytotoxic compounds and radiation treatment [43-45]. Each of these characteristics are of importance to cancer relevant proteins by a cascade of ubiquitin ligases [36]. The cells escaping from the initial tumor site to form metastases, advantage of this functional assay approach is that it allows and as a consequence, EMP has been implicated in breast chemistry to explore interactions with multiple binding cancer recurrence and resistance to therapies [44]. interfaces in a single assay, in contrast to the standard HTS approach where the assay is often designed to detect The EMPathy network comprises several subgroups: compound binding at only a single interaction site. To date target discovery, preclinical and clinical validation, drug the functional unbiquitination assay platform has enabled discovery, mechanisms, diagnostics, and clinical trials; HTS of over 400,000 compounds against two E3 ligases, however, all subgroups are tightly integrated and information E6AP [37-39] & SIAH-2 [40, 41]. The utility of the assay flows freely between them. For three years the WEHI HTS platform is currently being extended to include de- Facility has been responsible for the early drug discovery ubiquitinating enzymes. component of this network, and for providing support to the preclinical validation group. The drug discovery work is Drug discovery is notorious for long time frames divided into two phases. The first phase is focused on between inception and conclusion. Nevertheless, CTx has developing cell-based phenotypic high content assays and already achieved several commercial successes. In 2012, two applying them to screen our focused libraries, e.g. the kinase programs delivering a highly selective FAK inhibitor and a inhibitors and known drug libraries to identify modulators of potent triple kinase inhibitor (FAK, VEGFR3 and FLT3) were licensed to commercial partners, and a third project has EMP. In addition, a set of compounds identified through a bioinformatics approach using the publicly available also recently been licensed. Connectivity Map from the Broad institute was screened 10-Years HTS Experience at the WEHI Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 249

[46]. The first phase of the drug discovery program is patent and commercial landscapes. Our Business Develop- designed to identify existing drugs that could be repurposed ment Office has access to patent databases (Patbase) and and quickly progressed to pre-clinical validation and clinical competitive intelligence databases (Global Data, Business trials. The second phase of the drug discovery program is a Insights and Datamonitor Healthcare). This experienced targeted approach, using targets identified and ranked by the business development team has supported all of the target discovery group as a basis for developing HTS assays collaborations with industry discussed above. and subsequently executing HTS campaigns. Compounds The primary objective for non-drug discovery colla- from the WEHI library of “lead-like” compounds will be borations is to generate publications. This funding model is screened against relevant targets from the list using cell- open to both WEHI researchers and external organizations. based and/or molecular-based assays. Hits from those Generally, a WEHI researcher will subscribe to this scheme screens will likely provide excellent starting points for the by funding a proportion of one of the HTS Facility personnel discovery and development of drugs in collaboration with to engage on a specific project. We generally recommend CTx or other biotechnology or pharma organizations. that a minimum of 0.3 FTE per collaboration is funded via this mechanism, so that our personnel are not spread too MECHANISMS FOR ENGAGEMENT thinly across multiple projects. The WEHI HTS Facility engages on multiple levels with Our final major financial mechanism for engagement is many of WEHI’s 14 scientific Divisions, national universi- cost recovery. This model has been implemented to ties and research organizations, national biotechnology com- accommodate short term users, and external parties such as panies, and in some instances international universities and Biotech companies, who are less willing to enter into a pharmaceutical companies. Establishing the Parkville based revenue sharing arrangement with WEHI. Cost recovery also Assay Development Laboratory in 2012 facilitated ease of works particularly well for accessing boutique libraries as access to liquid handling automation and other HTS enabling well as our Assay Development Laboratory. Much of the technologies for the ~10,000 biomedical researchers in that technology we have available within our Facility is not precinct. accessible to academic or industry researchers anywhere else A wide variety of boutique libraries stored in a purpose within Australia. built compound management facility enables repurposing Analysis of our project portfolio from the last ten years and chemical biology approaches to research. We are able to reveals that of our 92 collaborations, over 54% originated supply assay ready 96, 384 or 1,536 microtitre plates with or involved WEHI researchers, illustrating the value of containing these libraries worldwide. To date these libraries platforms like the HTS Facility to the host institute. have been used extensively not only within Australia, but Interestingly the vast majority of projects (89%) originated also in Japan and New Zealand. from academic institutes, and we were able to support Our main HTS Facility engages with researchers as early approximately 25% of these with funding from industry or as possible. In many instances funding is not immediately translational granting schemes, again illustrating the colla- available for the proposed HTS campaigns, and we work in borative and translational benefit of this type of infra- partnership with the researchers to develop HTS amenable structure. An analysis of our entire project portfolio by pro- assays and perform pilot screening campaigns, generating ject originator, project collaborator/sponsor and collaborative the necessary proof of concept data to support subsequent model is presented in Fig. (4). grant applications. We now have a proven track record with a number of our national granting schemes. In other CONCLUSIONS instances our biotech partners are in position to fund full- HTS screening has become established in academia over scale HTS campaigns, and we work with them at all stages to the past decade, but faces the challenge of justifying its place ensure desired outcomes. – not just a pale imitation of industrial screening, but Currently the HTS group supports three types of funding something more. Chemical Biology, the development of models: (i) collaborative drug discovery, (ii) non-drug probe compounds and boutique libraries, to be deployed in discovery and (iii) cost recovery. With regards to collabo- phenotypic assays with the express objective of biological rative drug discovery projects, a collaboration/revenue exploration is one key area of differentiation from industry. sharing agreement must be entered into with WEHI and the However, in the context of Australia’s national setting the basis of funding agreed prior to commencement of the WEHI HTS Facility has also established a proven track project. Generally, the collaborating partner funds the direct record in delivering translational research outcomes. The project costs (staff costs + consumables) and WEHI current era of risk aversion and decreased R&D resources in contributes the cost of providing infrastructure access and Pharma will ultimately create increased demand for new institutional overheads in exchange for a future revenue validated projects for in-licensing as companies strive to fill stream if the project is commercially successful. Capture of their development pipelines. Our strong ties with academia intellectual property is of utmost importance within this type provide us with the opportunity to respond to cutting edge of collaboration, and both the HTS Facility and Medicinal biology, and to rapidly test the translational value of Chemistry group benefit from using industry-standard hypotheses arising from these new discoveries. Groups like Electronic Laboratory Notebooks. Research teams have the the WEHI HTS Facility, WEHI Medicinal Chemistry and direct support of Dr. Julian Clark, a technology transfer CTx are ideally placed to bridge the gap between biological professional, and two experienced in-house patent counsels hypotheses and pre-clinical proof of concept. It could be from WEHI’s Business Development Office to monitor the argued that in the current climate, groups like this are a 250 Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 Lackovic et al.

ProjectType

MedChem Support 9%

Assay Development Screens 10% 71%

ProjectOrigins MechanismofEngagement

Industry 10% Australian Academic Collaborative 27% Drug Discovery Nondrug 36% Discovery 50% International WEHI54% Academic 9% Cost Recovery 14%

Fig. (4). Analysis of our entire project portfolio by project type, project origin and mechanism of engagement. The total number of collaborative projects engaging our Facility since inception in 2003 is 92. necessity, and not an option, if we are to achieve effective ACKNOWLEDGEMENTS translation of our basic research effort into improved health We gratefully acknowledge the Victorian State outcomes. Government for their initial investment via the Strategic Over the last ten years we have been affiliated with three Technology Initiative and Bio21 Australia Ltd. The early WEHI Divisions, Structural Biology, Chemical Biology, and and ongoing support of Professor (WEHI most recently Systems Biology and Personalized Medicine. Director from 1996-2009) and Professor Douglas Hilton The culture of each division has strongly influenced the (WEHI Director from 2009-Present) is also warmly evolution of the HTCS Facility. For example, during our time acknowledged. We gratefully acknowledge the contribution embedded in the Structural Biology division (2003-2010) of all members of the WEHI HTS and Medicinal Chemistry there was a strong emphasis on drug discovery and links with Groups, past and present, particularly Keith Watson and structural and computational biology. In this period we John Parisot. We thank our division heads, , developed the methodology for building our diversity David Huang and Liam O’Connor, along with Julian Clark libraries, established infrastructure for screening larger and the WEHI Business Development Office for their libraries and engaged in very successful collaborations with guidance and support over the years. Projects discussed have Pharma and Biotech companies. When we were affiliated with benefited from various granting schemes including the WEHI’s Chemical Biology Division (2010-2013), a strong NH&MRC Program, Project and Development granting interest in deploying chemistry as a tool to explore biology funding schemes, the Leukemia and Lymphoma Society, the drove development of our Boutique libraries, and National Breast Cancer Foundation, Cancer Australia, the establishment of the Assay Development lab. Most recently Australian Cancer Research Foundation, Australian Federal we have joined Systems Biology and Personalized Medicine. Government’s Cooperative Research Centre scheme, This move is part of an Institute wide reorganization of high UNICEF/UNDP/World Bank/WHO special program for technology platforms, bringing all groups with an interest in research and training in tropical diseases, Drugs for the use of advanced technologies in research together under Neglected Diseases Initiative and Therapeutic Innovation one umbrella. Other technology focused groups in this newly Australia, and the Australian Government’s Super Science expanded Division include proteomics, next generation Initiative financed from the Education Investment Fund. We sequencing, imaging, FACS and high performance computing. are appreciative of Moana Simpson and the Queensland It is early days yet, but many synergies are apparent, and we Compound Library, which is a recipient of funding from the are looking forward to exciting times ahead. Queensland Government “Smart State Research Facilities Fund” and Australian Government funding, provided under CONFLICT OF INTEREST the “Super Science Initiative” and financed from the Education Investment Fund. Authors declare that they do not have any conflict of interest. 10-Years HTS Experience at the WEHI Combinatorial Chemistry & High Throughput Screening, 2014, Vol. 17, No. 3 251

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Received: October 4, 2013 Revised: December 16, 2013 Accepted: December 17, 2013