Abstract
The conference was the first of its kind in a series of trend-setting conferences conceived and organized by Select Biosciences. The word ‘probe’ is a broad term that can be interpreted to mean any one of a wide variety of agents. These include active chemistries discovered in academic screening laboratories or in government organizations (Molecular Libraries Probe Production Centers Network or the US NIH), commercially available probes (i.e., dyes, antibodies or fluorescent proteins), failed drug candidates from the pharmaceutical industry, whole-body or cellular imaging agents, specific biomarkers or tool molecules from chemogenomics and/or systems biology efforts. The conference attendees represented academia, biotech and pharma, the speaker list was stellar, and the conference organizers succeeded in bringing together all of the various incarnations of probe hunters in order to share their experiences and to network with a common purpose.
The Probe Discovery World Congress was designed to address how best to approach the one issue that has been at the forefront of drug-discovery endeavors: who is best suited among academia, biotechnology and pharmacuetical industries to discover chemical probes against key therapeutic targets and how best the process could be implemented. There were a total of 22 high-quality podium presentations by acknowledged experts, critically exploring various aspects of probe discovery over a period of 2 days; each day started with a keynote presentation.
Drug-discovery research is often described as searching for a needle in a haystack. Large compound libraries, corporate databases, virtual compound collections and supplier’s databases are routinely screened for drug candidates. The situation here is worse than finding a needle in a haystack, as we do not exactly know the size or shape of the needle. We have very limited knowledge about the ‘probe’ we need to find. Often we do not know how it looks or even if it is there. However, there is a distinct possibility that we may end up finding more than one.
The discovery of safe and efficacious drugs to combat unmet medical needs is at an all time low. While the discovery of new therapeutic drug targets in the post-human-genome sequencing era is at an all time high, the introduction of new molecular entities (NMEs) against therapeutic targets is at an all time low. Over the last decade, the NME introductions per year have almost halved, from a historical perspective. Just 21 NMEs were approved by the US FDA in 2008. By any account, the pharmaceutical industry is at a critical juncture at present, with R&D costs escalating by leaps and bounds, and the productivity, as indicated by investigational new drug filings, lagging far behind. This disconnection could be attributed to the difficulty in translating the disease biology-derived therapeutic target discoveries to the discovery and development of chemical probes and their subsequent development as preclinical candidates. In this context, the relevance of the Probe Discovery World Congress could not be overemphasized. Despite unprecedented investment (almost US$50 billion/year being spent on research), it is quite alarming to see that the drug-discovery ‘industry’ is in such a pathetic situation. To achieve sustainability of new NME introductions over time, it is important that we critically analyze this conundrum and identify ways and means to conquer the valley of death attributable to the discovery and successful development of chemical probes. An important factor in achieving this goal is the need for a strategic partnership between the public and private sectors and the biologists and the medicinal chemists at these institutions, in navigating translational research. The need for a radical paradigm shift in how we approach probe discovery and development was echoed by the majority of the speakers at this conference.
Probe development & drug discovery in academia
The session started with a keynote presentation by myself (University of Kansas, USA). The title of my talk “Discovery and development of molecular probes in academia – easier said than done”, aptly highlighted the difficulties faced by academic investigators in implementing this lofty goal. Academia has traditionally been involved in understanding the fundamental aspects of disease biology and associated biochemical targets, and in developing tool molecules against these therapeutic targets to explore their therapeutic relevance. Probe discovery has now gained unprecedented momentum in academia with the availability of vendor-supplied, diversity-based chemical libraries and an easy and ready access to oncampus high-throughput screening (HTS) laboratories. Academic researchers, who long held the view that HTS is an anti-intellectual endeavor, have been experiencing a change of mindset, and have since come to appreciate the strength of HTS in new lead discovery and in the development of molecular probes as tool molecules. With the flow of top pharmaceutical drug discovery scientific talent into academia and the industrialization of small-molecule library synthesis, academia is poised to take new lead drug discovery to greater heights. The challenge that remains to be addressed by academia is defining the end goal of HTS. Is it simply to find a ‘tool’ molecule, or is it to discover and develop a ‘drug‘? What processes and collaborations have been set in place to transform a hit to a lead? With the ‘publish or perish’ dogma that is prevalent in academia, protection of intellectual property rights is a formidable task even if one finds the proverbial ‘needle in a haystack’. Technology-transfer offices in academia continue to experience difficulty in licensing these molecular probes to pharamaceutical companies during these very early stages of medicinal chemistry explorations. I advocated the need for a better understanding of the changing landscape at the pharma–academia interface, the role of academia in translational research and what constitutes a win–win scenario for technology transfer, to strengthen opportunities for close collaboration between pharmaceutical companies and academia.
The stochastic nature of screening for probes is now a recognized discipline in academia and many of the academic screening centers (each with different specializations) have the potential to develop to a level of maturity and robustness that will allow them to contribute meaningfully to the development of chemical probes in the era of chemical genomics. For the academic drug-discovery initiatives to succeed, expert medicinal chemistry is critical. There are 143 well-defined substituents reported in the literature. If we use all of them in just three positions, we would have 1433 or 2,924,207 possible compounds. This just hints at the fact that we can never be certain. An experienced medicinal chemist might consider making a drastic structural change, based on historical perspective prior hands-on knowledge and chemoinformatics. It is in this area that the academic drug-discovery programs are at their weakest, due to lack of adequate medicinal chemistry support. This was echoed by Andy Mesecar (University of Illinois, IL, USA) in his talk entitled “Lead-discovery and probe development in smaller academic-based HTS laboratories”. I also further discussed the drug discovery and development initiatives at the University of Kansas fostering a matrix team approach and a pharmaceutical industry-based best practice, to bridge the gap between in vitro drug-target screening and chemical-probe development, in order to make drug discovery in academia a reality.
This session brought together several academic ‘screening center’ directors, as well as faculty involved in ground-breaking research related to probe discovery and development. Thomas Chung, Burnham Institute for Medical Research, provided an overview of their experiences in the network from the pilot Molecular Libraries Screening Centers Network (MLSCN) to the production Molecular Libraries Probe Production Centers Network (MLPCN) phase, including overcoming technical and organizational challenges and forming a worthwhile national resource to academic, not-for-profit and commercial research organizations. With almost 1900 HTS programs, 1300 bioassays, over 37 million data points against 171 molecular targets deposited in the PubChem database, the NIH roadmap initiatives, along with the MLSCN/MLPCN programs, have identified approximately 62 probes with submicromolar potency, at a cost of approximately US$6.2 million per probe, thus illustrating the enormous difficulty in identifying probes against novel therapeutic targets. One could also attribute this to the inefficiency in coordinating therapeutic biology with medicinal chemistry, a rare combination of skills in the biomedical science community that is more biology-oriented.
Peter Brown (Structural Genomics Consortium, University of Toronto, Canada) discussed the emerging technology pertaining to the discovery of chemical probes for epigenetic targets. Epigenetic signaling is responsible for the control of gene expression through the regulation of chromatin packing. At the molecular level, this is accomplished by proteins that catalyze methylation/demethylation and acetylation/deacetylation of histone tails. In addition, a large family of proteins recognizes or ‘reads’ the epigenetic ‘marks’. In order to understand the role of these proteins in diseases, potent and selective inhibitors of these processes are highly desired. He discussed a variety of strategies that are being employed to discover epigenetic chemical probes, including HTS and structure-based design.
The Scripps lead-identification facility in Florida is a state-of-the art, big pharmaceutical companies equivalent HTS laboratory at a nonprofit institution. Dimitry Minond highlighted the comprehensive nature of their probe discovery and development efforts against four therapeutic targets (matrix metalloproteases, SF-1 nuclear receptor, neuropeptide-Y receptor and Rock II kinase). The probe reports are publicly available, with some of the probes being made available through commercial sources. Larry Sklar, director of the University of New Mexico Center for Molecular Discovery for the NIH Roadmap Molecular Libraries Initiative, described the unique capabilities of flow cytometry towards homogeneous detection of molecular assembly and analysis of binding. He described the discovery and development of small molecule probes using HTS flow cytometry for several targets, including FPR family receptors, GPR30 estrogen receptor, low molecular-weight GTPases, quorum sensing and prostate cancer cell granularity. This renewed appreciation offers exceptional opportunities for flow cytometry, relating to the discovery of probes against biochemical targets that were not previously amenable for HTS applications.
Likewise, Michelle Arkin described the efforts at University of California, San Francisco’s (UCSF) Small Molecule Discovery Center (SMDC) in developing screening platforms against challenging targets and the technologies – from fragment-screening to high-content – which she applies in addressing them. She illustrated the usefulness of surface plasmon resonance technology, detecting small changes in molecular mass, using a Fuji AP3000 to identify fragment-based weak ligands against targets without a validated biochemical assay. This technology platform has become quite instrumental for the Fragment Discovery Center at UCSF, working with the Cancer Biology Consortium (∼11 academic drug-discovery laboratories) of the National Cancer Institute (NCI) to identify the next generation of NCI-sponsored cancer drugs with the focus on harvesting ‘high-hanging’ cancer targets.
Protein–protein interactions are essential for diverse physiological processes. Dysfunction of critical protein–protein interactions has been associated, directly or indirectly, with pathological development of a variety of human diseases. Understanding protein–protein interactions involved in intracellular signal transduction and apoptosis may provide opportunities for drug intervention in a variety of human diseases, including cancer. No drugs have yet been reported as targeting this important biological function. Thus, the protein–protein interaction interface represents a fertile ground for molecular probe discovery and has emerged as an attractive target class for drug discovery. Haian Fu, Emory University, highlighted the efforts of the Emory chemical biology center in the discovery of potential protein–protein interaction modulators.
Robert Damoiseaux (University of California, CA, USA) discussed an emerging technology platform, high-throughput nanotoxicology, while raising awareness of the toxicological issues pertaining to nanomaterials. Nanotechnology is one of the fastest growing markets, with approximately 100,000 projected nanoproducts within the next 10 years. It is expected to grow by approximately 50% each year and expected to reach US$1 trillion within the next decade. With nanomaterials now being widely used in everyday items, as well as pharmaceuticals, it is paramount to understand the biological ramifications of their use. Toward this end, the Molecular Screening Center at UCLA has employed reporter-gene based, ten pathway-arrayed HTS approaches toward biological characterization of nanomaterials. The oxidative stress response paradigm was highlighted as one of the key culprits in inducing toxicity by nanoparticles. Weibo Cai (University of Wisconsin-Madison) described the development of a multifunctional nanoplatform, a novel quantum dot- and single-walled carbon nanotube-based and single-modality and dual-modality molecular imaging probes targeting integrin avβ3, which is overexpressed on the neovasculature during tumor angiogenesis. Given the promise of this work, multifunctional nanoplatform appears to have a promising future for multimodality molecular imaging and cancer nanomedicine.
Hakim Djaballah, Memorial Sloan Kettering Cancer Center (MSKCC), gave a very charged talk, “Lessons learned from HTS: the short and long journey to probe discovery”, about the role the MSKCC HTS center has played over the years in enriching the drug-led pipeline at MSKCC. The strategic location, meaning the physical proximity, of the HTS center within the MSKCC complex has played a key role in fostering collaborations and alleviating bottlenecks in a timely fashion. This could be a model setup for emulation by academic institutions in advancing investigator-driven drug discovery approaches.
Innovative screening technologies for probe discovery
Doug Auld, NIH Chemical Genomics Center (NCGC), gave his keynote presentation on the second day of the conference, describing the chemical library profiles at NCGC and the quantitative HTS paradigm, a titration-based approach that efficiently identifies biological activities in large chemical libraries. HTS of chemical library holdings aims to identify modulators and probes of gene, pathway and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal, he articulated, will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Auld argued that traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and negatives and requires extensive follow-up testing. To alleviate this problem, his group at NCGC has developed a paradigm, quantitative HTS (qHTS), to generate concentration–response curves in an efficient and cost-effective manner, and demonstrated the feasibility of qHTS for accurately profiling every compound in large chemical libraries. qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery. While the efforts at NCGC are impressive in developing the qHTS platform, the fact remains, the fact remains that HTS of random/diverse chemical libraries is by far to generate null data since HTS is a binary modality in identifying approximately 99.9% inactives and 0.1% actives.
Sanjit Nirmalanandhan (University of Kansas) suggested that the current practice in cancer drug discovery of employing 2D cell-based assay systems may be one of the reasons for the lack of predictability in preclinical to clinical translation of new molecules and probes. His team is exploring more predictive and physiologically relevant 3D in vitro screening systems using cancer cells and cell lines, which seems to hold promise.
Terry Riss, Promega, illustrated how three different luminescent assay chemistries have been exploited for use in probe-discovery screening platforms. The firefly luciferase reaction can be used for three different types of assays by formulating reagents to quantify the amount of luciferase, ATP or luciferin. Recent developments in all three luminescence assay technologies and applications for multiplexing assays for probe discovery were presented. He described the development of short half-life luciferases for use as improved genetic reporters, mutant luciferases emitting at different wavelengths, rearranged gene version used for biosensor and a stable luciferase used as a reagent in ATP and luciferin assays.
Optical imaging facilitated by near-infrared (NIR) dyes appears promising for in vivo detection of molecular disease signatures. Studies in animals have shown that the technique is specific, sensitive and safe. However, this area of research is hindered dramatically by the lack of robust chemistry with which to obtain such dyes. Wellington Pham, Vanderbilt University, discussed methods for designing NIR dyes to support efforts to generate molecular beacons to be used in noninvasive imaging applications.
The histone code comprises many post-translational modifications that occur mainly in histone tail peptides. The identity and location of these marks are read by a variety of histone-binding proteins that are emerging as important regulators of cellular differentiation and development and are increasingly being implicated in numerous disease states. Tim Wigle, University of North Carolina, described the development of the first HTS assay for the discovery of inhibitors of methyl-lysine-binding proteins that will be used to initiate a full-scale discovery effort for this broad target class. His group focused on the development of an AlphaScreen™-based assay for malignant brain tumor (MBT) domain-containing proteins, which bind to the lower methylation states of lysine residues present in histone tail peptides. This assay takes advantage of the avidity of the AlphaScreen beads to clear the assay development hurdle presented by the low micromolar-binding constants of histone-binding proteins for their cognate peptides. The assay is applicable to other families of methyl-lysine-binding proteins and it has the potential to be used in screening efforts towards the discovery of novel small molecules, with utility as research tools for cellular reprogramming and, ultimately, drug discovery.
The final talk of the congress was given by Kal Ramnarayan, Sapient Discovery, who described structure-guided methods aided by computational chemistry techniques to discover peptidic and nonpeptidic probes against protein targets, with specific examples against targets such as enzymes, GPCRs and protein–protein interaction targets. The strategy is based on using drug-target structures (proteins), derived from either physical methods (x-ray or NMR) or computer-derived models in discovering lead molecules (probes) and subsequent optimization in combination with medicinal chemistry, exploratory biology and preclinical studies. Sapient’s platforms, genes to leads and fragments to leads, were successfully used in the discovery of SHP-2-, IKK-b kinase, anthrax lethal factor inhibitors and small-molecule antagonists for TNF receptor and Id1 targets.
aDREAM Conference
In conjunction with the Probe Discovery Conference, Select Biosciences graciously sponsored a pilot Development of Robust Experimental Assay Methods (aDREAM) workshop, organized by the NIH/NCGC. The purpose of this workshop was to provide and establish a forum where pharmaceutical industry-trained experts, now entering academia, can impart their knowledge to their newfound colleagues, about the pharmaceutical industry best practices in early stage drug discovery. The goal of this free workshop was also to create awareness of the need to continuously update the Assay Guidance Manual on assay standards and best practices in HTS, and to recruit authors to contribute new chapters and updates to existing chapters on the NCGC Wiki site. The workshop started with an introductory presentation by Doug Auld, NIH NCGC, addressing the goals of the conference, followed by presentations dealing with issues concerning the Assay Guidance Manual. The discussions were moderated by Jeff Weidner (Eli Lilly) and Lisa Minor (Johnson & Johnson).
In closing, the pharmaceutical industry has come to recognize that early-stage drug discovery is the key bottleneck in its quest to take drug molecules in to preclinical and clinical development. The pharmaceutical industry is now faced with its key products at the edge of patent expiration in the ensuing years and the need for innovative paradigm(s) towards the discovery of drug leads is urgent more than ever. The inaugural Target Discovery World Congress organized by Select Biosciences in August 2009 highlighted the need to critically interrogate the druggable human genome in order to expand the target universe and facilitate the discovery of new, novel and safer drugs with greater selectivity and specificity. Likewise, the inaugural Probe Discovery World Congress, again organized by Select Biosciences, re-emphasized the role of academia, not only in the discovery of novel therapeutic targets, but also in the discovery and development of assay technology platforms and their usefulness in finding ‘chemical probes’ against these drug targets. Michelle Arkin at UCSF aptly said that academic investigators not only drive biological innovation in medicine, but can also drive technological innovation in drug discovery – making it possible through the enhanced funding opportunities through NIH’s roadmap initiative and the MLSCN and MLPCN programs. The talks presented at this conference are a clear example of the key role that academia has come to play in alleviating the drug discovery bottleneck by providing chemical probes, the starting points for lead optimization and the development of clinical candidates to address unmet medical needs. An effective partnership between academia and the pharmaceutical industry, with respect for the goals of these two diverse institutions, is paramount in our eternal quest for safe and efficacious drugs to fulfill the unmet medical needs. The conference organizers are to be commended for addressing a timely topic and bringing together experts in the field to share their views on the current status of probe discovery in finding new, novel and safer drugs.
Financial & competing interests disclosure
The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.