Features Tech News LY Leveraging the Promise of Chemical Genomics IO N S IS M Stuart Schreiber of Harvard University and the Broad Institute. Source: Len Rubenstein P E R inferring the effect on proteins, which are the typical interaction partners for small molecules. But that correlation is imperfect. “The main shortcoming today is that we don’t have a way to test therapeutic hypotheses in physiologically relevant conditions with small molecules,” Schreiber explains. One challenge here is developing small molecule libraries that represent biological diversity. The chemical space of small molecules is nearly infinite, exceeding 1060, more molecules than could be synthesized or screened. Still, many biologically relevant classes of molecules are not wellrepresented in current libraries, especially complex natural products (See sidebar: Library bias and enhancing diversity). As a result, research efforts continue in the attempt to fill the gaps in biologically relevant chemical space. Schreiber’s group at the Broad Institute, as well as other groups, are now using one approach called diversity oriented synthesis to construct new libraries, with the NIH supporting a variety of this research through their chemical methodology and libraries development (CMLD) initiative. Part of the idea is to tap into the creativity and resources of synthetic organic chemists, who previously might not have been involved in biology, says Jeffrey Aubé of the University of Kansas, a principal investigator on one of the CMLD grants. Working initially with several PIs whose groups develop new synthetic methods and P R IN T W IT H High-throughput screening (HTS) was once the exclusive domain of large pharmaceutical companies. But with the NIH’s Roadmap initiative, the Molecular Libraries Program (MLP), alongside publicly available libraries, today academic and government laboratories have become centers for looking at small molecules as probes of biological function, and are even taking steps toward developing lead molecules for drug discovery. In many ways this shift makes sense, this herculean effort feeds on an open cross collaboration among chemists, biologists, and informatics experts. Initial work that married synthetic chemistry and biological assays with HTS is beginning to pay dividends, both in innovations that allow researchers to directly test the therapeutic benefit of molecules in physiologically relevant systems and in the development of small molecules that tweak activity within individual cells. “I think it’s stunning. The progress of the MLP during this short period of time is remarkable,” says Stuart Schreiber of Harvard University and the Broad Institute. Although these efforts are showing promise, real challenges lie ahead. With the vastness of chemical space, researchers are trying to increase the diversity of chemical structures available for screening. And as more HTS datasets emerge, further chemistry is needed verify and optimize those hits to produce biological probes or lead compounds for drug discovery. O N NIH’s Molecular Libraries Program is giving chemical genomics researchers the tools to develop molecules to probe cellular function. But will all pieces of this herculean effort be enough to bridge the chasm toward translational research? R E Mining chemical diversity According to Schreiber, most compounds fail in the clinic not because they didn’t engage and modulate a target of interest, but because that process either didn’t lead to efficacy or it produced unexpected toxicity. “We’re really bad at making predictions,” he acknowledges. Many of those early predictions came from looking at nucleic acid targets and Vol. 52 | No. 1 | 2012 17 synthesize compounds with novel structures, Aubé and his colleagues have the resources to further advance these efforts into compound libraries that can be used for high throughput screening. After verifying that the molecules and structure represent nominally new chemical space, a postdoc or graduate student from the synthetic group might work with his staff to produce a library of 120-140 compounds which are prepared under quality controlled conditions for screening applications. The information is deposited in the public database, the Molecular Libraries Small Molecule Repository (MLSMR). Screening at multiple concentrations While building library diversity is useful, researchers are also designing screens to elicit maximum information, and using cheminformatics to analyze and make predictions from the resulting data. Early drug discovery screens typically tested small molecules at a single cutoff concentration as a way to screen a maximum number of compounds quickly and inexpensively. James Inglese of the NIH’s Chemical Genomics Center questioned that approach. “My thought was, well, why are we screening at just one concentration?” he says. “You get a lot of false positives, or you miss things.” Instead, Inglese and his www.BioTechniques.com Features colleagues developed a new method, called quantitative high throughput screening (qHTS), which can screen across up to five orders of magnitude difference in the chemical concentrations. The result is a dataset of titration curves which allows researchers to begin to see pharmacological trends such as the IC50 and EC50 within their initial screens. In one recent application of qHTS, Inglese and his colleagues married qHTS a biological assay of the malaria parasite, Plasmodium falciparum, developed by Inglese’s colleagues at the National Institute of Allergy and Infectious Disease (NIAID) that can be used with their low volume 1536-well microtiter plates. By using this strategy to examine the effect of library compounds on parasite viability, and because of the genetic variability of parasites from different locations, the data has helped the researchers pinpoint the basis of some of these response differences and look at resistance factors to particular drugs (1,2). Chemistry to follow up on the screen Chemistry is also important after the screen. An initial high throughput screen produces a number of hits, molecules that act on a target in some way. But this is just the beginning; these molecules need to be validated and optimized, and that’s where medicinal chemistry centers play a role in the Molecular Libraries Probe Production Centers Network (MLPCN): Aubé directs the Kansas Specialized Chemistry Center and Vanderbilt University has its Specialized Chemistry Center for Accelerated Probe Development Once that initial screen is complete, Aubé and his colleagues join forces with a team composed of researchers from the screening centers and assay developers to decide which initial hits should be pursued using confirmatory assays. It is then that the medicinal chemists will follow up on those hits and construct structure-activity relationships. “There are milestones and targets of potency and selectivity and physical properties that are associated with each of the probes,” Aubé says. Probe molecules can also feed back into investigations of the molecular mechanisms that generated the hits in the first place. Many of the screens are phenotypic: researchers are looking for a desired cellular response and identify hit molecules that generate that response. But most researchers would also like to go beyond phenotype to better understand the biochemical pathways that generated a particular response. Vol. 52 | No. 1 | 2012 After all this work is complete, the results are published as probe reports on the MLP website and purified samples are kept available for other researchers to use. One probe report published by the group last year details a molecule with a novel structure that blocks the activity of CDC42, a GTPase that regulates the cell cycle. Since late February, Aubé estimates he’s received a dozen requests for this particular probe. “As the network has gone forward, we’ve been making a deliberate effort to ensure that we’re providing not only compounds that are going to inform basic biology, but, when it’s reasonable to do so, also provide compounds that might be the first step of a lead compound for drug discovery or translational research.” The follow-up gap Outside the specialized groups that NIH has funded to carry out medicinal chemistry work, finding the resources to follow up on initial screening hits can present a bottleneck in chemical genomics research, says Hakim Djaballah, Director of the High Throughput Drug Screening Facility at Memorial Sloan-Kettering Cancer Center. Even with these NIH funded centers, the resources devoted to this piece of the puzzle are relatively small compared with the number of screening centers that are producing hits from high throughput screening. Following up on screening hits is risky and is not necessarily hypothesis driven, which can make it difficult to secure funding. “I’m involved in at least 3 or 4 projects like that that are tangibly parked because we don’t have any money to move them forward,” Djaballah says. Even once medicinal chemistry is done there’s still the question of doing secondary and tertiary screening on the optimized compounds. “The professor who develops the assays then has to come up with the funding for the secondary and tertiary assays and the functional assays to discern whether these [optimized] compounds are biologically relevant,” notes Rathnam Chaguturu, director of the High Throughput Screening Laboratory at the University of Kansas. Non-profit foundations with an interest in a particular rare disease can form one potential source of funding for projects aimed at questions in particular disease areas. Inglese, who studying Charcot-MarieTooth disease, a rare peripheral neuropathy, has funding from a foundation that supports this research. Another approach that could shorten the process of lead optimization is to start with a smaller pharmaceutical library of known drug compounds, such as the one NCGC assembled and published in Science Translational Medicine (3). That process of repurposing known drugs, whose potency and activity have already been thoroughly Library bias and enhancing diversity In the early days of high-throughput screening (HTS), a lot of potential hits turned out to be artifacts. Brian Shoichet and his colleagues at UCSF saw similar patterns in their own structure-based screening work and started to wonder if the problem was with the methods being employed or in the molecules that were being used for screens. Around the same time, Shoichet became aware of articles estimating the vastness of chemical space, which led him to another question: with the limited number of compounds available, why does high throughput screening ever work at all? In 2009, Shoichet and his colleagues published a computational analysis revealing that existing libraries are biased toward biogenic molecules (4). Molecules in existing libraries tend to be particularly good for targeting G-protein coupled receptors, ion channels, and kinases. Therefore, if researchers blindly increase chemical diversity, the likelihood of hits might actually get worse. A better strategy is to think in terms of the molecules and scaffolds organisms likely have in their environments, such as natural products. The challenge with natural products is that many have not been synthesized, and it turns out they are often difficult to isolate in sufficient quantities from the organisms that produce them. One emerging solution is to use natural products extracts, mixtures of compounds that are isolated from microorganisms (5). But producing extracts that are clean enough and homogenous enough for screening is complicated. Such extracts might include thousands of compounds of similar molecular weight, yet only a handful might be active in a screen. According to Brian Bachmann of Vanderbilt University isolation is the real problem. “That’s where we’re putting our energy right now. If you had a method that could beam compounds out of an extract and put them in tubes, that’s what we need.” -SW 18 www.BioTechniques.com Discernable results, discover EXPOSE IHC Quantitative high throughput screening (qHTS) allows researchers to obtain dose-response relationships during their initial screen of compounds. By varying the concentrations of compounds being screened, researchers can quickly construct response curves. Based on these patterns of activity, they can group compound classes based on their activity profiles and build structure-activity relationships. Vol. 52 | No. 1 | 2012 1. Yuan et al. 2009. Genetic mapping of targets mediating differential chemical phenotypes in Plasmodium falciparum. Nature Chemical Biology 5, 765. 2. Yuan et al. 2011. Chemical genomic profiling for antimalarial therapies, response signatures, and molecular targets. Science 333, 724. 3. Huang et al. 2011 The NCGC Pharmaceutical Collection: A comprehensive resource of clinically approved drugs enabling repurposing and chemical genomics. Sci Trans Med. 3, 1. 4. Hert et al. 2009 Quantifying biogenic bias in screening libraries. Nature Chemical Biology 5, 479. 5. Cruz et al. 2011. Titration-based screening for evaluation of natural product extracts: Identification of an aspulvinone family of luciferase inhibitors. Chemistry & Biology 18, 1442. • Reduced background with a biotin free detection system • Flexible options HRP/DAB, FRP/AEC and AP red • Economical less antibody required Written by Sarah Webb, Ph.D. BioTechniques 52:17-19 ( January 2012) doi: 10.2144/000113796 To purchase reprints of this article, contact:biotechniques@fosterprinting.com Order any EXPOSE IHC or polymer IHC kit from our range before January 31st 2012 and receive a FREE IHC Methods book Enter the following promotion code at time of purchase: BIOTIHC-Y7TS6 19 www.abcam.com/biofreeihc 164_11_GM tested, for new indications provides a fast track to the clinic. “A disease foundation wants that,” says Inglese. “They could use that information to begin a clinical trial with patients.” For those molecules that might have clinical applications, researchers and institutions would like to hold on to the intellectual property rights, but use of the NIH Molecular Libraries Program resources requires researchers to deposit the structures of the molecules that they discover into a public database. “By definition and by mandate, once done you have to post the structures into Pubchem. And once you’ve posted in Pubchem database, you’ve lost your intellectual property,” notes Chaguturu. “That’s one of the biggest drawbacks.” Although currently funded through 2014, questions surrounding future funding for the Molecular Libraries Program loom. High throughput screening is expensive, and it’s a kind of business transaction in the academic world, says Djaballah. “The initial investment is written off by a university, but maintaining [HTS centers] is expensive.” “This network has put into place some really fantastic infrastructure and has enabled some tremendously talented scientists from all over the country to engage in this work,” says Aubé. “It would be wonderful to see those capabilities leveraged in the future, and I’m not exactly sure how that will happen.” • Increased sensitivity a smaller antibody complex