When the human genome was sequenced in 2003, trumpets metaphorically blew throughout the realm of biomedical research. Surely with this road map of humanity’s genetic code, new drugs would quickly be developed that effectively targeted the genetic underpinnings of disease.
Now, the 15th anniversary of that historic occasion is less than six months away, and it’s been obvious for quite some time that mapping the genome, in terms of the journey to widespread genetically targeted therapies, represented something like reaching the first base camp on Mt. Everest. (Or maybe just picking up supplies in Kathmandu.) Drugs currently on the market target less than 10% of the “druggable genome”—the subset of genes that express proteins able to bind drug-like molecules. (Small molecule drugs can target only four types of macromolecule: proteins, polysaccharides, lipids and nucleic acids. For various reasons including toxicity and specificity, the latter three are limited in drug utility and proteins are the prime targets for new drugs.)
Of the estimated 20,000 genes in the human genome, approximately 3,000 of them encode proteins that are capable of being targeted by small molecule drugs. In 2013, through its Common Fund, the National Institutes of Health launched Illuminating the Druggable Genome, a program focused on exploring targets found in the four key protein families that account for the vast majority of the druggable genome: G-protein-coupled receptors, nuclear receptors, ion channels, and kinases. The first grants were made in 2014, and 2017 marked the completion of the IDG’s three-year pilot phase.
“There was a lot of discussion around choosing these families,” says Aaron C. Pawlyk, Ph.D., co-coordinator of the Illuminating the Druggable Genome project. “They are each well established in the pharmaceutical industry but they clearly have a large number of proteins—roughly 25% in each family—that are clearly and chronically understudied over time. There are members of these families that nobody ever looks at, and it’s challenging to get grant funding without some type of preliminary data or disease association.”
The idea of the druggable genome project is to be catalytic...
The idea of the druggable genome project is to be catalytic—to use a highly focused infusion of funding to generate enough data and tools that will spark disease associations in these understudied genes and proteins, the kind of associations that are required for everything from R01 grants to investments from industry.
The pilot funded two main research areas: the development of a knowledge management center, and the adaptation of scalable technologies. Funded investigators in these two areas have already published more than 60 articles in the literature related to their findings within the last three years, and all of the informatics pulled together thus far is publicly available via Pharos, the IDG’s user interface portal. In December, the IDG will announce further plans for the main implementation phase of the project.
“Several sets of reagents have already been produced by our funded investigators in the pilot phase,” says Dr. Pawlyk.
Bryan Roth, Ph.D., Michael Hooker Distinguished Professor in the department of pharmacology and division of chemical biology and medicinal chemistry at the Eshelman School of Pharmacy at the University of North Carolina, proposed to validate a platform to look at all orphan G-protein coupled receptors and generate tools and reagents for those, while Michael McManus, founder and director of the W.M. Keck Center for Noncoding RNAs at the University of California San Francisco, applied CRISPR-CAS technology to unlock the hidden kinome. Dr. Roth’s contributions are available through AddGene by searching on investigator name; additional contributions can be found on the IDG resources page.
RNA interference
Long the mainstay of drug treatment, small molecules do have their drawbacks when it comes to targeting the genome. “They target a certain step in the biochemical cascade, but unfortunately, they’re a little promiscuous in terms of off-target activities—leading to impressive package inserts,” observes Geert Cauwenbergh, Dr. Med. Sc., president and CEO of RXi Pharmaceuticals, which currently has several novel therapeutic compounds in development in dermatology, ophthalmology, and oncology.
More selective approaches to playing with druggable targets lie with messenger RNA, which transmits genetic information from DNA to the ribosome, where the proteins produced by gene expression get their sequences, and with RNA interference (RNAi), the process by which RNA molecules neutralize specific mRNA molecules and knock down gene expression or translation. RXi’s candidate drugs are based on its RNAi therapeutic platform, which includes self-delivering RNA (sd-rxRNA®) compounds that Dr. Cauwenbergh says have the potential to highly selectively block the expression of any target in the genome.
“RNAi compounds can be developed as highly selective drugs,” says Dr. Cauwenbergh. “Because these compounds are designed to target and cleave the messenger RNA of a protein through the RNAi mechanism, as long as the sequence of the mRNA for the protein is known, you have very clear targets and they are virtually all druggable.”
The company’s technology is based on RNAi compounds that can enter the cell without the need for a delivery vehicle. “sd-rxRNA compounds are designed so that cellular delivery properties are built into the compound itself. Our proprietary structure allows for uniform cell uptake and robust gene silencing in all cell types that we have tested,” Cauwenbergh says. “We have several ongoing clinical and consumer development programs based on our platform. Additionally, the technology has also been out-licensed to other drug development companies.”
Several leading pioneers in RNA interference have released a series of highly customized, genome-wide siRNA reagents, each with their own unique chemical modifications.
Dharmacon’s ON-TARGETplus siRNA reagents, for example, have been modified to provide higher specificity to the target gene. “siRNA is a two-stranded molecule, but only one strand is designed to target the gene of interest,” says senior product manager Louise Baskin. “There are two potential primary sources of off-targets. First is the opposite strand, that’s not supposed to align to anything; we modify our chemistry to prevent that from being incorporated into the silencing complex. On the other side, we’ve made modifications to the opposite strand, the strand that’s directing the targeting, preventing a small region of it from acting like a microRNA and having regulatory effects on many, many other genes. Our novel seed-region modification prevents off-target effects caused by both the sense and antisense strands, while maintaining high silencing potency.”
That premium specificity comes at a price, of course; ON-TARGETplus costs about 20-30% more than the company’s unmodified parallel product, siGenome. “Some customers may do an initial screening with siGenome and then go for something more specific with ON-TARGET,” Baskin says. “Sometimes, there is a level of comfort in having a larger hit list at first and really being able to scrutinize them at a closer level. People who go from siGenome to ON-TARGET are sometimes shocked. ‘Instead of 7,000 genes, I have 70—where did all my hits go?’ Well, they weren’t hits, and that’s why they aren’t there anymore. In general, academics tend to lean toward the lower-budget product and industry is more interested in higher specificity, although there’s plenty of crossover.”
Another industry leader, Thermo Fisher Scientific , offers two options within its genome-wide or predefined siRNA libraries such as druggable Silencer™ and Silencer™Select siRNA libraries. “Our Silencer Select siRNAs overcome some of the biggest challenges in identifying valid targets by employing powerful support vector machine design algorithms and third generation LNA™ chemical modifications to limit potential off-target effects that can occur with siRNA technologies,” says Ayaz Majid, Ph.D., global senior product manager-RNAi. Thermo Fisher’s Invitrogen™ siRNA libraries include pre-designed siRNAs and validated siRNAs functionally tested to reduce target gene expression. Thermo Fisher Scientific predominantly provides siRNA libraries with three siRNAs per target gene and with one siRNA per well (arrayed format) versus traditional pooled formats. However, custom pooled options are made available upon request.
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“Historically, vendors will take three or four siRNAs and put them into one well. While you may not have to do as many transfections in this format, with a pooled library, the challenge is that you don’t know which or if all of the siRNA sequences knocked down target gene expression,” says Dr. Majid. “Arrayed siRNA libraries means less data deconvolution from primary screens. If, for example, during a primary drug screen a specific kinase seems essential to virus infection, in an arrayed format you’ll be able to see very cleanly how many of your relevant siRNAs target that in the endpoint assay, giving you better assurances of a true hit.”
Dr. Majid said that the chemical modifications made to the Silencer™ Select library ensure that screening “hits” have a better chance of being real, versus in unmodified siRNA libraries. “We’re the only vendor in the marketplace that has proprietary designs for locked nucleic acid (LNA) modification for siRNA specifically. With this third-generation chemistry, we improve potency and you don’t have to use as much, which means less cytotoxicity in the cells during the screen.”
What’s truly druggable, anyway?
The druggable genome may turn out to be larger than previously thought, as genes and proteins that have long been thought to be “undruggable” may turn out to be more amenable to targeting after all. One example is TP53 (p53), the most frequently mutated gene in cancer, altered in about half of all human malignancies. Despite its important role in cancer, attempts to target p53 directly have often hit dead ends. But California-based Quark Pharmaceuticals recently announced the successful completion of a randomized, double-blinded, placebo-controlled multicenter Phase II trial of QPI-1002, a synthetic chemically modified siRNA acting to temporarily reduce p53 expression as a preventive therapy for acute kidney injury following cardiac surgery—one of the major complications associated with that type of surgery.
And a multidisciplinary research collaboration in the U.K. has identified an end run that may be applicable not only to p53, but a host of other undruggable targets as well. The group, brought together by Cancer Research UK’s Therapeutic Discovery Laboratories and including scientists at FORMA Therapeutics, the Universities of Oxford and Liverpool, and the MRC Laboratory for Molecular Biology in Cambridge, focused on the ubiquitin-proteasome system (UPS), which regulates the turnover of many proteins, including p53. In research published in Nature on October 18, the investigators identified two compounds that could target p53 by exploiting a unique binding site in the enzyme that controls it, ubiquitin-specific protease 7 (USP7).
“Our study shows that we can target these undruggable proteins by specifically targeting the enzymes that control them,” said lead investigator Andrew Turnbull, one of the lead researchers at the Cancer Research UK Therapeutic Discovery Laboratories, in a statement. “Combining this revelation with detailed three-dimensional structures of these enzymes, and their potential targets, means this could be the starting point to develop drugs that target them and the proteins they control.”
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