Quorum sensing synthetic biology project http://bit.ly/LO1ynR

Way back in May 2000, Decision Resources published my short report entitled “New approaches to small-molecule antibacterial drug discovery” as part of its Spectrum Life Sciences series. As might be expected, the report is now out of print.

The report was a brief review of then-novel approaches to antibacterial drug discovery, in the face of the increasing level of antibiotic resistance in pathogenic bacteria. These approaches included genomics and such technologies as high-throughput screening against bacterial-specific targets.

However, the most interesting part of the report was a section on using the study of bacterial physiology to identify targets that are important for the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival. The hypothesis behind these studies was that it might be possible to develop compounds that prevent these bacteria from causing disease, without selecting for resistant strains of the bacteria.

Antibiotics typically kill or prevent proliferation of bacteria by targeting biomolecules involved in such essential processes as cell wall synthesis, DNA proliferation, or protein synthesis. Treating large populations of bacteria with such agents inevitably selects for a few resistant mutant cells. These proliferate, mutate further, and give rise to antibiotic resistant populations. However, if a therapeutic targets a nonessential pathway that is involved in pathogenesis, resistant populations might not be selected for. That was the hypothesis.

This field of bacterial physiology for drug discovery focused on two related areas–virulence factors and quorum sensing. Virulence factors are not expressed by a strain of pathogenic bacteria in vitro, but are expressed only when the bacteria infect a host. Once expressed, they enable the bacteria to colonize the host and cause disease. Examples of such virulence factors include secretion systems that deliver bacterial effector proteins into host cells. These effector proteins may, for example, kill host cells, inhibit cytokine production or phagocytosis, or may mediate bacterial entry into the host cells.

Quorum sensing is a system by which certain bacteria can monitor their own population density. They accomplish this by secreting specific autoinducer molecules. When the concentration of an autoinducer reaches a critical threshold value (as the result of an increase in bacterial population density), it triggers specific response systems, causing the induction of sets of genes that are only expressed at high population density.

For example, many gram-negative bacteria (e.g., Pseudomonas aeruginosa, Vibrio cholerae, and Escherichia coli) use specific acyl homoserine lactones (AHSLs) as their autoinducers. P. aeruginosa has two quorum sensing systems that use the AHSL autoinducers butyrylhomoserinelactone and 3-oxododecanoylhomoserinelactone, respectively. These systems (operating via specific receptors for the auotoinducers and interacting with each other) control the induction of several genes, some of which are virulence factors. Some of these genes enable the bacteria, when they are at sufficient density, to form biofilms (slimy mats of bacteria and polysaccharide matrix).

P. aeruginosa is an opportunistic pathogen, causing infection in the lungs of people with cystic fibrosis, burn patients, and other hospitalized patients. These infections cause death in over 80% of cystic fibrosis patients. The ability to form biofilms renders the bacteria resistant to antibiotics and to the patient’s own immune system.

Other gram-negative bacteria that form biofilms have been implicated in dental caries, peridontitis, osteomyelitis, and numerous nosocomial infections. Bacterial biofilms can also form on the surface of implanted medical devices, such as catheters and mechanical heart valves, and cause device-related infections.

The gram-positive human pathogen Staphylococcus aureus also has a quorum sensing system. However, it does not use an AHSL as an autoinducer. The S. aureus autoinducers are peptides that contain an unusual thiolactone structure (i.e., a thol ester-linked cyclic structure). The S. aureus quorum sensing system controls the synthesis of virulence factors responsible for the pathogenicity of this organism in vivo. Although specific peptides induce virulence factors in a given strain of S. aureus, there are other specific peptides that inhibit the induction of virulence in strains of the organism other than the one secreting the inhibitory peptides. That finding suggested that researchers should be able to develop specific agents to shut down S. aureus pathogenesis by targeting the quorum sensing system.

Interestingly, quorum sensing-based systems have been used in projects for the International Genetically Engineered Machine (iGEM) competition, an annual undergraduate synthetic biology competition. See the figure above, which was taken from the 2009 Chiba University (Japan) iGEM project.  [http://2009.igem.org/Team:Chiba/Project/Signaling-system]

Quorum Sciences and Vertex Pharmaceuticals’ research on quorum sensing

At the time of the writing and publication of our antibacterial drug discovery report, there was a company, Quorum Sciences (Iowa City, IA) that had been established to commercialize the findings of leading researchers on bacterial quorum sensing. As the result of two successive acquisitions in 2000 and 2001, Quorum Sciences passed into the hands of Vertex Pharmaceuticals (Cambridge, MA). In 2006, Vertex researchers and their academic collaborators published a report on the discovery of novel specific inhibitors of the P. aeruginosa quorum sensing system. The last author of this report was quorum sensing pioneer E. Peter Greenberg, formerly of the University of Iowa and chief scientific officer at Quorum Sciences, and from 2005 to the present at the University of Washington School of Medicine. The compounds identified in the 2006 report, discovered via high-throughput screening of a diverse 200,000-compound chemical library, resembled the natural AHSL that binds to the P. aeruginosa quorum sensing receptor LasR. (LasR is a transcription factor that when bound to its specific AHSL, mediates the expression of a set of downstream genes, including those that encode virulence factors.) The researchers concluded that the novel quorum sensing inhibitors might be useful chemical tools, but not drug leads.

In 2010, other academic researchers published a report on the discovery of novel antagonists and agonists of the P. aeruginosa quorum sensing receptor LasR, which were of lower molecular weight and otherwise structurally distinct from the natural P. aeruginosa AHSL. However, these compounds were still deemed to be scaffolds for chemical tools, not drug leads. Nevertheless, the researchers speculated that the compounds “could, with further development, provide a pathway for the design of novel antivirulence agents”. Other researchers are continuing studies aimed at discovery of quorum sensing receptor antagonists, whether synthetic organic molecules or natural products. These involve studies with quorum sensing systems of both gram-positive and gram-negative bacteria.

The 2006 report appears to be the last Vertex publication on quorum sensing. However, Vertex continues to conduct research on antibacterial agents. And the company has a facility in the University of Iowa BioVentures Center (Coralville, IA),  which is a continuation of the old Quorum Sciences Iowa facility. As of 2009, Vertex’s Iowa-based team consisted of seven full-time scientists, working on development of antibacterials, and agents to treat hepatitis C and cystic fibrosis, among other areas. The Iowa group participated in the development of Vertex’ now-marketed anti-hepatitis C virus (HCV) agent Incivek (telaprevir).

The May 2012 article “Freezing Time” in The Scientist, and discovery of novel quorum sensing inhibitors

The May 2012 issue of The Scientist contains an article entitled “Freezing Time”, by Vern L Schramm, Ph.D. (Albert Einstein College of Medicine (Bronx, NY). The article focused on design of “transition state analogues”, i.e., compounds with a chemical structure that resembles the transition state of a substrate in an enzyme-catalyzed reaction. Transition state analogs usually act as enzyme inhibitors by blocking the enzyme’s active site. They are exquisitely potent and specific inhibitors, which act at extremely small doses. This makes these compounds potentially attractive as drugs.

A transition state analogue inhibitor that was designed by Dr. Schramm and his colleagues in the early 2000s as an early proof-of-concept molecule is immucillin-H, or forodesine. This is a transition-state analog inhibitor of purine nucleoside phosphorylase.  Forodesine is being developed by BioCryst Pharmaceuticals for treatment of relapsed B-cell chronic lymphocytic leukemia, and the results of a Phase 2 trial were published in 2010.

As described in Dr. Schramm’s May 2012 article, his laboratory has been applying their transition-state analogue technology to the field of quorum sensing in bacteria. Instead of targeting the recognition of AHSLs by quorum sensing receptors such as LasR, the researchers targeted the key enzyme in the AHSL biosynthesis pathway in gram-negative bacteria, known as 5′-methylthioadenosine nucleosidase (MTAN). The biosynthetic pathway for the production of AHSLs, including the key role of MTAN, had been elucidated by Dr. Greenberg and his colleagues in the late 1990s.

Dr. Schramm and his colleagues published the results of studies of three transition state analogues that potently inhibited MTANs of gram-negative bacteria. For example, they inhibited the Vibrio cholerae MTAN with dissociation constants of 73, 70, and 208 pM, respectively. They inhibited MTAN in cell of a virulent strain of V. cholerae with IC50 values of 27, 31, and 6 nM respectively, disrupting autoinducer production in a dose-dependent manner without affecting bacterial growth. The compounds were also potent inhibitors of autoinducer production in an enterohemorrhagic strain of Escherichia coli. The transition-state analogues did not inhibit growth in either V. cholerae or E. coli, but one such compound reduced biofilm production by 18% in E. coli and 71% in V. cholerae.

Moreover, the MTAN inhibitors did not appear to select for bacterial resistance in vitro. When V. cholerae bacteria were grown for 26 generations in the presence of a large excess of MTAN inhibitors, subsequent generations of these bacteria were equally sensitive to inhibition by these compounds as bacteria that had not been previously exposed to the inhibitors. These results are consistent with the hypothesis that agents that inhibit targets that are important in the ability of bacteria to cause disease, but are not essential for bacterial proliferation or survival might not select for drug resistance.

As Dr. Schramm said in the May 2012 article in The Scientist, it remains to be seen whether the MTAN-targeting transition-state analogs developed in his laboratory can translate into novel antibiotics that do not select for resistant pathogens. As of March 2009, Dr. Schramm’s team had developed over 20 potent MTAN inhibitors, which will be specific for bacteria and should have no effect on human metabolism. These compounds have been licensed to Pico Pharmaceuticals (Melbourne, Australia), which plans to develop and initiate clinical trials. Dr. Schramm is a Pico Pharmaceuticals co-founder and chairman of its scientific advisory board. Pico claims that one of its quorum sensing inhibitors, designated as PC0208, has demonstrated proof-of-concept in preclinical studies, and now has “pre-IND” status.

Lessons from these studies

Dr. Schramm’s discovery of novel quorum sensing inhibitors was made possible by a strategy that involved a combination of biology-driven drug discovery and sophisticated chemistry technology. The biology-driven drug discovery strategy involved a combination of 1. Building on the quorum sensing studies of Dr. Greenberg and others, and adopting the strategy, as reviewed in our 2000 Spectrum report, of targeting the quorum sensing system in order to discover agents that would have the possibility of not triggering resistance, and 2. Targeting a critical, bacterial-specific pathway enzyme that is upstream of the recognition of AHSLs by quorum sensing receptors (the usual target of most researchers in this area). This enzyme, MTAN, has a key role in the biosynthesis of AHSLs.

The sophisticated chemical technology employed by Dr. Schramm and his colleagues was of course the transition state analogue technology developed in his own laboratory. Combined with the biology-driven strategy described in the last paragraph, Dr. Schramm’s approach has succeeded in the discovery of compounds that are potential drug candidates, while approaches based on high-throughput screening for AHSL antagonists have so far failed to produce any such compounds. Dr. Scharamm’s laboratory has also obtained evidence that treatment with their compounds should not result in the selection of resistant strains of pathogenic bacteria.

It is possible that other chemistry approaches might be successfully employed to discover quorum sensing inhibitors, both for gram-negative bacteria and gram-positive organisms such as S. aureus.

As we have discussed in numerous articles on this blog, biology-driven drug discovery strategies, often coupled with innovative approaches to chemistry (in the case of small-molecule drug discovery) are applicable to very many different targets involved in a whole range of human diseases. (Biology-driven drug discovery has also been central to discovery and development of many successful large-molecule drugs.) The quorum sensing case study in this article is a simple, understandable, and elegant example of such a strategy.

In addition to the scientific, clinical, and medical aspects of antibacterial drug discovery, the other major issue is the business of antibacterial discovery and development. The economics of drug discovery and development have shifted pharmaceutical industry investment away from the development of drugs targeting short course therapies for acute diseases (such as antibacterials) and towards long-term treatment of chronic conditions.  At the same time, discovery of novel antibacterials has gotten more difficult. As a result, during the 2000-2010 period, such companies as Wyeth, Aventis, Eli Lilly, GlaxoSmithKline, Bristol-Myers Squibb, Abbott Laboratories, Proctor & Gamble, and Merck have either deprioritized anti-bacterial R&D or left the field altogether. Meanwhile, antibiotic resistance, which was a problem in 2000, has become an even greater problem in 2012, in some cases reaching crisis proportions [e.g, methicillin resistant S. aureus (MRSA) that is also resistant to the drug of last resort, vancomycin].

As a result of these economic, scientific, and medical challenges, a €223.7 consortium of five pharmaceutical companies and leading academics, called NewDrugs4BagBugs (ND4BB) was launched in Europe in May 2012. The program is envisioned to involve a three-stage approach – to improve the understanding of antimicrobial resistance, to design and implement efficient clinical trials, and finally, to take novel drug candidates through clinical development.

And at least one venture capitalist has observed that biotechs that specialize in antibacterial drug development (as well as those that specialize in other areas that have been deemphasized by Big Pharmas) have provided “contrarian opportunities” in biotech venture. According to a June 2 2012 article by Bruce Booth of Atlas Venture published in Forbes, what has been deprioritized by some (or several) Big Pharmas, are likely be re-prioritized by others several years later. Such antibacterial drug developers as Calixa, Cerexa, Novexel, Neutec, Paratek, Pennisula, Protez, and Vicuron have produced some of the best returns in biotech venture capital from merger/acquisition exits. These biotechs included companies that were built around compounds outlicensed from Big Pharma, and others that conducted new research on novel targets, especially for MRSA and other resistant bacteria.  By taking advantage of a strategic depriorization in Pharma, these biotechs and their venture backers were able to create considerable value in the past decade out of antibacterial drug development.

Meanwhile, antibiotic specialist Cubist Pharmaceuticals (Lexington, MA) remains an independent, and profitable, biotech company that is continuing to conduct R&D, including on discovery and development of agents to treat pathogens that are resistant to current antibiotics. It has expanded into development and marketing of peripheral mu-opioid receptor antagonists (including via acquisition of Adolor in 2011), and has recently expanded its R&D facilities.

Can Pico Pharmaceuticals (which has oncology programs in addition to antibacterials) experience similar success?

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please contact us by phone or e-mail. We also welcome your comments on this or any other article on this blog.

Brown fat in humans

The CNS-targeting “Class of 2010” drugs

We have not had an article on obesity therapeutics on this blog since February 1, 2011. At that time, we had an article entitled “That’s all, folks!”, complete with the old Warner Brothers Porky Pig graphic. As of that date, all three of the obesity drug candidates that came up for FDA review in 2010-–Vivus’ Qnexa, Arena’s lorcaserin, and Orexigen’s Contrave–were rejected for approval by the FDA, and sent back for further studies. Also in 2010, the then-marketed antiobesity drug sibutramine (Abbott’s Meridia) was withdrawn from the market at the FDA’s request. All of these agents targeted the central nervous system (CNS).

Concern about long-term safety was the major consideration in the rejection of the NDAs for Qnexa, lorcaserin, and Contrave, and safety issues were also the reason for the withdrawal of sibutramine. That left only one anti-obesity drug approved by the FDA for long term use– orlistat (Roche’s Xenical), with no new drugs In sight. The outlook for obesity drugs was gloomy indeed.

However, as of May 2012, after the further studies prescribed by the FDA in 2010, two of the obesity drug Class of 2010–Qnexa and lorcaserin have received positive votes by the FDA’s Endocrinologic and Metabolic Drugs Advisory Committee, and are awaiting final FDA action later this year. Contrave, after a February 6, 2012 agreement with the FDA, appears to be on track for possible NDA resubmission in 2014.

We shall continue to follow progress with the consideration of the resubmitted NDAs for Qnexa and lorcaserin in 2012.

Novel approaches based on the physiology of brown fat

Meanwhile, there is renewed interest in earlier-stage, alternative obesity therapies based on the physiology of brown fat, also known as brown adipose tissue (BAT). The May 1, 2012 issue of The Scientist has an article by the publication’s associate editor Edyta Zielinska entitled “Treating Fat with Fat: Is brown fat ready for therapeutic prime time?”  This article focuses on new discoveries in brown fat physiology, and on entrepreneurial companies that are attempting to develop these discoveries into therapeutics.

On the Biopharmconsortium Blog, we also have an article on brown fat physiology and companies attempting to develop therapeutics based on these findings. The article is dated November 17, 2010. As we state in that article, brown fat researchers and companies are seeking to develop therapeutics that work by increasing energy expenditure, rather than the usual approaches of decreasing appetite (as with the Class of 2010 CNS-targeting antiobesity drugs) or blocking absorption of fat in the gut (as with orlistat).

More specifically, these researchers and companies intend to discover and develop drugs that increase the amount and/or activity of BAT, which is a type of mitochondria-rich adipose tissue that oxidizes fat and dissipates the resulting energy as heat rather than storing it. The mitochondrial protein UCP1 (uncoupling protein 1) is the key biomolecule that makes this process possible. BAT has long been known to be central to non-shivering thermogenesis in rodents, for example to maintain body temperature when they are exposed to cold.

Until recently, researchers believed that in humans, significant populations of BAT cells were found only in infants. However, in recent years researchers found that adult humans possess reservoirs of brown fat in the neck region and other areas of the upper body as well as in skeletal muscle. Adult human BAT can be stimulated by acute exposure to cold and via the sympathetic nervous system, and by various pharmacological agents.

Energesis’ autologous brown adipose tissue transplantation program

Our November 17, 2010 article in particular focused on the Boston-based early-stage company Energesis Pharmaceuticals. Energesis was confounded by Olivier Boss, PhD (formerly of Sirtris Pharmaceuticals), Brian Freeman, MD (former Venture Partner at GreatPoint Ventures), and Jean-Paul Giacobino, MD (Professor Emeritus, University of Geneva Medical School, Switzerland). Dr. Boss serves as Energesis’ Chief Scientific Officer, and Dr. Freeman as its Chief Operating Officer.

Energesis is also mentioned in the new article in The Scientist. According to that article, Energesis is using brown fat “stem cells” (which are precursor cells found in skeletal muscle that can differentiate into either muscle or brown fat) to identify novel targets that activate brown fat. Energesis researchers then work to discover new drugs that address these targets. They are also investigating transplantation of brown fat “stem cells” as an obesity therapy.  According to the article, Energesis is planning to initiate clinical trials of their therapies within 2 to 3 years.

In October 2011, Energesis was awarded a U.S. Department of Defense Small Business Technology Transfer (STTR) grant to develop therapeutics based on autologous BAT transplantation. The project is a feasibility study to define a source and culture system for the generation of human BAT for autologous transplantation therapy. It will involve isolating and characterizing the best brown adipocyte progenitor sub-population from human muscle biopsies, expanding these cells, and establishing the optimal culture conditions for in vitro differentiation to generate approximately 50 grams of BAT cells for transplantation. This project is being conducted in collaboration with Dr. Stephen R. Farmer of the Boston University School of Medicine; Boston University is Energesis’ academic partner on the STTR grant.

According to a January 31, 2012 article in Wired magazine, the U.S. Army’s interest in Energesis’ technology is the result of the growing incidence of overweight and obesity in the Army’s recruit pool, as in young Americans in general. The Army is funding the Energesis/Boston University researchers in the hopes of using autologous BAT transplantation to boost weight loss in military personnel.

According to Brian Freeman, an autologous cell transplantation therapy might also be commercialized for treatment of severely obese individuals in lieu of bariatric surgery. Such an autologous cellular therapy would be analogous to the FDA-approved Genzyme cell transplantation therapy products Carticel and Epicel. It may be easier and faster for Energesis to gain FDA approval for an autologous BAT transplantation product than to develop and gain approval for a drug based on the company’s BAT research. Energesis will therefore pursue both drug discovery and autologous cell transplantation programs, with the strategy to gain early approval and revenues for a transplantation product while it continues to pursue drug discovery and development. Success in development of an autologous transplantation product should also boost the company’s prospects for funding, which would enable its wider R&D programs.

Other approaches to brown adipose tissue-based therapies

The May 1 2012 Edyta Zielinska article begins with a discussion of metabolic diseases start-up Ember Therapeutics. As stated in the article, Ember was founded by Third Rock Ventures partner Lou Tartaglia, a scientist by background who was formerly the Vice President of Metabolic Diseases at Millennium Pharmaceuticals. Ember was launched with $34 million in financing from Third Rock. The company plans to work both on therapeutics based on BAT biology, and on developing a new generation of safer insulin sensitizers for treatment of type 2 diabetes. The latter area of focus is based on studies by Ember scientific founders Dr. Bruce Spiegelman (Dana-Farber Cancer Institute and Harvard Medical School, Boston MA) and Patrick R. Griffin (Scripps Research Institute, Scripps FL) We discussed that work on our blog in an August 29, 2010 article, which was followed by two additional articles on September 16, 2010 and September 21, 2011.

In the January 11 2012 issue of Nature, Dr. Spiegelman’s group reported the discovery of a myokine hormone (i.e., a cytokine produced by muscle cells), which the researchers named irisin. Irisin is named after the Greek goddess Iris, the messenger of the gods. It acts on white adipose cells in culture and in vivo to stimulate what appears to be development into brown fat-like cells. Specifically, irisin stimulates expression of UCP1 and an array of other brown fat genes. Mildly increased blood levels of irisin results in an increase in energy expenditure in mice with no changes in movement or food intake, as would be expected with an increase in brown fat levels. This results in improvements in obesity and glucose homeostasis. Exercise increases levels of blood irisin in mice and humans, leading to the hypothesis that irisin is an “exercise hormone” that mediates at least some of the beneficial metabolic effects of exercise. Irisin is therefore a potential therapeutic for metabolic diseases such as type 2 diabetes and obesity. Ember entered into an exclusive license agreement with Dana-Farber Cancer Institute for the irisin technology, and is optimizing and developing a proprietary molecule based on this technology. This molecule is designed to augment and activate the body’s brown fat. This research constitutes the company’s lead BAT biology program.

On March 28, 2012, Ember also exclusively licensed technology from the Joslin Diabetes Center (Boston, MA) covering bone morphogenetic protein 7 (BMP7), and its role in BAT development. The role of BMP7 in BAT biology was discovered by Ember scientific co-founder C Ronald Kahn, M.D. and his colleagues, who published their findings in Nature in 2008.

In addition to its lead irisin program, Ember is developing a pipeline of biologics (including those based on BMP7) and small molecules designed to increase BAT levels and to activate BAT-specific pathways. According to the article in The Scientist, among the pathways being investigated by Ember are those involving the PRDM-16 transcription factor and FoxC2.

Zafgen’s beloranib (ZGN-433)

Meanwhile, the other obesity start-up founded by Brian Freeman, Zafgen (Cambridge, MA) has been making progress in developing its lead drug candidate, beloranib (ZGN-433). Beloranib, a methionine aminopeptidase 2 (MetAP2) inhibitor, was originally discovered by the Korean company CKD Pharmaceuticals, and was being developed as an angiogenesis inhibitor for treatment of solid tumors. However, the drug was poorly efficacious for this indication in animal models. At much lower concentrations, however, beloranib exerts an antlobesity effect. Zafgen therefore licensed the compound from CKD, and has been developing it as an agent to induce weight loss in severely obese patients.

Beloranib targeting of MetAP2 in vivo results in downregulation of signal transduction pathways within the liver that are involved in the biosynthesis of fat. Animals or humans treated with the drug oxidize fat to form ketone bodies, which can be used as energy or are excreted from the body. The result is breakdown of fat cells and weight loss. Obese individuals do not usually have the ability to form ketone bodies.

In January 2011, Zafgen reported top-line data from a Phase Ib multiple-ascending dose study in which 24 obese women were given 0.9 milligrams/meter(2) of beloranib twice-weekly intravenous. The subjects had a median reduction in body weight of 1 kg/week or 3.1% over 26 days. Treatment with beloranib also reduced triglycerides by 38% and LDL cholesterol (“bad cholesterol”) by 23% from baseline. These results were statistically significant  (p<0.05).

Patients (who were given no instructions regarding diet or exercise) also showed a decline in hunger, and showed no treatment-related serious adverse effects. If sustained (e.g., over a 6-9 month course of treatment in individuals requiring a 20-40 percent reduction in weight) the degree of weight loss seen in this study would be comparable to bariatric surgery.

On July 7, 2011, Zafgen secured a $33 million Series C financing, which was led by the company’s original investor syndicate, including Atlas Venture and Third Rock Ventures. Proceeds from the financing were to be used to support development of Zafgen’s pipeline and especially to advance its lead compound beloranib for the treatment of severe obesity into Phase 2 clinical studies. Zafgen, like Energesis, is operated as a lean virtual company, with only 5 employees. Thus Zafgen should have sufficient cash to advance its beloranib program to the next stage.

Inducing brown fat via modulation of TGFβ signaling

In our November 17, 2010 article, we also mentioned Acceleron Pharma (Cambridge, MA), and its R&D program aimed at brown fat induction via inhibition of signaling by members of the TGFβ (transforming growth factor beta) superfamily. Acceleron is continuing to investigate this approach, and has published a report on this research in the online version of the journal Endocrinology in May 2012. Novartis researchers also published a report on their studies in this area in the online version of the journal Molecular and Cellular Biology.

Conclusions

Despite the doom-and-gloom atmosphere of the obesity drug field in late 2010 and early 2011, with investment bank and business press analysts declaring the field to be “dead”, obesity drug R&D has shown definite signs of life in recent months. NDAs for two of the “Class of 2010” CNS-targeting antiobesity drugs, Qnexa and lorcaserin, have been resubmitted and are up for reconsideration by the FDA later this year. Meanwhile, R&D efforts aimed at producing therapeutics to increase energy expenditure via brown fat induction are progressing, mainly in small entrepreneurial biotech companies. The latter approach, if confirmed by future clinical trials, appears to have a greater likelihood of inducing the degree of weight loss needed to reverse even severe obesity.

Regulatory hurdles–especially safety concerns–were the most significant factor in the failure of the initial NDA submissions of the “Class of 2010” CNS-targeting drugs. The developers of these drugs are working to overcome these hurdles via performing the additional studies mandated by the FDA followed by NDA resubmission. We shall see how well this approach is working when the FDA rules on marketing approval of Qnexa and lorcaserin later this year. Meanwhile, developers of brown-fat targeting therapies are attempting to target severe obesity rather than the general obese population. They are positioning their therapeutics as alternatives to bariatric surgery. They expect that the regulatory hurdles to treating this population will be lower than for the general obese population.

As discussed in several articles on the Biopharmconsortium Blog, the need for antiobesity agents is great, and with the fast accelerating incidence of obesity and its complications, the need is also accelerating. Moreover, our understanding of the pathogenesis of obesity is limited. Thus both continuing basic research and development of agents with novel mechanisms are sorely needed.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

RAS/BRAF/PI3K pathways. Source: Source BioScience

Two previous articles on this blog have included discussions of the “co-clinical mouse/human trial” strategy for improving mouse models of human cancer, and simultaneously improving human clinical trials of drugs for these cancers. Now comes an article on the use of a co-clinical trial strategy in personalized treatment of non-small cell lung cancer (NSCLC) in the 29 March 2012 issue of Nature. In the same issue of Nature is a News and Views article by Genentech’s Leisa Johnson Ph.D. that provides a minireview of the research article.

As we discussed in our April 15, 2010 article on this blog, the co-clinical trial strategy has been developed by Pier Paolo Pandolfi, MD, PhD (Director, Cancer and Genetics Program, Beth Israel-Deaconess Medical Center Cancer Center and the Dana-Farber/Harvard Cancer Center) and his colleagues.

As discussed in that article, these researchers constructed genetically engineered transgenic mouse strains that have genetic changes that mimic those found in human cancers. These mouse models spontaneous develop cancers that resemble the corresponding human cancers. In Dr. Pandolfi’s  ongoing co-clinical mouse/human trial project, researchers simultaneously treat a genetically engineered mouse model and patients with tumors that exhibit the same set of genetic changes with the same experimental targeted drugs. The goal of this two-year project is to determine to what extent the mouse models are predictive of patient response to therapeutic agents, and of tumor progression and survival. The studies may thus result in validated mouse models that are more predictive of drug efficacy than the currently standard xenograft models.

The human clinical trials being “shadowed” by simultaneous studies in mice included Phase 3 trials of several targeted therapies for lung and prostate cancer. Xenograft models in which tumor tissue from the patients had been transplanted into immunosuppressed mice were also being tested in parallel with the genetically engineered mouse models. This project represents the most rigorous test to date of how well genetically engineered mouse models of cancer can predict clinical outcomes.

Our October 28, 2011 blog article, which is mainly a review of a 29 September 2011 Nature article by Nature writer Heidi Ledford, Ph.D., focuses on ways to fix the clinical trial system. Our article includes a discussion of a co-clinical trial published in January 2011. This trial utilized two genetically-engineered PDGF (platelet-derived growth factor)-driven mouse models of the brain tumor glioblastoma multiforme (GBM), one of which had an intact PTEN gene and the other of which was PTEN deficient. In this trial, researchers tested the Akt inhibitor perifosine (Keryx Biopharmaceuticals, an alkylphospholipid) and the mTOR inhibitor CCI-779 (temsirolimus; Pfizer’s Torisel), both alone and in combination, in vitro and in vivo. The drugs and drug combinations were tested in cultured primary glioma cell cultures derived from the PTEN-null and PTEN-intact mouse PDGF-driven GBM models, and in the animal models themselves.

The studies showed that both in vitro and in vivo, the most effective inhibition of Akt and mTOR activity in both PTEN-intact and PTEN-null cells in animals was achieved by using both inhibitors in combination.  In vivo, the decreased Akt and mTOR signaling seen in mice treated with the combination therapy correlated with decreased tumor cell proliferation and increased cell death; these changes were independent of PTEN status. The co-clinical animal study also suggested new ways of screening GBM patients for inclusion in clinical trials of treatment with perifosine and/or CCI-779.

The new co-clinical trial reported in the March 2012 issue of Nature

The March 2012 Nature report describes research carried out by a large, multi-institution academic consortium, which included Dr. Pandolfi. It focuses on strategies for treatment of patients with non-small-cell lung cancer (NSCLC) with activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog). These mutations occur in 20–30% of NSCLC cases, and patients whose tumors carry KRAS driver mutations have a poor prognosis. Moreover, KRAS is a “hard” or “undruggable” target, and no researchers have thus been able to discover inhibitors of oncogenic KRAS.

Because of the intractability of oncogenic KRAS as a target, researchers have been attempting to develop combination therapies for mutant-KRAS tumors (including, for example, colorectal cancers as well as NSCLCs) that address downstream pathways controlled by KRAS. We discussed examples of these strategies in our book-length report Multitargeted Therapies: Promiscuous Drugs and Combination Therapies, published by Cambridge Healthtech Institute/Insight Pharma Reports in 2011. Strategies discussed in that report are based on the finding that KRAS controls signal transduction via two key pathways: the B-Raf-MEK-ERK pathway and the PI3K-Akt pathway. This is illustrated in the figure at the top of this article. As discussed in our 2011 report, researchers are attempting to develop treatments of mutant-KRAS tumors that involve combination therapies with an inhibitor of the mitogen-activated protein kinase (MEK) together with an inhibitor of phosphatidylinositol 3-kinase (PI3K). Researchers are also attempting to develop combination therapies of MEK inhibitors with standard cytotoxic chemotherapies, which if successful will avoid having to use combinations of two expensive targeted therapies.

In the co-clinical trial that is the focus of the 29 March 2012 Nature research report and News and Views commentary, researchers developed a genetically-engineered mouse model to study treatment of mutant-KRAS NSCLCs with either the antimitotic chemotherapy drug docetaxel alone, or docetaxel in combination with the MEK kinase inhibitor selumetinib (AZD6244, AstraZeneca). In the parallel human clinical trial, researchers are also studying treatment of patients with mutant-KRAS NSCLC with docetaxel alone or docetaxel plus selumetinib. (There is no treatment arm in the human clinical trial in which patients are treated with selumetinib alone, since selumetinib monotherapy of NSCLC patients had shown no efficacy in a previous Phase 2 study; this was confirmed in mouse model studies.)

In humans with mutant-KRAS NSCLC, many tumors with mutations in KRAS have concomitant genetic alterations in other genes that may affect response to therapy. Therefore, the co-clinical trial researchers wished to design mouse models with lung tumors with either Kras mutations alone or with mutations in both Kras and another gene that is often concomitantly mutated in mutant-KRAS NSCLCs in humans. The researchers therefore constructed mouse models with cancers bearing the activating Kras(G12D) mutation, either alone or together with an inactivating mutation in either p53 or Lkb1. The researchers achieved this via a conditional mutation system using nasal instillation of specifically genetically-engineered adenoviruses. As result, a small percentage of lung epithelial cells harbored these mutations. It is from these cells that the NSCLC-like tumors arose, analogous to the clonal origin of sporadic lung tumors in humans.

Of the two tumor suppressor genes that are frequently mutated in human mutant-KRAS NSCLCs and that were modeled by the co-clinical trial researchers, p53, often called the “guardian of the genome”, is familiar to most of you. The other gene, Lkb1 [liver kinase B1, also known as serine/threonine kinase 11 (STK11)], was discussed in an earlier article on the Biopharmconsortium Blog, entitled “The great metformin mystery–genomics, diabetes, and cancer.”

LKB1 (whether in regulation of gluconeogenesis in the liver or in its role as a tumor suppressor) acts by activation of AMPK (AMP-activated kinase, a sensor of intracellular energy status.) In lung cancer (as shown by the same group that performed the 2012 co-clinical trial), LKB1 acts to modulate lung cancer differentiation and metastasis.  Germline mutations in LKB1 are associated with the familial disease Peutz-Jegher syndrome, in which patients develop benign polyps in the gastrointestinal tract. Studying a mouse model of mutant-LKB1 Peutz-Jeger syndrome, Reuben J. Shaw (Howard Hughes Medical Institute, The Salk Institute for Biological Studies, La Jolla, CA, who was prominently mentioned in our “great metformin mystery” article) and his colleagues showed that the LKB1-AMPK pathway downregulates the mTOR pathway–specifically the rapamycin-sensitive mTOR complex 1 (mTORC1) and its downstream effector hypoxia-inducible factor-1α (HIF-1α). HIF-1α expression in turn upregulates the expression of its downstream effectors hexokinase II and glucose transporter 1 (GLUT1), which are involved in cellular utilization of glucose. LKB1-deficient polyps in this mouse model thus show increased expression of hexokinase II and GLUT1, resulting in dramatically increased glucose utilization.

In the new co-clinical trial, genetically-engineered mice that showed established lung tumors [as determined via magnetic resonance imaging (MRI)] were randomized to receive either docetaxel, selumetinib, or a combination of the two drugs. For tumors with only a Kras mutation, treatment with docetaxel alone resulted in a modest rate of response, with 30% of mice showing a partial response. Mice that bore mutant-Kras tumors that also had mutations in either p53 or Lkb1 had much lower rates of response to docetaxel monotherapy (5% and 0%, respectively), and more of these mice showed progressive disease on MRI or died of their disease. Of mice treated with the docetaxel/selumetinib combination, those with single-mutant Kras tumors showed a 92% overall response rate, and those with mutant Kras/p53 tumors showed a 61% overall response rate. However, mice with mutant Kras/Lkb1 cancers showed only a modest response to the docetaxel/selumetinib combination; 33% of mice achieved a partial response. The difference in response rate of mice with Kras/Lkb1 tumors to docetaxel/selumetinib compared to the other two genotypes was found to be statistically significant.

Using the genetically-engineered NSCLC mouse model in biomarker development

In human patients in clinical trials or in treatment for their cancers, performing repeated biopsies to monitor treatment is difficult. The co-clinical trial researchers therefore wished to develop less invasive means of monitoring both co-clinical and clinical trials of docetaxel/selumetinib treatment of NSCLC. They therefore tested the use of positron emission tomography (PET) with 18F-fluoro-2-deoxyglucose (FDG-PET) as an indicator of early response to therapy that could be used in the clinic.  Prior to its radioactive decay (109.8 minute half -life), 18F-FDG is a nonmetabolizable glucose analogue that moves into cells that is preferentially taken up by high-glucose utilizing cells. The researchers found that both Kras/p53 and Kras/Lkb1 tumors showed higher FDG uptake in vivo in the mouse model than did single-mutant Kras tumors. As expected from the earlier study, GLUT1 expression was elevated in Kras/Lkb1 mutant tumors. In human patients, pre-treatment, mutant KRAS/LKB1 tumors also showed a higher FDG uptake that did KRAS tumors negative for LKB1.

Treatment of the mice with docetaxel alone gave no significant changes in FDG uptake in Kras, Kras/p53, or Kras/Lkb1 tumors in vivo. However, within 24 hours of the first dosing of docetaxel/selumetinib, FDG uptake was markedly inhibited in Kras and Kras/p53 tumors. In contrast, treatment of mice with Kras/Lkb1 mutant tumors gave no appreciable decrease in FDG uptake in these tumors. These results show that early changes in tumor metabolism, as assessed by FDG-PET, predict antitumor efficacy of docetaxel/selumetinib treatment.

The FDG-PET study in this mouse model supports the use of this imaging method as a biomarker to monitor the course of treatment in humans.

Cellular signaling in mutant Kras, Kras/p53, and Kras/Lkb1 tumors

The researchers assessed activation of relevant intracellular pathways in tumors in treated and untreated mice with mutant Kras, Kras/p53, and Kras/Lkb1 lung cancers. They performed these studies using two different methods–immunostaining of cancer nodules for phosphorylated ERK, and immunoblotting of tumor lysates. In untreated mice, Kras/Lkb1 tumors show less activation of ERK than do Kras and Kras/p53 tumors, with Kras/p53 tumors showing the greatest amount of activation of the MEK-ERK pathway. Docetaxel had no discernible effect on signaling via the MEK-ERK pathway. Selumetinib alone resulted in decreased ERK activation in Kras and Kras/p53 tumors, but there was still residual activity. The docetaxel/selumetinib combination, however, was more effective in eliminating ERK activation. Pharmacokinetic studies indicated that selumetinib levels were higher in both serum and tumors of mice treated with docetaxel/selumetinib that in those treated with selumetinib alone; this might account for the more potent suppression of MEK-ERK signaling by the combination therapy as compared to selumetinib monotherapy. The researchers studied MEK-ERK activation (as determined by phospho-ERK staining) in  a set of 57 human NSCLC tumors with known RAS, p53 and LKB1 mutation status. As with the tumors in the mouse model, of seven patients whose tumors harbored the KRAS activating mutation, the three patients with concurrent p53 mutations showed higher levels of ERK activation.

The decreased activation of ERK in Kras/Lkb1 tumors suggested that these tumors utilize other pathways to drive their proliferation. On the basis of their prior studies of signal transduction in mutant-Lkb1 lung tumors, the researchers focused on AKT and SRC. Immunoblotting studies showed that Kras/Lkb1 mutant tumors had elevated activation of both AKT and SRC. As one can see from the figure at the top of this article, AKT is a downstream effector of PI3K; since the PI3K/AKT pathway regulates expression of GLUT1 and hexokinase, increased activation of the PI3K/AKT pathway is consistent with the increased uptake of FDG of mutant Kras/Lkb1 tumors. In the figure, SRC (which is not shown) represents one of the major “other effectors” controlled by RAS. These results indicate that concomitant mutation of Lkb1 in mutant-Kras NSCLCs may shift the signaling pathways that drive tumor proliferation from MEK-ERK to PI3K/AKT and/or SRC. This shift would result in primarily resistance of Kras/Lkb1 tumors to treatment with docetaxel/selumetinib.

Long-term benefits of treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel/selumetinib as opposed to docetaxel monotherapy

The researchers studied long-term treatment of mice with mutant-Kras and Kras/p53 tumors with docetaxel monotherapy versus docetaxel/selumetinib. In mice with mutant-Kras tumors, treatment with docetaxel monotherapy gave stable disease for several weeks, while docetaxel/selumetinib treatment resulted in tumor regression and slower regrowth of tumors. The addition of selumetinib to docetaxel significantly prolonged progression-free survival in these mice. In mice with Kras/p53 tumors, treatment with docetaxel alone resulted in progressive disease, but docetaxel/selumetinib treatment resulted in initial disease regression followed by progression, resulting in prolonged progression-free survival. These results indicate that treatment with combination therapy as opposed to docetaxel alone results in improved progression-free survival, but not cure, in mice with Kras– and Kras/p53-mutant tumors.

The researchers also investigated mechanisms of acquired tumor resistance in mice with mutant-Kras and Kras/p53 tumors, which had been treated long-term with docetaxel/selumetinib. In moribund animals that had received this treatment, all tumor nodules examined showed a recurrence of ERK phosphorylation. This suggested that acquired resistance could be at least in part due to reactivation of MEK–ERK signaling despite ongoing treatment with selumetinib. Evaluation of resistant tumor nodules suggested that more than one mechanism for pathway reactivation was occurring; study of these mechanisms is ongoing.

Conclusions of the new co-clinical study

The results of this co-clinical study predict that docetaxel/selumetinib combination therapy will be more effective than docetaxel monotherapy in several sub-classes of mutant-KRAS NSCLC. This prediction is consistent with the early results of a Phase 2 clinical trial of these two drug combinations in second-line treatment of patients with KRAS-mutant NSCLC.

However, the co-clinical trial also predicts that concurrent mutation of LKB1 in mutant-KRAS  tumors will result in primary resistance to docetaxel/selumetinib combination therapy, perhaps via activation of parallel signaling pathways such as AKT and SRC. Since LKB1 status is not being prospectively assessed in the ongoing human clinical trial, the presence of patients with cancers having concurrent LKB1 mutations may diminish the differences between treatment arms based solely on KRAS status. The results of the co-clinical trial suggests that researchers perform retrospective analysis of p53 and LKB1 status in samples from the concurrent human clinical trial. Future clinical trials should then be designed that involve prospective analysis to ensure sufficient enrollment of patients with all three genotypes to enable sufficiently powered sub-group analyses.

Although the results of the co-clinical trial indicate that patients with mutant KRAS/LBK1 tumors be excluded from trials of docetaxel/selumetinib treatment, the research group that has been conducting the co-clinical trial has also been conducting studies that may lead to treatment strategies for KRAS/LBK1 tumors.

The co-clinical trial also allowed researchers to design and validate biomarker strategies, specifically the potential use of the less-invasive FDG-PET to predict efficacy and to monitor treatment. The co-clinical animal-model study also enabled the discovery of mechanisms of both primary and acquired resistance that might benefit future clinical trials and discovery/development of drugs. (The studies on acquired resistance are in a early stage and are ongoing). Any mechanisms of acquired resistance discovered in co-clinical studies should be confirmed in human clinical trials by examining biopsy samples from patients who relapse on therapy. The ability to assess mechanisms of resistance in preclinical or co-clinical animal studies may enable researchers to design rational drug combination strategies that can be implemented in future clinical studies.

The results of the new co-clinical trial strengthens the contention that co-clinical trials in genetically-engineered mice can provide data that can predict the outcome of parallel human clinical trials. Co-clinical trials can also be used to generate new hypotheses for use in analyzing concurrent human trials, and for design of future clinical studies. Moreover, co-clinical trials can result in the validation of improved animal models for human cancers, which can be used in research and preclinical testing of oncology agents, and in validation of biomarkers for clinical studies in oncology. Given the inadequacy of “standard” xenograft models, which is a major factor in the high attrition rate of pipeline oncology drugs, the availability of validated genetically-engineered animal models may be expected to enable improved oncology drug development.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Eltrombopag

On April 13, 2012, Informa’s Scrip Insights the publication of a new book-length report, Advances in the Discovery of Protein-Protein Interaction Modulators, by Allan B. Haberman, Ph.D.

Protein-protein interactions (PPIs) are of central importance in biochemical pathways, including pathways involved in disease processes. However, PPIs have been considered the prototypical “undruggable” or “challenging” targets. The discovery of small-molecule drugs that can serve as antagonists or agonists of PPIs, and which are capable of being successfully taken into human clinical trials, has been extremely difficult. Among the theoretical reasons for this is that contact surfaces involved in PPIs are usually large and flat, and lack the types of cavities present in the surfaces of proteins that bind to small-molecule ligands.

Nevertheless, over the last twenty years, researchers have developed a set of technologies and strategies that have enabled them, in a several cases, to discover developable small-molecule PPI modulators. One direct PPI agonist, the thrombopoietin mimetic eltrombopag (Ligand/GlaxoSmithKline’s Promacta/Revolade), has reached the market. The chemical structure of this compound is illustrated in the figure above. Several other small-molecule PPI modulators are in clinical trials. Despite this progress, the discovery and development of small-molecule PPI modulators has been one-at-a-time, slow and laborious.

The new strategic importance of protein-protein interactions as drug targets

Meanwhile, PPIs as potential drug targets have acquired a key strategic importance for the success of the pharmaceutical industry. Over at least the last decade, pharmaceutical R&D has failed to develop enough high-valued new drugs to make up for or exceed revenues from blockbusters that are losing patent protection. As we have discussed in previous publications and in , this low productivity is mainly due to pipeline attrition. There are several factors (ranging from target selection through drug design, preclinical studies, identification and use of biomarkers, and design of clinical trials) that can influence pipeline attrition.

However, increasing numbers of industry leaders and analysts identify target selection as the key factor that is limiting the productivity of pharmaceutical R&D. For example, I served as a workshop leader at Hanson Wade’s   last summer, which took that point of view. There are at least several such conferences throughout the year, which are organized at the request of industry leaders.

Industry experts who identify poor target selection as a major cause of pharma R&D’s productivity woes conclude that the main issue is that companies are running out of “druggable” targets that have not already been addressed by marketed drugs. As of 2011, only 2% of human proteins have been targeted with drugs. Most of the remaining disease-relevant proteins, including transcription factors and many other types of signaling proteins, work via interacting with other proteins in PPIs. Therefore, in order to reverse its R&D slump, the pharmaceutical industry needs to develop technologies and strategies to address PPIs and other hitherto “undruggable” targets.

Contents of the report

Our report discusses technologies and strategies that enable the discovery of drugs targeting PPIs, including both small-molecule and synthetic peptidic modulators. It includes case studies on the discovery of compounds that address specific target classes, with emphasis on agents that have reached human clinical studies. This includes addressing the issue of the need to produce PPI modulatory agents that have pharmacological properties that will enable them to be good clinical candidates.

The report also includes discussions of second-generation technologies for the discovery of small-molecule and peptidic PPI modulators, which have been developed by such companies as Forma, Ensemble, and Aileron, and by academic laboratories. The field of PPI modulator discovery has represented a “premature technology”, i.e., a field of biomedical science in which consistent practicable therapeutic applications are in the indefinite future, due to difficult technological hurdles. We have discussed premature technologies on earlier on this blog. The second-generation technologies are designed to overcome the hurdles and to thus enable a more accelerated and systematic approach to PPI drug discovery and development.

In part as the result of the development of these technologies, and of the increasing strategic importance of PPI modulator development, companies have been moving into the field. Examples include Bristol-Myers Squibb, Pfizer, Novartis, and Roche. A key issue is to what extent the new technologies for PPI modulator R&D will enable this area to be commercially successful, and to meet the strategic needs of the industry for expanding the universe of targets for which drugs can be developed.

To see the report Advances in the Discovery of Protein-Protein Interaction Modulators, please click here.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.

Atlas!

This is Part 2 of the article on RaNA Therapeutics that we began on March 29, 2012.

Jeannie Lee’s research and RaNA’s technology platform

Jeannie Lee’s laboratory focuses on the study of the mechanism of X-chromosome inactivation in mammals. In X-chromosome inactivation, one of the two copies of the X chromosome present in the cells of female mammals is inactivated. The inactive X chromosome is silenced by packaging into transcriptionally inactive heterochromatin.  X-chromosome inactivation results in dosage compensation, the process by which cells of males and females have the same level of expression of X-chromosome genes, even though female cells have two X chromosomes and male cells have only one. In placental mammals such as mice and humans, the choice of which X chromosome will be inactivated is random, but once an X chromosome is inactivated it will remain inactive throughout the lifetime of the cell and its descendants.

The Lee laboratory has focused on genes encoded by the X-chromosome whose actions coordinate X-chromosome inactivation. These genes  are contained in the 100 kilobase long X-inactivation center (Xic). One of these genes, Xist, encodes the lncRNA XIST, which as discussed in Part 1 of this article inactivates an X-chromosome by spreading along the X chromosome and recruiting the silencing factor PRC2. XIST is regulated in cis by TSIX, an antisense version of XIST which works to keep the active X-chromosome active. Tsix is in turn regulated by Xite (X-Inactivation intergenic transcription element), an upstream locus that harbors an enhancer that enables the persistence of TSIX expression on the active X chromosome. The mechanism by which Xite acts (including whether it acts via its RNA transcripts) is not clear. Xite and Tsix appear to regulate pairing between the two X chromosomes in a female cell, and determine which X chromosome will be chosen for inactivation. Several other recently discovered genes in the region of the Xic, which work via lncRNAs, also serve as regulators of XIST function. For example, the Rep A and Jpx genes, work via lncRNA transcripts to induce Xist. Thus Xist is controlled by positive and negative lncRNA-based switches–TSIX for the active X chromosome and JPX and REPA for the inactive X. Of these lncRNAs, REPA, XIST, and TSIX bind to and control PRC2.

In late 2010, the Lee laboratory published an article in Molecular Cell in which the researchers identified a genome-wide pool of over 9000 lncRNA transcripts that interact with PRC2 in mouse ES cells. Many of these transcripts have sequences that correspond to potentially medically-important loci, including dozens of imprinted loci (i.e., loci that are epigenetically modified such that only the paternal or maternal allele is expressed), hundreds of oncogene and tumor suppressor loci, and multiple genes that are important in development and show differential chromatin regulation in stem cells and in differentiated cells. The researchers obtained evidence that at least in one case, an RNAs works to recruit PRC2 to a disease-relevant genes, similar to PRC2 recruitment by XIST and HOTAIR. This case of specific PRC2 recruitment has not been previously known, suggesting that the researchers’ methodology could be used to discover new examples of PRC2 recruitment by lncRNAs.

Some of the PRC2-associated lncRNAs identified in the Molecular Cell report may be potential therapeutic targets and/or biomarkers. Overexpression of PCR2 proteins have been linked to various types of cancer, including metastatic prostate and breast cancer, and cancers  of the colon, breast, and liver. Pharmacological inhibition of PRC2-mediated gene repression was found to induce apoptosis in several cancer cell lines in vitro, but not in various types of normal cells. Induction of apoptosis in this system is dependent on reactivation of genes that had been repressed by PRC2. There is also evidence that PRC2-mediated gene repression may be linked to the maintenance of the stem-cell properties of cancer stem cells. These results suggest that at least in some cases, inhibition of PRC2-mediated gene repression–including via targeting lncRNAs that recruit PRC2 to critical genes–is a potential strategy for treating various types of cancer.

RaNA’s R&D strategy

Not much information is available about RaNA’s strategy.  However, according to the January 2012 Mass High Tech article, RaNA Therapeutics has licensed technology from Mass General Hospital based on Dr. Lee’s research. The company has also filed several patent applications, some of which are described as being very broad. This includes patent applications on the existence and method of use of thousands of lncRNA targets. However, Dr. Lee’s currently include only three items involving the X-chromosome inactivation system or TERC. Presumably, the patent applications mentioned in the Mass High Tech article will be published at the end of the 18-month publication period for U.S. patent applications.

According to the Mass High Tech article, RaNA is in the process of narrowing down the diseases it will initially focus on. Likely areas will include genetic diseases, including diseases that result from haploinsufficiency. In haploinsufficiency, one allele of a gene is nonfunctional, so all of the protein coded by the gene is made from the other allele. However, this results in insufficient levels of the protein to produce a normal phenotype. RaNA intends to use its technology to increase expression of the functional gene, resulting in a adequate dosage of the protein for a normal phenotype.

RaNA intends to choose one indication out of a short list of 20 diseases for internal R&D, and to seek collaborations for other indications. Dr. Krieg says that he hopes to have a collaboration by the end of 2012, and also to have Investigational New Drug (IND)-enabling safety studies on its internal drug candidate by the end of the year as well.

As one might expect, RaNA will target the appropriate lncRNAs using oligonucleotides, similar to how RNAi companies target mRNAs. Dr. Krieg, an oligonucleotide therapeutic development veteran, recruited some of his old oligonucleotide team from Pfizer into RaNA, according to a Fierce Biotech article. Thus Dr. Krieg and his team can quickly get up and running in designing and testing oligonucleotide therapeutics, once RaNA selects the targets for its initial focus.

In the Mass High Tech article, Dr. Krieg says that he believes that “oligonucleotides are on the cusp of being recognized as the third leg of drug development,” along with small-molecule and protein therapeutics. However, as we discussed in our August 22, 2011 article on this blog, oligonucleotide drug development, as exemplified by RNAi and microRNA-based therapeutics, has run into several technological hurdles, especially those involving drug delivery. The August 2011 article cites an editorial by Dr. Krieg, in which he voices his optimism despite these hurdles.

Nevertheless, large pharmaceutical companies and investors have been moving away from the oligonucleotide field. This is exemplified by Alnylam’s January 20, 2012 restructuring, which cut one-third of its work force and focused the company on two of its Phase 1 programs. Having exhausted its ability to capture major Big Phama licensing and R&D deals, Alnylam has had to become a normal early-2012 biotech company and focus its strategy. (However, Alnylam did a $86.9 million public offering in February 2012.)

The emergence of RaNA, and its $20.7 million funding, thus swims against the tide of the general pessimism about oligonucleotide therapeutics of Big Pharmas, investors, and stock analysts. However, at least some oligonucleotide therapeutics will eventually emerge onto the market, and lncRNA regulation is likely to be crucial to many disease pathways. RaNA is thus the pioneering company in this field.

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As the producers of this blog, and as consultants to the biotechnology and pharmaceutical industry, Haberman Associates would like to hear from you. If you are in a biotech or pharmaceutical company, and would like a 15-20-minute, no-obligation telephone discussion of issues raised by this or other blog articles, or of other issues that are important to  your company, please click here. We also welcome your comments on this or any other article on this blog.