11 June 2012

Developing resistance-free antibiotics by targeting quorum sensing

By | 2012-06-11T00:00:00+00:00 June 11, 2012|Chemistry, Drug Development, Drug Discovery, Haberman Associates, Infectious Disease, Strategy and Consulting, Synthetic biology|

 

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.

30 December 2010

2010: Breakthroughs, Newsmakers, and Deals of the Year

By | 2010-12-30T00:00:00+00:00 December 30, 2010|About Our Blog, Animal Models, Drug Development, Drug Discovery, Strategy and Consulting, Synthetic biology|

2010

Breakthroughs of the Year

As it does every year, Science published its “Breakthrough of the Year” for 2010 in the 17 December issue of the journal.

For its “Breakthrough of the Year”, Science chose a non-life science innovation, the first quantum machine. Interestingly, the same issue of Science included a Perspective on biophysicist Britton Chance, who died last November at the age of 97. Among his many accomplishments, Dr. Chance discovered that biological electron transfer operates via quantum tunneling, a mechanism central to photosynthesis, respiration, and many oxidoreductase enzymes. Mitochondria, chloroplasts, and oxidoreductase enzymes thus constitute biological quantum machines of a sort.

Interestingly, Dr. Chance continued to work on metabolism all of his long working life, including in the era of molecular biology when interest in that field waned. By doing so, he made many important contributions, including the mechanism for the generation of the reactive oxygen species (ROS) superoxide and peroxide during normal mitochondrial respiration, and the  use of near-infrared (NIR) light for noninvasive diagnostics.

Although Science chose a non-life science advancement as its “Breakthrough of the Year”, the journal’s runners-up for “Breakthrough of the Year” were replete with life science items. The first runner-up was the synthetic Mycoplasma mycoides genome constructed by the J. Craig Venter Institute, which they used to create “the first synthetic cell”. As we discussed in a series of two articles on this blog (see here and here) although the creation of the synthetic genome and the “synthetic cell” represented a technical tour de force, it did not represent a true breakthrough. Many leading scientists, including leaders in the field of synthetic biology, agreed with us. However, at least several bioethicists and philosophers hailed this work as a milestone, calling it “the end of vitalism”. (As we noted in another blog post, however, not all bioethicists agree.)

Moreover, policy-makers were sufficiently alarmed by the “synthetic cell” that (as noted in the Science “Breakthrough of the Year” runners-up article) the Presidential Commission for the Study of Bioethical Issues held hearings on policy implications of this research. Nevertheless, the report of this commission (issued in December 2010) concluded that the Venter research “does not amount to creating life as either a scientific or a moral matter” and that synthetic biology remains “in the early stages,” with any dangers well into the future. The commission recommended continuing White House oversight, but a relatively mild set of regulatory measures.

As we said in our second article on the “synthetic cell”, we are much more impressed by the metabolic engineering studies of Jay Keasling, and by George Church’s automated method for optimizing metabolic engineering pathways, which we had discussed in an earlier blog post. The Science “Breakthrough of the Year” runners-up article mentioned Dr. Church’s automated system, among other synthetic biology advances made in 2009 and 2010.

Meanwhile, in a review of metabolic engineering published in the 3 December 2010 issue of Science, Dr. Keasling says that although minimal bacterial hosts such as Dr. Venter’s “synthetic” mycoplasma may be of scientific interest, they are not suitable to use in metabolic engineering studies whose goal is scale-up for industrial production of medicines, chemicals, or biofuels. This agrees with our statement that such applications require  “workhorse” organisms that can take the extensive genetic manipulation needed to engineer new metabolic pathways, and which are capable of scale-up.

We therefore believe that the “synthetic cell” is not the life science breakthrough of the year, despite its placement at the top of Science‘s “Breakthrough of the Year” runners-up article.

Our nominee for the life science breakthrough of the year is listed right under the “synthetic cell” in the Science “Breakthrough of the Year” runners-up article. It is the determination of the sequence of approximately two-thirds of the Neandertal genome by Svante Pääbo (Max-Planck Institute for Evolutionary Anthropology, Leipzig, Germany.) and his colleagues. This achievement is something that only a few years ago seemed completely impossible. Moreover, this work is of great cultural significance, since it indicates that Neandertals contributed some 1-4 percent of the genome sequences of non-African present-day humans. More recently, Dr. Pääbo and his colleagues followed up the Neandertal studies by using their DNA synthesis methods to identify a third species of humans, known as Denisovans. Denisovans, who were more closely related to Neandertals than to modern humans, were alive at the same time as modern humans emerged from Africa and also encountered the Neandertals. Dr. Pääbo’s new studies indicate that the Denisovans contributed some 4–6% of the genome sequences of present-day Melanesians.

Despite the importance of the Pääbo Neandertal studies, we have not blogged on this work simply because it has nothing to do with drug discovery and development. However, perhaps someday, for example, some of the products of genes that are found in present-day humans but not in Neandertals could emerge as potential drug targets. As discussed in the Science “Breakthrough of the Year” runners-up article, researchers have begun studying some of these gene products in cell culture systems.

Moreover, the types of advanced, next-generation DNA sequencing methods used by Dr. Pääbo and his colleagues are being applied to studies that are relevant to drug discovery. These include the 1000 Genomes Project, which seeks to find all single-nucleotide polymorphisms (SNPs) present in at least 1% of humans. This and other next-generation genomics projects were listed in the Science “Breakthrough of the Year” runners-up article, as the third runner-up. The 1000 Genomes Project, as well as genome-wide association studies (GWAS) that use high-throughput DNA sequencing methods, may enable researchers to identify rare mutations that are involved in complex human diseases. This might in turn lead to the discovery of novel drugs and diagnostics.

Among other life science items in the Science “Breakthrough of the Year” runners-up article was the production of knockout rats. We discussed knockout rats in an October 1, 2010 blog post.

Newsmaker of the Year

Nature also had an end-of-2010 special article, “The Newsmaker of the Year”, in its 23/30 December issue. Unfortunately, Nature chose a U.S. government official as its Newsmaker of the Year.

We would prefer that Nature stick to what it does so very well, and stay out of U.S. politics, whether in its “opinionated editorials” [sic] or elsewhere. Perhaps the low point in Nature‘s political forays was its November 2010 editorial calling for what amounts to a new version of Prohibition. This is despite the ample evidence that moderate consumption of red wine (for example) is healthy for most adults. Readers would be well advised not to believe everything they read in Nature editorials.

Our nominee for Newsmaker of the Year in the life sciences is Dr. Svante Pääbo, for the reasons we discussed earlier.

Deals of the Year

Also as an end-of-year feature, the IN VIVO blog has been running a Deal of the Year competition. The nominees are grouped in three categories: M&A Deal of the Year, Alliance Deal of the Year, and Exit/Financing Deal of the Year.

Only one of the nominees had been featured in an article on our blog: the Celgene/Agios alliance (April 23, 2010).

The IN VIVO Blog invited readers to vote on the Deal of the Year in each of the three categories, by going to their website. The voting closed at 12:00pm on 6 January 2011 (Eastern Standard Time).

The winners of the vote were:

  • M&A Deal of the Year: Celgene/Abraxis (50.31% of 1,799 votes)
  • Alliance Deal of the Year: Celgene/Agios (55.32% of 3,176 votes)
  • Exit/Financing Deal of the Year: Ablexis (46.54% of 1,631 votes)

Congratulations to all the winners, especially Agios and Celgene, which were featured in our blog post.

Happy New Year!

This is our own version of an end-of-year special article, and will be our last blog post of 2010. Best wishes to all of you for a happy, productive, and innovative New Year in 2011.

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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.

22 December 2010

Will intermediary metabolism be a hot field of biology again?

By | 2010-12-22T00:00:00+00:00 December 22, 2010|Cancer, Drug Discovery, Metabolism, Strategy and Consulting, Synthetic biology|

Citric acid cycle

The 3 December issue of Science featured a Special Section on metabolism, headed by an introductory article entitled “Metabolism is Not Boring”.

Way back in the 1920s through the 1950s, intermediary metabolism was a hot field of biology. This culminated in the awarding of the Nobel Prize in Physiology or Medicine in 1953 to Hans Krebs “for his discovery of the citric acid cycle” and Fritz Lipmann “for his discovery of co-enzyme A and its importance for intermediary metabolism”.

As most of you know, that same year, 1953, Watson and Crick published the structure of DNA, which won them the Nobel Prize in Physiology or Medicine in 1962. This began the great era of molecular biology. As the result of the overwhelming success of molecular biology, the study of intermediary metabolism receded into the background. Answering most questions in leading-edge biology required little or no attention to intermediary metabolism. However, as discussed in the review article by Steven L. McKnight included in the Special Section, metabolism is coming to the forefront of biomedicine again. Research problems that require both consideration of molecular biology and of metabolism now appear as interesting and important challenges.

Considerations of intermediary metabolism have always been important in the study of what are known as metabolic diseases, especially type 2 diabetes and obesity and such related conditions as dyslipidemia. However, as detailed both in the McKnight article and in an article by Arnold J. Levine and Anna M. Puzio-Kuter, the study of intermediary metabolism has now also become important in cancer, with the discovery that alterations in metabolic enzymes can result in the production of “oncometabolites” that support the growth of cancer cells.

In an article on this blog dated December 31, 2009, we discussed research in cancer metabolism that is behind the technology platform of Agios Pharmaceuticals (Cambridge, MA). In that article, we highlighted the discovery that mutations in a metabolic enzyme, cytosolic isocitrate dehydrogenase (IDH1) are a causative factor in a major subset of human brain cancers. The wild-type form of IDH1 catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate. However, the mutant forms of IDH1 catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). 2HG appears to be an oncometabolite that is involved in the progression of low-grade gliomas to lethal secondary glioblastomas. Agios researchers and their academic collaborators later implicated mutations in isocitrate dehydrogenase enzymes and the production of the oncometabolite 2HG in the pathogenesis of acute myelogenous leukemia (AML).

Also discussed in our article was the Warburg effect, in which cancer cells carry out aerobic glycolysis (conversion of glucose to lactate, with the production of 2 molecules of ATP even in the presence of oxygen). In contrast, most normal mammalian cells metabolize glucose to CO2 and water via glycolysis coupled to the mitochondrial citric acid cycle, generating 36 molecules of ATP. Agios scientific founder and signal-transduction pioneer Lewis Cantley showed that there is a connection between growth factor-mediated signal transduction and aerobic glycolysis in cancer cells. In particular, Dr. Cantley and his colleagues found that pyruvate kinase M2 (PKM2) is a link between signal transduction and aerobic glycolysis. PKM2 binds to tyrosine-phosphorylated signaling proteins, which results in the diversion of glycolytic metabolites from energy production via mitochondrial oxidative phosphorylation to anabolic processes required for rapid proliferation of cancer cells.

The McKnight and Levine and Puzio-Kuter papers also discuss the Warburg effect in cancer cells, and the role of mutations in several metabolic enzymes that contribute to malignant phenotypes. The McKnight article notes that in addition to dominant mutations in isocitrate dehydrogenates, rare recessive mutations in fumarate hydratase and succinate dehydrogenase are also associated with cancer. Mutations in the genes for these enzymes, coupled with loss of the wild-type allele, result in elevated intracellular levels of fumarate and succinate, respectively. These appear to act as oncometabolites that can induce activation of the hypoxia response pathway, which triggers the induction of aerobic glycolysis (the Warburg effect) and angiogenesis.

The Levine and Puzio-Kuter paper also discusses the role of oncogenes and tumor suppressor genes and their signaling pathways in regulating metabolism and in particular in inducing the Warburg effect. For example, p53 regulation suppresses the Warburg effect and promotes mitochondrial oxidative metabolism. Thus the loss of p53 function seen in most human cancers tends to promote aerobic glycolysis. Other signaling pathways that have been implicated in cancer-associated changes in metabolism include the Akt and mTOR pathways, which are frequently altered by mutations in key genes (e.g., mutations in PTEN and amplifications of such growth factor receptors as Her2 and EGFR) in cancer.  Deregulation of these pathways activates the hypoxia response pathway, thus triggering the Warburg effect.

Levine and Puzio-Kuter suggest that research aimed at a deeper understanding of how cancer-associated signaling pathways regulate biochemical metabolic pathways and trigger the Warburg effect, and the role of the Warburg effect in the pathogenesis of cancer, may lead to novel drug discovery strategies in oncology.

The Special Section on metabolism also includes an article on autophagy, a process by which cells break down cellular components in order to eliminate damaged biomolecules and organelles or to provide substrates for metabolism in case of starvation. Although autophagy promotes the health of cells and can prevent degenerative diseases, it can also enable cancer cells to survive in nutrient poor tumors.

There is also a review by Jay Keasling on metabolic engineering to produce such substances as natural product drugs, chemicals, and biofuels. Metabolic engineering is a branch of synthetic biology that engineers metabolic pathways to produce such substances, hence the inclusion of this review in the Special Section on metabolism. We have several articles on synthetic biology on this blog, most of which focus on metabolic engineering and its role in drug manufacture and drug discovery.

All in all, the 3 December Special Section on metabolism is worth reading by basic researchers, and by drug discovery and development researchers in biotechnology and pharmaceutical companies. It may broaden your perspectives, and lead to new ideas for R&D or partnering, especially in oncology drug discovery.
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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.

27 July 2010

A bioethicist says that the “synthetic cell” does not refute vitalism

By | 2010-07-27T00:00:00+00:00 July 27, 2010|Epigenetics, Synthetic biology|

In our last two blog posts–dated July 12, 2010 and July 18, 2010, we discussed the “synthetic cell” that was recently constructed by researchers at the J. Craig Venter institute. As we discussed, at least several leading bioethicists and philosophers said that the construction of a “synthetic” microbial cell refuted vitalism–i.e., the contention that there is something special about processes in living organisms that cannot be artificially created from nonliving systems–once and for all. However, leading scientists (including Nobel Prize winners and leading synthetic biologists) disagreed with that assessment. We said that we agreed with the leading scientists, and gave our reasons why.

Meanwhile, a one-page essay by bioethicist Gregory Kaebnick, Ph.D. appeared In the July 2010 issue of The Scientist (registration required).  Dr. Kaebnick is the editor of the bioethics journal The Hastings Center Report, and a co-investigator of a Hastings Center research project on synthetic biology. Dr. Kaebnick agrees with the leading scientists, and with us, even though his friend and colleague Arthur Caplan is one of the bioethicists who says that the “synthetic cell” has refuted vitalism.

According to Dr. Kaebnick, what the Venter group created was not a synthetic cell, but a synthetic genome. (As we stated in our second article, the researchers had help from yeast in creating the “synthetic” genome–perhaps it is really a semi-synthetic genome.) But the Venter group says that since the genome took over the cell it was transferred into, and since the genome is synthetic, therefore the cell is synthetic. But that assumes a top-down control of a cell by its genome (i.e., genetic determinism). Dr. Kaebnick argues that one might instead say that the cell and the genome worked out their differences and collaborated, or that the cell “adopted” the genome. He goes on to assert that we may not know enough to say which of these two metaphors is most adequate.

Then Dr. Kaebnick goes on to ask whether even if the Venter group did create a synthetic cell, whether that really demystified life at all. You will have to read his article to follow that argument.

From our point of view, even if the “top down” control model is the most nearly correct, without a pre-formed cell it would have been impossible to use the synthetic genome to create a living organism. Researchers cannot, at least at present, create a cell, with its membranes, organization of biomolecules, biochemical systems, etc. that is necessary for a genome to work to express itself in a living system.

Moreover, with the discoveries on epigenetics in the last decade or so, researchers know that a “top down” control model–especially in multicellular eukaryotic organisms–does not fully account for how cells and organisms work. The environment can mediate changes in chromatin, such as DNA methylation and histone modification, which can be passed down from cell to cell and in some cases even to the next generation.

Thus the issue of “top down” genetic determinism versus collaboration between a cell and its genome has implications for cutting-edge biological research. Since some drug discovery researchers have been working on discovery and development of epigenetics-based drugs, it is of interest to the biotechnology/pharmaceutical industry as well. Several such drugs, including Celgene’s DNA methyltransferase inhibitors and histone deacetylase inhibitors that we mentioned in an earlier blog post, are already on the market.

19 July 2010

The “synthetic cell”: not such a big deal (part 2)

By | 2010-07-19T00:00:00+00:00 July 19, 2010|Synthetic biology|

In our previous (July 12) blog post, we began a discussion of the creation of the “synthetic cell” by J. Craig Venter and his colleague at the J. Craig Venter Institute (JCVI).  In this study, the researchers produced a synthetic version of the genome of bacterium Mycoplasma mycoides. They then transferred the synthetic M. mycoides genome into the closely related bacterium M. capricolum, where then new genome took over the cells, resulting in bacteria that expressed the proteome of M. mycoides. The resulting cells were dubbed “synthetic cells”.

We said that we agreed with scientists (including working synthetic biologists) who said that the “synthetic cell” project did very little to advance synthetic biology, beyond the demonstration of technical virtuosity. Here’s why we agree.

JCVI’s “synthetic genome” is merely a copy of the natural M. mycoides genome, with a few minor differences. Copying the natural genome does not advance our understanding of how the various genes of M. mycoides function. Even in the case of such a simple organism as M. mycoides, only about half the genes in the genome are understood in terms of their biological function.

Copying the natural genome of M. mycoides also does not tell us how the genes of this organism work together in biological processes. Researchers find it difficult to construct even simple synthetic biology devices, because it is difficult to get artificially-introduced genes to work together as planned even in prokaryotic systems. For example, in 2000 researchers constructed a simple biological clock based on a three-gene “repressilator network” in E. coli. The clock was designed so that the cells flash on and off, based on oscillating expression of a fluorescent protein. However, the operation of this simple clock was “noisy”; i.e., the period of these oscillations was highly variable. However, natural biological clocks are not noisy. Other researchers therefore designed a more complex synthetic clock to damp down the noise of the original clock. This example illustrates how the complexity of biology can affect the ability of researchers to construct even simple biological devices. However, constructing such devices and then working to improve them can help researchers gain a greater understanding of how genes work together in natural networks and processes.

Every year since 2004, there has been an international undergraduate synthetic biology competition, run out of MIT, called the International Genetically Engineered Machine (iGEM) competition. In this competition, students use “biological parts” from a “kit”, and/or develop new “parts”, to design a synthetic biology device. Many teams that participate in this competition find out that designing new synthetic biology systems is not a simple case of putting together “biological parts” like Lego blocks or components of an electrical circuit. Not all the “parts” deposited in MIT’s Registry of Standard Biological Parts (including the ones designed by iGEM competition participants themselves) are well characterized, and even if they are, the “parts” may not work together with each other, or with the organism that contains them, as planned.

A January 2010 news feature in Nature, entitled “Five hard truths for synthetic biology”, details the many challenges to the field of synthetic biology. The construction of the “synthetic cell” does little or nothing to help synthetic biologists to meet these challenges.

Our main interest in synthetic biology is in metabolic engineering for use in drug discovery and in production of natural product drugs that are difficult to obtain cheaply from their natural plant sources and are difficult to synthesize chemically. We have two articles on synthetic biology on this blog pervious to the July 12, 2010 article–one dated July 28, 2009 and the other dated September 7, 2009 .

We are much more impressed by some of the work discussed in the September 7, 2009 article than by the creation of the “synthetic cell”. These include, for example, the metabolic engineering studies of Jay Keasling, such as his engineering of yeast and E coli to produce precursors of the antimalarial drug artemisinin. (For example, see this 2006 Nature article.) Dr. Keasling and his colleagues had to contend with several genes that did not work together as envisioned, and to find ways to optimize their expression so that they would work together to produce significant quantities of the desired product. Dr. Keasling says that it took approximately 150 person-years to complete this project. The pharmaceutical company Sanofi-Aventis has been scaling up Dr. Keasling’s artemisinin yeast production system, and plans to sell its product cheaper than artemisinin produced by current methods (i.e., from its natural plant source) by 2012.

We are also impressed by George Church’s automated method for optimizing metabolic engineering pathways, which was the focus of our September 7, 2009 article. This might make the work of metabolic engineers, epitomized by the Keasling groups’s work on artemisinin, faster, easier, and less expensive.

Finally, engineered mycoplasma are unsuitable for making medicines. They are too fragile, and their genomes are too small. For making medicines, one needs a “workhorse” organism that can take the extensive genetic manipulation needed to engineer new metabolic pathways, without losing viability and while maintaining a reasonable growth rate. And cultures of engineered microorganisms must be capable of being scaled up to industrial levels. Metabolic engineers have used such “workhorse” organism as E. coli, Saccharomyces cerevisiae, Streptomyces lividans, and Myxococcus xanthus.

Dr. Venter, through his company Synthetic Genomics, Inc. (SGI, La Jolla, CA) intends to work on synthetic genomics solutions for the production of biofuels. SGI has an alliance with the Exxon Mobil Research and Engineering (EMRE) group to generate biofuels from algae. Dr. Venter claims that SGI will make use of the tools and technologies from the synthetic genome research (together with more traditional metabolic engineering methods) to build “an entire algae genome so we can vary the 50 to 60 different parameters for algae growth to make superproductive organisms.” An alga that would be capable of being scaled up to produce commercial amounts of biofuels would have to be a real “workhorse” organism. And engineering of such algae would be an impressive achievement, beyond the level of a mere technical tour de force as with the “synthetic” mycoplasma cell. We (along with the rest of the world) await further progress on this project.