On November 8, 2009, we posted an article entitled “Anti-aging biology: new basic research, drug development, and organizational strategy” on this blog. This article focused on new findings in anti-aging biology, their applications to drug discovery and development, and on how this field has affected the organizational strategy of GlaxoSmithKline (GSK).

GSK acquired Sirtris for $720 million in 2008. Later that year, GSK appointed Christoph Westphal, the CEO and co-founder of Sirtris, as the Senior Vice President of GSK’s Centre of Excellence in External Drug Discovery (CEEDD). The CEEDD works to develop external alliances with biotech companies, with the goal of acquiring promising new drug candidates for GSK’s pipeline. Michelle Dipp, who was the vice president of business development at Sirtris at the time of GSK’s appointment of Dr. Wesphal, became Vice President and the head of the US CEEDD at GSK. Thus GSK has been using its relationship with Sirtris to restructure its organizational strategy, attempting to become more “biotech-like” in order to improve its R&D performance.

Now we learn that several research groups and companies have been questioning whether resveratrol (a natural product derived from red wine which has been the basis of Sirtris’ sirtuin-activator platform), as well as Sirtris’ second-generation compounds, may not modulate the sirtuin SIRT1 at all. Thanks to Derek Lowe’s “In the Pipeline” blog for the information. This issue was also covered in a second post on the same blog. It was also covered by articles in the 15 January 2010 issue of New Scientist and in the January 26, 2010 issue of Forbes. Nature also covered this story in an online news article.

In a report published in December 2009, researchers at Amgen found evidence that the apparent in vitro activation of SIRT1 was an artifact of the experimental method used by Sirtris researchers. The Amgen group found that the fluorescent SIRT1 peptide substrate used in the Sirtris assay is a substrate for SIRT1, but in the absence of the covalently linked fluorophore, the peptide is not a SIRT1 substrate. Although resveratrol appears to be an activator of SIRT1 if the artificial fluorophore-conjugted substrate is used, resveratrol does not activate SIRT1 in vitro as determined by assays using two other non-fluorescently-labeled substrates.

More recently, researchers at Pfizer published a study of SIRT1 activation by resveratrol and three of Sirtris’ second-generation sirtuin activators (which the Pfizer researchers synthesized themselves, using published protocols). These researchers also found that although these compounds activated SIRT1 when a fluorophore-bearing peptide substrate was used, they were not SIRT1 activators in in vitro assays using native peptide or protein substrates. The Pfizer researchers also found that the Sirtris compounds interact directly with the fluorophore-conjugated peptide, but not with native peptide substrates.

Moreover, the Pfizer researchers were not able to replicate Sirtris’ in vivo studies of its compounds. Specifically, when the Pfizer researchers tested SRT1720 in a mouse model of obese diabetes, a 30 mg/kg dose of the compound failed to improve blood glucose levels, and the treated mice showed increased food intake and weight gain. A 100 mg/kg dose of SRT1720 was toxic, and resulted in the death of 3 out of 8 mice tested.

The Pfizer researchers also found that the Sirtris compounds interacted with an even greater number of cellular targets (including an assortment of receptors, enzymes, transporters, and ion channels) than resveratrol. For example, SRT1720 showed over 50% inhibition of 38 out of 100 targets tested, while resveratrol only inhibited 7 targets. Only one target, norepinephrine transporter, was inhibited by greater than 50% by all three Sirtris compounds and by resveratrol. Thus the Sirtris compounds have a different target selectivity profile than resveratrol, and all of these compounds exhibit promiscuous targeting.

Sirtris and GSK dispute the findings of the Amgen and Pfizer researchers. One issue raised by Sirtris is that the Sirtris compounds synthesized by Pfizer may have contained impurities, resulting in the toxicity and lack of specificity of the compounds in vivo. Researchers associated with Sirtris and GSK also contend that although the Sirtris compounds only work with fluorophore-conjugated peptides in vitro, they appear to increase the activity of SIRT1 in cells. However, other researchers assert that since resveratrol interacts with many targets in cells, the results of the cellular assays are difficult to interpret. In the Forbes article, GSK’s CEO Andrew Witty is quoted as calling the dispute over the activity of the Sirtris compounds “a bit of a storm in a teacup”. He says that the compounds that Pfizer tested in mice are not the same ones that Sirtris and GSK are currently testing in clinical trials for treatment of diabetes and cancer. (Sirtris’ compounds in clinical trials, discussed in the next paragraph, are in fact different from the ones tested by the Pfizer researchers.)

Currently, Sirtris is testing its proprietary formulation of resveratrol, SRT501, in a Phase IIa clinical trial in cancer. The company reports that SRT501 lowered blood glucose and improved insulin sensitivity in patients with type 2 diabetes in a Phase IIa trial. Sirtris is also testing a second-generation SIRT1 activator, SRT2104, in Phase IIa trials in patients with metabolic, inflammatory and cardiovascular diseases. SRT2104 was found to be safe and well tolerated in Phase I trials in healthy volunteers. Sirtris is also testing another second-generation SIRT1 activator, SRT2379, In Phase I trials. SRT2379 is structurally distinct from resveratrol and from SRT2104.

As we discussed in our original blog post, Elixir Pharmaceuticals is also developing various sirtuin inhibitors and activators for metabolic and neurodegenerative diseases and for cancer. One of Elixir’s products, the SIRT1 inhibitor EX-527, was in-licensed by Siena Biotech (Siena, Italy) in 2009, and was entered into Phase I clinical trials in January 2010. Siena Biotech is developing this compound for treatment of Huntington’s disease.

Despite the dispute over whether Sirtris’ compounds are real SIRT1 activators, the numerous studies on the biology of sirtuins are still valid. Companies with assays that use native peptide substrates and are amenable to high-throughput screening could use these assays to discover novel sirtuin activators. For example, Amgen published a report in 2008 describing such assays. The ability of companies such as Amgen and Pfizer to commercialize such novel sirtuin activators would depend on whether they could overcome the intellectual property position of Sirtris (and Elixir). Since Amgen and Pfizer are working in this area, this indicates that they believe that they can do so.

The efficacy of high doses of resveratrol in improving metabolic parameters of mice fed a high-calorie diet is also not invalidated by the Amgen and Pfizer studies. However these studies cast doubt on the mechanisms by which resveratrol exerts these effects. The apparent efficacy of SRT501 in improving metabolic parameters in patients with type 2 diabetes in a Sirtris Phase IIa trial is consistent with the mouse studies.

Finally, as we discussed in our November 8, 2009 blog post, longevity is controlled by numerous interacting pathways, which may provide at least several targets for drug discovery. Researchers are hard at work to gain additional understanding of these pathways, and some companies are working to discover and develop compounds that modulate these targets. For example, several companies are developing AMPK activators, as discussed in our original blog post. And numerous research groups are reportedly attempting to find drugs that act similarly to rapamycin in increasing lifespan in mice (the main focus of our November blog post), without rapamycin’s immunosuppressive effects.

In the December 15, 2009 issue of Neurology, a research report by Stephen Salloway and his colleagues at the Butler Hospital and Brown University (Providence, RI) and an editorial by Dan Kaufer and Sam Gandy (University of North Carolina at Chapel Hill) focus on a Phase II multicenter placebo-controlled clinical trial of Elan/Wyeth’s bapineuzumab (AAB-001) in patients with mild to moderate Alzheimer’s disease (AD). (Wyeth is now part of Pfizer.) (A subscription is required to read the full text of both of these articles.) Bapineuzumab is a monoclonal antibody (MAb) drug that is specific for amyloid-β (Aβ) peptide. The dominant paradigm among AD researchers and drug developers is that the disease is caused by aberrant metabolism of Aβ, resulting in accumulation of neurotoxic Aβ plaques. This paradigm is known as the “amyloid hypothesis”.

The overall result of the study by Salloway et al. was that there was no difference in cognitive function between patients in the drug-treated and the placebo groups. However, the study did not have sufficient statistical power to exclude the possibility that there was such a difference. About 10% of patients treated with the agent also experienced vasogenic edema (VE), which was reversible. (Cerebral VE is the infiltration of intravascular fluid and proteins into brain tissue, as the result of breakdown of the blood-brain barrier.)

Retrospective analysis of the data suggested that bapineuzumab-treated patients who were not carriers of the apolipoprotein E epsilon4 allele (ApoE4) showed improved cognitive function as compared to placebo treatment, and that they had a lower incidence of VE than ApoE4 carriers. The ApoE4 polymorphism is the only known, well-characterized genetic risk factor associated with the development of late-onset AD. Of the three common isoforms of ApoE, ApoE3 is the most common, followed by ApoE4 and ApoE2, respectively. Unlike ApoE4, the ApoE2 allele appears to protect against development of AD. Some researchers estimate that allelic variations in ApoE may account for over 95% of AD cases.

In the study by Salloway et al., nearly two-thirds of the AD patients carried one or more ApoE4 alleles; thus only the remaining one-third of patients appeared to show positive effects of bapineuzumab treatment according to the retrospective analysis. However, the idea that the drug is efficacious in ApoE4 noncarriers is only a hypothesis, which will require prospective clinical trials to confirm. Elan and Pfizer are now conducting large Phase III clinical trials of bapineuzumab, which have prospectively segregated enrollment into ApoE4 carrier and noncarrier groups.

The hypothesized association of ApoE4 noncarrier status of AD patients with bapineuzumab efficacy and safety has been used as a case study in workshops on stratified medicine sponsored by the FDA, MIT, and industry partners in 2009 and 2010. You can read about the October 2009 workshop here. The most recent workshop was held at MIT on January 19, 2010. In these workshops, two case studies were discussed: the use of diagnostic tests for the HER2 receptor in identifying breast cancer patients who are likely to benefit from treatment with trastuzumab (Genentech/Roche’s Herceptin), and the bapineuzumab/ApoE4 case. The HER2/ trastuzumab relationship is well known and well characterized, and is considered to be a paradigm of stratified medicine. This contrasts with the bapineuzumab/ApoE4 association, which remains a hypothesis pending the results of the Phase III prospective clinical studies.

A growing minority of researchers is skeptical that the amyloid hypothesis is sufficient to account for AD pathogenesis in all stages of the disease or in various disease subpopulations, and they are investigating other pathways that may contribute to the disease, either in combination with the amyloid pathway or as alternative mechanisms. We have discussed alternative hypotheses for AD pathogenesis in a 2004 article published in Genetic Engineering News (available on our website), and in book-length reports published by Cambridge Healthtech Institute in 2006 and in 2009.

The search for alternative hypotheses takes on added urgency because of the clinical failure of several AD drugs that had been designed based on the amyloid hypothesis. These include Neurochem’s (now Bellus Health) Alzhemed (3-amino-1-propanesulfonic acid) and Myriad Pharmaceuticals’ Flurizan (tarenflurbil), both of which failed in Phase III clinical trials. Based on the overall results of the Phase II trial of bapineuzumab, most researchers and industry commentators would add bapineuzumab to the list, unless the stratified Phase III trial shows that the drug is significantly efficacious and safe for ApoE4 noncarriers.

Since ApoE4 carrier status is such a prominent risk factor for developing late-onset AD, might ApoE4 itself be a target for drug discovery in AD? Drs. Kaufer and Gandy suggest that such an approach might be fruitful, whatever the outcome of the Phase III trial of bapineuzumab. Several academic laboratories have been investigating mechanisms by which ApoE4 may be involved in the pathobiology of AD. You may read two recent papers on this subject here and here. ApoE4 may contribute to AD pathogenesis via multiple mechanisms, including by causing synaptic deficits and mitochondrial dysfunction in neurons, and by inducing endoplasmic reticulum stress leading to astrocyte dysfunction.

Given the prominence of ApoE4 expression as a risk factor for AD, the study of the mechanistic basis of ApoE4’s role in AD pathobiology needs greater attention. Hopefully, this research will lead to the development of novel therapeutic strategies for AD.

In the December 10 2009 issue of Nature, researchers at Agios Pharmaceuticals (Cambridge, MA) and their academic collaborators published an article implicating mutations in a metabolic enzyme, cytosolic isocitrate dehydrogenase (IDH1) as a causative factor in a major subset of human brain cancers.

The mutated forms of IDH1 are found in around 80% of human grade II-III gliomas and secondary glioblastomas. The mutations occur in arginine 132, which is usually mutated to histidine. (In other less common mutations, arginine 132 is mutated to serine, cysteine, glycine, or leucine.) Typically, only one allele of IDH1 is mutated. These mutations appear to occur early in the process of tumorigenesis, and often appear to be the first mutation that occurs. The mutant forms of IDH1 are also found in a subset of acute myelogenous leukemia (AML).

The wild-type form of IDH1 catalyzes the NADP+-dependent oxidative decarboxylation of isocitrate to α-ketoglutarate. However, the researchers found that the mutant forms of IDH1 no longer catalyzes this reaction, but instead catalyzes the NADPH-dependent reduction of α-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). This is the result of changes in the active site of the enzyme, as demonstrated by structural studies carried out by the researchers. Tumors that harbor the mutant form of IDH1 have elevated levels of 2HG. The researchers therefore hypothesize that these elevated levels of 2HG are a causative factor in tumorigenesis and/or tumor progression in human gliomas.

This hypothesis is supported by the effects of the familial metabolic disorder 2-hydroxyglutaric aciduria. This disease is caused by a deficiency of 2-hydroxyglutarate dehydrogenase, an enzyme that converts 2HG to α-ketoglutarate. Patients with this metabolic disease have elevated levels of 2HG in bodily fluids and in the brain, and an increased risk of developing brain tumors.

The mechanism by which 2HG might contribute to tumorigenesis is unknown. The authors advance several hypotheses, including increasing reactive oxygen species (ROS) levels, serving as an NMDA (N- methyl-D-aspartate) receptor agonist, and competitive inhibition of enzymes that use glutamate and/or α-ketoglutarate resulting in the induction of hypoxia-inducible factor-1α, a transcription factor that facilitates tumor growth under conditions of hypoxia.

According to the authors, these results suggest that in patients with low-grade gliomas containing mutant forms of IDH1, therapeutic inhibition of 2HG production may slow or halt progression of these tumors to lethal secondary glioblastomas. 2HG levels may also be used as a prognostic test for IDH1 mutations, since patients with these mutations tend to live longer than patients with gliomas that have other mutations.

The company that led this research, Agios Pharmaceuticals, is developing a pipeline of oncology drugs based on targeting metabolic pathways in cancer cells. Interestingly, Agios means “holy” in Greek.

Way back in 1924, Otto Warburg demonstrated a difference between cancer cells and normal adult cells in glucose metabolism. In the presence of oxygen, most normal adult cells metabolize glucose to pyruvate via the process of glycolysis, generating two molecules of ATP (the energy currency of the cell) per glucose molecule. In the mitochondria, they then utilize oxygen to catabolize pyruvate to CO2 and water, in the process generating 36 molecules of ATP per glucose molecule. Cancer cells, however, predominantly carry out aerobic glycolysis, in which they carry out glycolytic conversion of glucose to pyruvate, followed by reduction of pyruvate to lactate. Despite the presence of oxygen, cancer cells generate the bulk of their ATP from glycolysis, not mitochondrial oxidative phosphorylation, in the process consuming large amounts of glucose. The reliance of cancer cells on aerobic glycolysis for their metabolism is known as the “Warburg effect”.

Agios’ platform is based in part on the work of signal-transduction pioneer Lewis Cantley (Beth Israel Deaconess Cancer center/Harvard Medical School, Boston MA). It is Dr. Cantley’s work on the connection between growth factor-mediated signal transduction and aerobic glycolysis that is the basis for Agios’ platform. 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 mitochondria oxidative phosphorylation to anabolic processes required for rapid proliferation of cancer cells.

Agios closed a $33 million Series A financing in July 2008, co-led by Third Rock Ventures, Flagship Ventures and ARCH Venture Partners. In June 2009, Fierce Biotech named Agios to the 2009 FierceBiotech “Fierce 15” list. On December 21, 2009, Agios received funding from the nonprofit organization Accelerate Brain Cancer Cure (ABC2), to supplement Agios’s research on the development of IDH1-based therapeutics and diagnostics. Agios expects to have a lead compound in the clinic some time in 2010.

The Agios website calls cancer metabolism “one of the most exciting new areas of cancer research”. But the study of cancer metabolism, and especially the Warburg effect, is not new—the Warburg effect is a classic observation going back 85 years. Moreover, biotechnologists working in such areas as production of recombinant proteins in CHO cells have been familiar with aerobic glycolysis, which is carried out by most mammalian cell lines in culture, for decades. Nevertheless, cancer metabolism has been well out of the mainstream of cancer drug discovery. It was Dr. Cantley’s work, which links the classic Warburg effect to the mainstream area of signal transduction and protein kinases, which has made Agios’ platform possible.

Similarly, it was Julian Adams’ work on the biology of the proteasome in the 1990s, through a series of biotechnology company mergers that eventually led him to Millennium Pharmaceuticals (now Millennium: The Takeda Oncology Company), which resulted in Millennium’s proteasome inhibitor Velcade (bortezomib). Velcade, the only proteasome inhibitor on the market, is now approved by the FDA for the treatment of multiple myeloma and mantle cell lymphoma. Prior to Dr. Adams’ work, proteasome biology and protein degradation were out of the mainstream of cancer drug discovery. Now Joseph Bolen, the chief scientific officer of Millennium, sees “protein homeostasis” as one of the most exciting areas of cancer research.

Finally, although the development of protein kinase inhibitors to target signaling pathways in cancer is now well within the mainstream of oncology drug discovery, prior to the discovery and development of imatinib (Novartis’ Gleevec/Glivec) (approved by the FDA in 2001), specific targeting of protein kinases was though to be unlikely, since all of these enzymes have a high degree of similarly in their ATP binding sites. Thus the field of protein kinase inhibitors did not enter the mainstream until the late 1990s-early 2000s.

The take-home lesson is that drug developers may find fertile areas for innovation in seemingly obscure or out-of-the mainstream areas of biology (or of chemistry, as we have discussed in previous blog posts). Some of these areas may be technologically premature, and not quite ready for exploitation by drug developers. However, as demonstrated by our blog post on monoclonal antibodies, even some technologically premature areas may yield to innovators who are willing and able to develop enabling technologies to move these areas up the development curve.

In our November 27th blog post, we discussed an innovative new technology, stapled peptides, for use in targeting intracellular protein-protein interactions. In the example we gave, the target was a transcription factor complex in the Notch pathway. As we stated, protein-protein interactions are deemed to be “undruggable”, since they cannot be readily addressed with small molecule drugs.

Nevertheless, in some cases, small molecules have been discovered that do address key protein-protein interactions, and which may become clinical candidates.

Back in February 2006, Decision Resources published our report, “Protein-Protein Interactions: Are They Now Druggable Targets?” Among the case studies we discussed in that report was one in which researchers were attempting to discover small-molecule agents that targeted the Wnt pathway. The researchers discovered small-molecule agents that, as with the stapled-peptide example we discussed in our previous blog post, targeted a transcription factor complex. As of late 2009, two of these compounds are in preclinical development for treatment of various cancers.

Mutations that mediate deregulation of the Wnt pathway are causative factors in several types of cancer, most notably colorectal cancer, as well as multiple myeloma (MM), hepatocellular carcinoma (HCC), and B-cell chronic lymphocytic leukemia B-CLL). In the canonical Wnt pathway, soluble extracellular factors that are members of the Wnt family activate the pathway. A complex that includes the protein adenomatous polyplosis coli (APC) is central to the Wnt pathway. When Wnt receptors are not engaged by their ligands, kinases in the APC complex phosphorylate β-catenin, a multifunctional protein that is involved both in signal transduction and in adhesion between cells. Phosphorylation targets β-catenin for degradation.

When Wnt proteins bind to their receptors, the kinase activity of the APC complex is inactivated. This results in the accumulation of β-catenin, which moves into the nucleus. There it binds to proteins of the T cell factor (Tcf) family. β-catenin binding changes Tcf from a transcriptional repressor into a transcriptional activator. Downstream genes controlled by the β-catenin/Tcf complex include the oncogene Myc and other genes that mediate cell proliferation.

In precancerous colonic adenomas or the colorectal cancers that they may evolve into, APC is usually mutated. This results in constitutive stabilization of β-catenin and constitutive activation of Tcf and its downstream genes. In other types of cancer that involve constitutive Wnt pathway activation, β-catenin also becomes stabilized, via other means. This makes the Tcf/β-catenin a tempting target for drug discovery. However, it is a protein-protein interaction, and is thus deemed “undruggable”.

In 2004, A group led by Ramesh Shivdasani (Harvard Medical School, Dana-Farber Cancer Institute, and Brigham and Women’s Hospital, Boston MA), including researchers from the Novartis Institutes for BioMedical Research (Cambridge, MA), discovered several small-molecule inhibitors of the interaction between human Tcf4 and human β-catenin.

Dr. Shivdasani’s group, among others, had previously determined crystal structures of Tcf-β-catenin complexes. The interaction between the two proteins occurs over a large surface area. It is the large, and usually hydrophobic, interface between proteins in protein-protein interactions that forms the theoretical basis for the difficulty of addressing these interactions with small molecules. However, there is a small hydrophobic pocket that is critical for binding (as also confirmed by site-specific mutation studies), which might accommodate a small molecule inhibitor.

Therefore, the researchers screened approximately 7,000 purified natural products from public and proprietary libraries using an enzyme-linked immunosorbent (ELISA) assay involving release of a labeled Tcf4 binding fragment from its complex with a β-catenin fragment absorbed to an ELISA plate. Eight compounds were found that gave reproducible, concentration-dependent release of the Tcf4 fragment at less than 10 micromolar concentration. The structures and purity of these compounds (most of which are complex, multi-ringed planar compounds with multiple hydroxy groups) were then determined. The sources of these compounds include fungi, actinomycetes, and a marine sponge.

The researchers performed several additional biochemical assays to confirm the compounds’ specific disruption of the Tcf/β-catenin complex, and also performed cellular assays and an in vivo assay in the Xenopus (frog) embryo to study the activities of these compounds against β-catenin-mediated cellular effects. Each of the eight compounds shows different levels of potency in the different assays used in this study, and the compounds differ from each other in their activities in the different assays.

Two fungal-derived compounds, PKF115-854 and CGP04909, gave the best results in all the assays. It is those compounds that have been tested in preclinical studies as potential oncology drug candidates. In a study published in PNAS in 2007, researchers at the Dana-Farber and at Brigham and Women’s Hospital tested PKF115-584 in human MM cells in vitro and in xenograft models. The compound blocked expression of Wnt target genes, induced cytotoxicity in MM cells in vitro, and inhibited tumor growth and prolonged survival in the xenograft model. In a study in HCC at the Asian Liver Center at Stanford University School of Medicine, PKF115-584, CGP049090, and another of the Shivdasani group’s compounds, PKF118-310, also induced cytotoxicity in human HCC cell lines in vitro, and suppressed tumor growth and induced apoptosis in tumor cells in a human HCC xenograft model. Finally, in an abstract presented at the American Society of Hematology (ASH) meeting in December 2009, researchers at the Novartis Institute for Biomedical Research in Basel and their academic collaborators presented data that showed that CGP04090 and PKF115-584 potently inhibited the survival of primary human B-CLL cells in vitro and in vivo. In all three cases, the compounds showed no significant cytotoxicty against normal cells.

In the conclusion of the ASH meeting abstract, the authors stated that further investigations are warranted to determine the feasibility of testing these compounds in human clinical trials.

Many medicinal chemists remain skeptical about the ability of researchers to develop small-molecule drugs that target protein-protein interactions, which have satisfactory pharmacokinetics and can advance through clinical trials and reach the market. However, at least one nonpeptide small-molecule compound that targets a protein-protein interaction, the thrombopoietin receptor agonist eltrombopag (Ligand/GSK’s Promacta), has reached the market. (The FDA approved it in November 2008.) Several other small-molecule drugs that target protein-protein interactions are in clinical development. And Cambridge Healthtech Institute will be sponsoring a conference on this subject, which is scheduled for April 2010. This conference is in its third year. Thus, as also shown by the development of stapled peptides, there is renewed interest in discovering and developing drugs that address these “hard targets”.

In the 2 October issue of Science (the “Ardipithecus ramidus issue”), there was a Perspective (authored by Matt Kaeberlein and Pankaj Kapahi) and a Report (authored by Colin Selman and his colleagues) on recent findings in anti-aging biology.

Since the late 1980s, researchers have found that caloric restriction (CR) (reduction in caloric intake while maintaining essential nutrients) slows aging in a variety of organisms—yeasts, nematodes, fruit flies, mice, and most recently rhesus macaques. In the recently published 20-year study in rhesus macaques, CR not only increased lifespan, but also delayed the onset of a suite of aging-related disease conditions—diabetes, cancer, cardiovascular disease, and brain atrophy. This parallels the studies with other organisms.

Researchers who have been studying the CR model have been attempting to elucidate the mechanisms by which CR works to slow the aging process and to retard aging-related disease. They hope to find targets for drugs to mimic the effects of CR in humans, since long-term CR is not practical for most people. Over the years, researchers have discovered several pathways by which CR appears to exert its effects. The Report describes new research results on one such pathway, the mammalian target of rapamycin (mTOR) pathway. The Perspective reviews this research in the context of related recent studies.

In a report published in Nature earlier this year (16 July 2009), researchers found that rapamycin administered in food increased the median and maximal lifespan of genetically heterogeneous laboratory mice, whether it was fed to middle-aged (600 days old) or young adult (270 days old) mice. Rapamycin feeding beginning at 600 days of age led to an increase in lifespan of 14% for females and 9% for males, on the basis of age at 90% mortality.

Rapamycin targets mTOR (mammalian target of rapamycin), a kinase that regulates signaling pathways that affect many cellular processes. mTOR forms two protein complexes that are active in intracellular signaling—mTORC1 and mTORC2. It is mTORC1 that is most sensitive to rapamycin. mTORC1 works to coordinate cellular growth and survival responses induced by changes in the availability of nutrients, and also responses to cellular stresses (e.g., hypoxia, DNA damage and osmotic stress). Genetic inhibition of TORC1 in yeast and invertebrates has been found to extend their lifespan. In particular, in the nematode Caenorhabditis elegans, TORC1 interacts with the insulin pathway (via raptor, a component of TORC1) to control lifespan. The role of the insulin pathway in the enhancement of lifespan by CR in C. elegans has been known for many years. The role of mTORC1 at the junction of nutrient and stress sensing pathways, together with these results in invertebrates and now mice, has led researchers to hypothesize that the mTORC1 pathway may be involved in CR-mediated enhancement of lifespan, and that drugs that modulate this pathway may substitute for CR in lifespan extension.

In other studies, inhibition of the mTOR pathway in mice was found to retard development of such aging-related conditions as cancer, metabolic disease, and cardiovascular disease. This effect has also been seen in studies of CR in mice and in nonhuman primates, as stated above.

Rapamycin is an immunosuppressant that is marketed as Wyeth’s (now Pfizer’s, since the October 2009 merger) Rapimmune, to prevent organ transplant rejection. More recently, a derivative of rapamycin, temsirolimus (Wyeth/Pfizer’s Toricel) has been approved for treatment of renal cell carcinoma. The authors of the Nature paper therefore hypothesized that rapamycin may have extended lifespan in the mice either by working via CR-related pathways that control lifespan, by postponing death from cancer, or both.

The finding that oral rapamycin can retard aging in mice, even when fed to 600-day-old mice (the equivalent of 60 years old in humans) raises hope for the development of anti-aging drugs for human use. However, rapamycin itself cannot be used for this purpose because of its immunosuppressant effects. (In the mouse rapamycin feeding studies, the mice were kept under specific pathogen-free conditions.) If researchers were to attempt to modulate the mTORC1 pathway to extend lifespan, they would therefore need to discover other drugs that modulate that pathway without rapamycin’s side effects. Learning more about specific pathway components that may be targeted to increase lifespan may help researchers discover such drugs.

In the new Selman et al. report, researchers endeavored to learn more about how the mTORC1 pathway might extend lifespan in mice. They constructed knockout mice that lacked S6 protein kinase 1 (S6K1). S6K1 is a downstream target of mTORC1, which upregulates mRNA translation and protein synthesis in response to mTORC1 signaling. The researchers found that deletion of the gene for S6K1 resulted in a 19% increase in median lifespan in female mice (as compared to wild-type females), and also increased maximum lifespan. S6K1 deletion had no effect on the lifespan of male mice. This was in contrast to the study with rapamycin feeding, which showed lifespan extension in both sexes, even though the effect in female mice was greater. However, the results of the two studies are not strictly comparable, since mice of different genetic background were used in the two studies.

Female S6K1 knockout mice also showed improvement in several biomarkers of aging (e.g., motor and neurological function, level of physical activity, insulin sensitivity, glucose tolerance, fat mass, immunological parameters). Hepatic gene expression in 600-day-old female S6K1 knockout mice resembled that of wild type mice subjected to CR. Female S6K1 knockout mice showed increased hepatic, muscle, and adipose tissue expression (as compared to wild-type mice) of genes associated with other pathways associated with longevity, including genes for sirtuin-1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK).

Selman et al. went on to obtain evidence that the effect of S6K1 knockout on lifespan in female mice is due to activation of AMPK. The gene expression profile of muscle tissue of long-lived female S6K1 knockout mice resembled the profile of wild-type mice treated with the AMPK activator aminoimidazole carboxamide ribonucleotide (AICAR). Hepatocytes from S6K1 knockout mice also showed enhanced AICAR activation of AMPK as compared to hepatocytes from wild type mice. A parallel study in C. elegans showed that deletion of the aak-2 gene, which encodes a subunit of AMPK, suppresses lifespan extension in mutants that lack rsks-1, the nematode homolog of S6K1. These results suggest that S6K1 knockout may exert its pro-longevity effects via activation of AMPK.

AMPK is found in all eukaryotic organisms, and serves as a sensor of intracellular energy status. In mammals, it also is involved in maintaining whole-body energy balance, and helps regulate food intake and body weight. AMPK has been implicated in metabolic response to CR in eukaryotic organisms from yeasts to humans, and it mediates the effects on lifespan of at least one type of CR regimen in C. elegans. Thus the hypothesis that lifespan extension via the mTORC1-S6K1 pathway works via AMPK activation is an attractive one.

However, it is not known how deletion of S6K1 (or its inhibition via mTORC1 in rapamycin-treated mice) might activate AMPK. Moreover, as pointed out by Kaeberlein and Kapahi, there are other downstream targets of S6K1 that might play a role in anti-aging effects of SK61 deletion or inhibition. Among these is hypoxia-inducible factor-1α (HIF-1α). Moreover, there are other biomolecules and pathways that have been implicated in the effects of CR on retarding aging. These especially include the sirtuins, an evolutionarily conserved family of nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylases.

As shown by the Perspective and Report in the 2 October issue of Science, anti-aging research is an exciting area of basic biological research, and researchers still have much to learn about pathways that mediate the effects of CR on longevity. However, this field is already being applied to drug discovery and development. A basic issue in applying anti-aging research to the development of drugs is that one clearly cannot use increased lifespan as an endpoint in clinical trials. Companies must test putative anti-aging drugs against one or more diseases of aging. The hope is that any “anti-aging” drugs approved for treatment of one disease of aging will have pleiotropic effects on multiple diseases of aging, and will ultimately be found to increase lifespan or “healthspan” (the length of a person’s life in which he/she is generally healthy and not debilitated by chronic diseases).

The two principal types of “anti-aging” drugs currently in company pipelines are sirtuin modulators and AMPK activators. Sirtris Pharmaceuticals (Cambridge, MA, a wholly-owned subsidiary of GlaxoSmithKline [GSK]) is developing the SIRT1 activators SRT501 (a proprietary formulation of the natural product resveratrol) and SRT2104 (a novel synthetic small-molecule SIRT1 activator that is structurally unrelated to resveratrol and is up to 1000-fold more potent). SRT501 is in Phase II clinical trials in type 2 diabetes. SRT2104 has been tested in Phase I trials in healthy volunteers, and was found to be safe and well tolerated. Elixir Pharmaceuticals (Cambridge, MA) is developing a preclinical-stage SIRT1 inhibitor for treatment of Huntington’s disease and certain cancers, and a preclinical-stage SIRT1 activator for treatment of type 2 diabetes and obesity. Elixir also has a research-stage SIRT2 inhibitor under development for treatment of type 2 diabetes and obesity.

Companies developing AMPK activators include a collaboration between Metabasis Therapeutics (La Jolla, CA; about to be acquired by Ligand Pharmaceuticals, San Diego, CA) and Merck–preclinical oral AMPK activators, for treatment of type 2 diabetes and hyperlipidemia), Mercury Therapeutics (Woburn, MA)–research and preclinical-stage oral AMPK activators for treatment of type 2 diabetes, and Betagenon (Umea, Sweden)–the preclinical-stage oral AMPK activator BG8702, for treatment of type 2 diabetes.

The relationship between sirtuin-modulator developer Sirtris and GSK represents a prime example of the attempt of large pharmaceutical companies to become more “biotech-like” in order to improve their R&D performance. We discussed this strategy in our recent report, Approaches to Reducing Phase II Attrition. GSK acquired Sirtris for $720 million in June 2008. In December 2008, GSK announced that it had appointed Christoph Westphal, the CEO and co-founder of Sirtris, as the Senior Vice President of GSK’s Centre of Excellence in External Drug Discovery (CEEDD). The CEEDD works to develop external alliances with biotech companies, with the goal of acquiring promising new drug candidates for GSK’s pipeline. Michelle Dipp, who was the vice president of business development at Sirtris at the time of GSK’s appointment of Dr. Wesphal, is now Vice President and the head of the US CEEDD at GSK. Dr. Westphal, who is also a former venture capitalist, remains as CEO of Sirtris, and is based at Sirtris’ Cambridge location.

Thus anti-aging research, despite the fact that it is mainly in the basic research stage, is not only beginning to produce drug candidates, but has also been having an impact on the organizational strategy of one of the major pharmaceutical companies, GSK.