Pharma’s Cutting Edge

This is destined to be one of the key HIV papers of this decade, if not longer.

–Robert Gallo

I’m a real fan of unconventional thinking and scientists who are unafraid of venturing outside of their comfort zones.  So, when I read the story of the discovery of new potential drug targets against HIV in Science recently, I was thrilled.  I believe the full article behind the news piece requires a subscription; if you have one it’s worth at least a quick read-through.

Stephen Elledge is a pioneer in the use of “forward genetics” to screen for host factors that influence (suppress and or promote) the malignant phenotype (see this for an example of his group’s creative scientific approach).  In Elledge’s brand of forward genetics applied to cancer target screening, he and his team apply massive pools of interfering RNA to cells of various types to determine whether blocking endogenous gene transcription modulates the malignant cell phenotype. 

In the recent Science paper, Elledge and his collaborators used the same approach with a foreign invader–HIV.  I don’t know whose idea that was, but it was brilliant, and I give Elledge lots of credit for not shying away from a complex, costly, time-consuming HIV study despite his previous lack of experience working with the virus.  In short, the labs grew transformed epithelial cells (HeLa) transfected with some 21,000 pools of siRNAs in pools of 4 siRNAs per gene (i.e. siRNAs against all expressed human genes with transcription-suppresion redundancy to help gaurd against false positives and negatives) and then infected the cells with HIV-1.  When HIV-1 was able to replicate despite host-gene transcription inhibition, the scientists could conclude that the host gene probably wasn’t important for HIV replication, but if HIV replication was disrupted, they could surmise that a host protein involved in HIV replication had been discovered.  As you’ll read, the group found 273 host proteins that HIV relies upon for replication, only 36 of which had been previously discovered, thus explaining how HIV is able to so effectively parasitize its human hosts despite having a genome of just 9000 RNA bases.

As this is a scientific paper, the authors themselves have respectfully avoided aggrandizing this work and their screening approach in general, but this type of study is potentially very clinically relevant in the near-should be a more fruitful strategy to eliminate the virus from the body.  This is because HIV can easily mutate in the face of selection pressure from drugs targeting its own proteins, but it will be much harder for HIV to mutate to circumvent a blockade of a host target (or two) that it requires for reproduction. 

The accompanying news story expresses some skepticism from a Novartis researcher regarding the ease of discovering drugs targeting HDFs and the willingness of pharma to fund such work.  I frankly can’t understand such skepticism, but perhaps the story excerpted the interview in order to provide a bit of contrast to the enthusiasm from Dr. Gallo that opened the piece.  Yes, the HDFs are mostly internal cellular targets, and yes, they probably have some important roles to play in normal host functions, but let’s remember that we already use lots of drugs that fit this description, particularly in oncology, and although side effects are common, so are benefits.  As for pharma funding the work needed to capitalize on this work, let’s remember that it’s one thing to gauge the suits’ interests in the face of established pathway opportunities, and it’s quite another to anticipate their interest in the face of potential therapeutic breakthroughs with large commercial potential.   In my mind, this is a must-do opportunity for any company with virology capabilities and experience.  From a platform perspective, I’d be surprised if most large discovery outfits aren’t already taking advantage of genome-wide functional screening to identify the host factors enabling invaders to live at our expense, whether those invaders be infectious organisms or our own cells gone awry.

Obesity-associated improvements in metabolic profile through expansion of adipose tissue

Obesity is bad for you, right?  Not so quick.  It’s been known for decades that deficiencies of fat in unusual to rare syndromes known as lipodystrophies (aka lipoatrophic diabetes) can cause early-onset severe insulin resistance and eventually diabetes.  Based on these cases, clinical researchers surmised that fat isn’t necessarily bad for you–in fact you need some minimum amount of fat to have normal metabolism–and abnormal patterns of fat accumulation, such as an acquired loss of fat in subcutaneous tissues, can trigger insulin resistance and diabetes. 

Since those early clinical studies of lipoatrophic diabetes, many clinical and animal studies have demonstrated that the above is consistently true, and have recently led to a hypothesis that visceral fat (i.e. the fat surrounding internal organs, particularly in the abdomen) is harmful, whereas subcutaneous fat is not and may even help protect against diabetes.  The prevailing theory devised to account for the visceral/subcutaneous fat dichotomy is that triglyceride accumulation within subcutaneous fat serves as a kind of sink that shields vital organs from the sometimes harmful metabolic consequences of intracellular triglycerides (I say sometimes because elite athletes accumulate intracellular fat in muscle tissue without apparent harmful consequences).  In contrast, visceral fat accumulation leads to harmful metabolic consequences because venous drainage of visceral fat feeds fatty acids and glycerol to the liver, where they are reformed in hepatocytes into triglycerides.  Hepatic steatosis results, leading to insulin resistance.  For some unknown reason, says the lipotoxicity theory of diabetes, some people who consume an excess of energy relative to their energy expenditure, are able to accumulate visceral fat more effectively than subcutaneous fat and develop insulin resistance and beta cell failure (as a consequence of fat accumulation in the pancreas), leading to diabetes.  Aside from the “unknown reason” bit, it’s an elegant theory supported by a large body of observational and experimental data in multiple species, including humans.  What has been lacking, however, is an experimental model demonstrating that unconstrained subcutaneous fat expansion allows for development of obesity uncoupled from harmful metabolic consequences (i.e. insulin resistance, beta cell failure and diabetes).

In yesterday’s JCI (link above), Kim et al from Philpp Scherer’s group at UT Southwestern have published detailed descriprions of just such a model.  They created the unusual obesity model by breeding a transgenic mouse that constitutively overexpresses the hormone adiponectin onto an ob/ob mouse background.  The ob/ob mouse is a well described model of congenital leptin deficiency that results in morbid obesity and insulin-resistant diabetes.  Adiponectin is one of the hormones whose expression increases in response to PPAR gamma agonists, like the TZDs pioglitazone (Actos) and rosiglitazone (Avandia), drugs that increase fat mass, primarily subcutaneously, and that also improve insulin resistance.  As it turns out overexpressing adiponectin in leptin-deficient mice leads to diminished food intake (relative to body size) and diminished calorie expenditure (lower activity and lower body temperature) relative to leptin-deficiency alone.  It also leads to fat accumulation and even more weight gain, resulting in massively obese mice.  The balance of energy expenditure and fat accumulation without a change in fat clearance rate suggests enhanced efficiency of fat generation as an important mechanism for obesity in the mice.  Despite the weight gain and fat accumulation, the mice were protected from insulin resistance, beta cell failure and diabetes.  Furthermore, the triglyceride accumulation seen in the organs of leptin-deficient animals was absent in the transgenic animals, as was the associated inflammation (thought to be related to the lipotoxicity of intracellular fat).

Of interest clinically, the transgenic animals, including those bred on a WT background, also developed cardiomyopathy without fat accumulation in heart muscle.  As you’ll recall, as of August, the TZDs now have black-box warnings in their US labels regarding an increased risk of heart failure.  The animal finding suggests that adiponectin might be mechanistically related to the observed heart failure risk.  Keep in mind, though, that Kim et al also found that adiponectin feeds forward to enhance synthesis of PPAR gamma, so it’s not possible to make a TZD-adiponectin link to CHF based on these observations alone.

Although this model probably doesn’t offer any direct uses to drug hunters, it does offer an example of the rescue of a mouse model of obesity-related diabetes through fat accumulation.  In that regard, it serves two important purposes relevant to drug discovery.  First, it adds substantial weight to the lipotoxicity theory of type 2 diabetes, providing a basis for continued drug research in that area.  Many questions remain:  Is it possible to promote subcutaneous lipogenesis without simultaneously promoting morbid obesity?  Can these experimental findings be replicated by manipulating the downstream effectors of adiponectin action in fat; can it be done without promoting cardiomyopathy?  What exactly are the mediators of the toxic effects of triglycerides in liver, muscle and pancreas, and can they be safely interrupted?  Second it provides an animal-model benchmark for drugs intended to disrupt the obesity-diabetes link.  As it happens, the mark for prevention is set pretty high; the mice were completely protected against insulin resistance, beta cell dysfunction, and diabetes resulting from increased food intake.  Of course, the cure also further worsened their already morbid obesity, so there’s still lots of room for improvement. 

In some future post, I’ll discuss what has already been done and what might be done in the future to further test the lipotoxicity/fat deficiency model of diabetes in humans.

Common virus may contribute to obesity in some people

Last September, I noted that the epidemiology of modern obesity (in the last couple decades) strongly suggests transmissible or pervasive environmental factors–more persavive even than junk food and sedentary behavior–at work in its etiology.  Now, Magdalena Pasarica, M.D., Ph.D., of the Pennington Biomedical Research Center and her colleagues are reporting at the American Chemical Society meeting that adenovirus-36 (Ad-36), a common form of this common human-infecting virus, is capable of differentiating adult human stem cells into fat:

In the current study, Pasarica and her associates obtained adult stem cells from fatty tissue from a broad cross-section of patients who had undergone liposuction. Half of the stem cells were exposed to Ad-36 and the other half were not exposed to the virus.  After about a week of growth in tissue culture, most of the virus-infected adult stem cells developed into fat cells, whereas the non-infected stem cells did not, the researchers say.  Funded by the National Institutes of Health (NIH), Dr. Dhurandhar’s group recently identified a gene in the Ad-36 virus that appears to be involved in causing fat accumulation observed in infected animals. That gene, called E4Orfl, is now emerging as a promising target for future human therapies, such as vaccines and anti-viral medicines, aimed at preventing or inhibiting the obesity virus, she says.

About 30% of obese people are infected with Ad-36, compared with 11% of nonobese people.  While this evidence doesn’t prove an etiologic link between Ad-36 and obesity–that ultimately requires evidence that eradication of the virus or its effectors ameliorates obesity–it is a necessary step up the proof ladder.  Personally, I’m thinking that environmental factors that alter the gut microbiome (antibiotics? environmental pollutants?), increasing the efficiency of intestinal energy absorption, are more likely culprits than adenovirus, but then again, multiple etiologic agents are likely contributing.

The AOP of the July Nature Medicine contains the article “Neuropeptide Y acts directly in the periphery on fat tissue and mediates stress-induced obesity and metabolic syndrome” by Kuo et al from Georgetown U.  I’ve only read the abstract and stories so far (like this one from the Scientist and the press release from GU), but it’s enough to have me feeling elated at the discovery.  Congratulations to the Georgetown group.

Recall my post back in October 2006, when I used the failure of orally administered MK-0557, Merck’s NPY anatagonist, to discuss the state of pharma R&D knowledge transfer.  At the time, I implied that the development of NPY antagonists was all but dead.  Seems I may have spoken too soon.  Actually, I stick by my call regarding orally adminstered NPY antagonists, but if Kuo et al’s discovery of the role of NPY in glucocorticoid-mediated fat accumulation holds up to further scrutiny, I’ll be happy to revise that pronouncement as follows:  NPY anaagonists will have no role as systemically administered drugs aimed at influencing feeding behavior and thus obesity, but they might have a role as localy administered drugs aimed at controlling accumulation of local fat depots. 

The stories linked above describe some of the potential therapeutic uses of local NPY antagonist (and agonist) therapy.  Cosmetic uses are likely to be the first attempted, as it’s easy to approach the subcutaneous space and assess drug response.  Later uses could include non-surgical ablation of visceral fat (N.B. the omental fat dual-source blood supply may approached transcutaneously or intrarterially by experienced vascular interventionalists), which could very well treat metabolic syndrome. 

Intriguingly, activation of the peripheral NPY system may explain in full or part why glucocorticoids cause accumulation of fat in selected anatomical locations, including viscerally.  Although this explanation isn’t needed to enjoy the benefits of blocking the regeneration of cortisol from cortisone (via inhibition of 11-betahydroxysteroid dehydrogenase 1), as is being attempted with AMG221, Amgen’s 11 HSD1 inhibitor, it might help explain a failure of this or a similar drug to have the desired effects on visceral fat.  For instance, CT-guided skinny needle biopsies of visceral fat may be used to determine adipose tissue NPY and NPYR expression in the presence and absence of 11HSD1 inhibitor.  As far as I know, Amgen hasn’t released efficacy findings with this drug, and I’m not suggesting that it will fail to have desired clinical benefits.  Of course, the NPY mechanism might also be used to judge the effectiveness of such drugs before they ever get into humans too–a no-go for drugs that don’t block NPY upregulation in stressed mice fed ad libitum? 

Bottom line; this newly discovered peripheral NPY mechanism has great promise in metabolism drug discovery, as both a biomarker and as a drug target. 

Amidst the depressing industry news that has been crossing my email lately–allegations of rampant fraud and public deception at BMS and Lilly, respectively, among the more disturbing–I’ve decided to focus one of my final posts of the year on some refreshingly positive basic research news of high therapeutic relevance.  I’d classify these two independent research findings, both in the metabolic-disease arena, as genuine breakthroughs.

The first breakthrough, based on research led by Michael Dosch and his colleagues at the Hospital for Sick Children in Toronto, opens up a very promising, novel approach for prevention and early treatment of autoimmune diabetes mellitus (Type 1) and some other autoimmune diseases and, even maybe for Type 2 diabetes mellitus.  The research is published in the December 15th issue of Cell.

In short, Dosch’s group, in collaboration with a group from the University of Calgary, has discovered that transient receptor potential vanilloid-1(TRPV1)-secreting afferent sensory neurons play a crucial role in islet inflammation and beta cell function.  Islet inflammation is characteristic of Type 1 diabetes, and impaired beta cell function with variable islet inflammation characterizes type 2 diabetes. 

Specifically, the group determined that removal of TRPV1+ neurons abrogates immune cell infiltration into the islets of mice genetically prone to developing autoimmune diabetes (called NOD mice), without affecting the autoimmune properties of the immune cells themselves.  In other words, the sensory neurons were found to be responsible for accumulation of the destructive autoimmune cells into the pancreatic islets.

Sequencing of the TRPV1 sequence of NOD mice revealed DNA mutations resulting in two amino acid exchanges compared with wild-type that resulted in hypofunctional afferent sensory neurons in NOD mice.  These neurons secrete the neuropeptide substance P (sP), which was found to have accumulated in the dorsal root ganglia of NOD mice, consistent with reduced secretion in these animals.  Treatment of the NOD mice with sP or selective removal of TRPV1+ neurons prevented immune cell infiltration and the other manifestations of autoimmune diabetes, suggesting that a partially functioning TRPV1-islet cell circuit is causing the disease, and that either replacing the missing neuropeptide(sP) or eliminating the neural circuit can essentially prevent the diease. 

This opens up an entirely new prevention/treatment strategy for type 1 diabetes (and prehaps some types of type 2 diabetes, especially the so-called LADA subtype) that has potential to be more effective and far less risky that generalized immunosuppression, which is currently being studied clinically.

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