Pharmtastic

I finally got around to setting up a proper website for this blog. I hope I can continue to add new content consistently from here on out. 

CD47, a five transmembrane domain, integrin-associated protein that is ubiquitously present throughout the human body. With respect to cancer, CD47 sends a “Don’t eat me” signal to macrophages and therefore considered to be one of the most common mechanisms for aggressive tumor growth. Modulating this signaling pathway has resulted in positive outcomes in almost all cancers studied. (see below)

Dr. Irv Weissman of Stanford Univ. presenting the list of human cancers under study via @CIRMnews 

Hu5F9-G4, the anti-CD47 antibody discovered by Dr. Weissman’s lab and now being developed further by Forty Seven Inc, a spin-off (backed by Google money!). They presented their data from the first-in-human trial in advanced cancers and concluded that Hu5F9-G4 was well tolerated at 3 mg/kg dosing. However, due to the ubiquitous nature of CD47, antibodies targeting this protein are expected to cause a number of side effects including anemia (RBC loss). To compensate for this adverse effect, they used a priming dose of Hu5F9-G4 to occupy all RBC CD47 receptors with “limited” side effects.

So what about the competition?

Obviously, a number of other biotech companies are also working on therapeutics targeting this pathway. Trillium Therapeutics ($TRIL) remains one of their biggest competitors, and are developing a SIRPαFc and human IgG1 Fc fusion protein (TTI-621) to act as a decoy receptor that elicits phagocytosis by macrophages followed by a T-cell response. They recently presented the results from their Phase 1a trail in Diffuse large B-cell lymphoma (DLBCL) and Hodgkin’s lymphoma patients (n=6 each). Make what you will of the data (below) but we can all agree it does not look like a “cure” (at least not as a monotherapy in really aggressive cancers). However, it is a start and it would be interesting to see how it works in combination with current drugs targeting antigens - CD20 (rituximab) in B cell lymphomas or HER2 (trastuzumab) in breast cancer.

Curiously, they managed to avoid binding to CD47 on RBCs and claim that this is due to “lack of CD47 membrane mobility combined with moderate affinity of TTI-621 for target”. However, they observed transient thrombocytopenia in their Phase 1a trial (Data below). Although they downplay this by saying “TTI-621 is generally well tolerated”, they are obviously a little worried about this adverse reaction, hence the development of second generation molecule TTI-622 that, unlike TTI-621, has a human IgG4 Fc fragment. They expect that “TTI-622 is less likely to deplete platelets, enabling higher exposures”.   

Further, they are working on other malignancies including solid tumors with results expected by the end of the year. More recently, they have had one big win by winning against Forty Seven Inc over patent infringement claims (below). 

What about the others?

OSE Pharma ($OSE) is taking the obvious alternate approach by blocking the SIRPαFc receptors on macrophages and hence preventing CD47-mediated “Do not eat me” signals. They claim that EFFI-DEM, an IgG4 specifically targeting SIRPαFc has shown promising data in solid tumor preclinical models. 

Celgene's CC-90002, anti-CD47 antibody licensed from Inhibrx in 2012 and might be the furthest along in the development process. Data from their Phase 1 trials expected at the AACR meeting in April this year. 

Novimmune’s NI-1701, anti-CD47/CD19 bispecific antibody and NI-1801 anti-CD47/Mesothelin bispecific antibodies are in pre-clinical and discovery phases respectively. 

Tioma Therapeutics (formerly Vasculox), a spin-off from William Frazier’s lab at Washington Univ., St. Louis. Interestingly, some of his recent papers show additional applications of anti-CD47 antibodies in organ transplants!  

Alexo Therapeutics, another Stanford spin-off, are developing “high-affinity soluble SIRPα monomers (Alexo compounds)” to block CD47-SIRPα interaction. These monomers are based on (some very cool) work previously reported from the Garcia lab in collaboration with Dr. Weissman et al.

Very few scientists would argue against looking into nature to find solutions for some of the core problems that we, as innovators and inventors, are trying to solve today. But the question remains - Where do we start? In my view the best resource for scientific innovation, especially for biotechnology, is Evolutionary Biology. 

A great example from recent past is the characterization of Cas9, a bacterial RNA-guided DNA endonuclease associated with CRISPR. It’s discovery timeline makes a clear case for how understanding prokaryotic evolutionary development can provide relevant solutions as potential therapeutic strategies. Unless you’ve been living under a rock, you probably are familiar with the implementation of this system for gene editing over the past 5 years. This technology is clearly on its way to providing solutions such as editing genes/mutations that cause debilitating diseases in humans, maybe even in early embryos.

Source: An updated evolutionary classification of CRISPR–Cas systems

While most of the early developers/adopters are still fighting over IP issues, other similar systems have also be identified. Apart from modifying the traditional CRISPR system to improve efficiency, the Zhang group has already identified the enzyme cpf1 to be a better option than Cas9 for DNA editing and C2c2 for potential RNA editing. Further, more compact versions of cas9 - casX and casY enzymes have been identified from other bacteria by the Doudna’s group. They have also reported potential alternatives from uncultured bacteria and even archaea. But it is not just the typical players in this field that are looking for alternatives. Natronobacterium gregoryi Argonaute (NgAgo), another DNA-guided endonuclease has also been shown (although no reports of being reproduced by others yet) by a Korean group, to be suitable for genome editing in human cells. All such “improvements” have come about from analysis of genome databases and understanding the evolutionary history of such endonuclease enzymes. 

Other examples include Nanobodies - single-domain antibodies developed from heavy chain antibodies produced by camelids (specifically Llamas). They are predicted to have increased brain bioavailability and hence provides opportunities to treat brain tumors. Ablynx already has a number of therapeutic agents that are in various stages of clinical development. Similarly, shark antibodies are under study for neurodegenerative diseases. 

Obviously all these examples may not all have been based on a deliberate attempt to use evolutionary biology data. However, it clearly makes a case for more intensive studies to understand evolutionary pathways and a more direct approach to leverage this information to innovation. 

Fig: FBDD-derived drugs that have entered the clinic.

In the past two decades since the advent of FBDD, over 30 drugs developed using this platform have reached the clinical stage and 2 have been approved (as you can see in the table). You can find the rest of this table here (Nature Review Drug Discovery access required).

It’s quite surprising how successful this approach has been over such a short period of time, especially in targets where high-throughput screening had failed previously.

One of the distinctive successes of FBDD is that it has identified tractable hits against targets for which HTS has failed. The most striking examples are for inhibitors of protein–protein interactions, wherein the binding sites are often fairly flat and initial hit compounds bind weakly....

Fragments identified by protein-observed NMR that bind to two distinct regions of BCL-XL were linked together, leading to a dual BCL-2 and BCL-XL inhibitor. Subsequent optimization gave the BCL-2 selective venetoclax, which gained the FDA's 'breakthrough therapy' designation for recalcitrant chronic lymphocytic leukaemia and was approved in the United States in April 2016.

Of course there are number of areas where FBDD can be improved further. The authors zero in on three key areas of potential - 

1) Content of fragment libraries - Time to weed out the ‘bad actors’

2) Fragment-monitoring techniques - More NMR and SPR over X-ray crystallography.

3) Optimization of fragments to leads - Off-rate screening.

Interesting interview with the senior management at BMS about Opdivo’s most recent failure as a first line therapy. This has obviously been a massive blow to both the company and the immuno-oncology sector on the whole. However, this does not necessarily mean that all the PD-1 compounds (current or ones in the future) would be ineffective as a first line therapy. They also seem to be very confident about their combination therapy trial (Opdivo + Yervoy). We will have to wait and see, but for now, Merck has the upper hand with the positive results from a more focussed clinical trial. 

I recently did some research to answer this question on Quora - “How was the mechanism of known checkpoint inhibitors elucidated?” I thought I might reblog it here with some additional comments at the end.

So here is the history of how it was done -

Step 1 - Understanding T-cell activation through co-stimulatory signals -

Until late 1980s, it was understood that Antigen-Presenting Cells (APCs) can present the antigens to T-cells and activate a T-cell response. However, Mueller et al.  [1] found that it is the costimulatory signals that determine the outcome of T-cell receptor-MHC interaction. An example of these costimulatory signals is the interaction between CD28 and B7 (B7–1 and B7–2) (see below).

Source

Step 2 - Identifying competing T-cell stimulatory and inhibitory signals -

Following CD28, researchers started looking for other similar cell surface glycoproteins. French researchers identified CTLA-4, a homologue of CD28, by screening mouse cytolytic-T-cell-derived cDNA libraries.  [2] The Oncogene division at BMS confirmed that CTLA-4 was an alternate ligand (but different binding kinetics than CD28) for B7 receptors and that it inhibits T-cell proliferation and response. [3]

Step 3 - Developing a method to effectively block inhibitory signals -

James Allison and colleagues at UC Berkeley, confirmed in murine studies that blocking CTLA-4 using anti-CTLA-4 antibodies resulted in complete rejection of tumors (also pre-established tumors) and even prevented tumor growth upon secondary exposure to tumor cells.  [4] In 2010, Medarex (later bought by BMS) completed a Ph III study and confirmed that this CTLA-4 antibody (Ipilimumab) improved overall survival in patients with previously treated metastatic melanoma. [5]

Source

Step 4 - What about other immune checkpoint targets? -

PD-1 is another immune checkpoint target (as seen in above) with approved drugs (Nivolimumab and Pembrolizumab) in the market and followed a similar development trajectory.

Source

Interestingly, both PD1 inhibitors have been such a huge success (and market) that there are over 16 discrete PD1/PD1L targeting programs under clinical study. For in depth analysis highly recommend this blogpost.

What about the other targets? As of 2015…

Source

Coming back to the original question - “How can we work out the mechanism of action for checkpoint inhibitors”. They all seem to work in more or less a similar fashion - physically block the interaction of ligands that exhibit tumor proliferative effects to their respective cell surface receptors. Alternatively, antibodies can be developed to activate costimulatory functions of T-cells. Interestingly, the industry seems to be moving towards the use of combination therapies with PD1 inhibitors with anti-CTLA-4 or other targets.  [6]

Looking into the future of immune checkpoint inhibitors there are a couple interesting questions that pop up straight away. How are all these players going to compete in this congested market (even if immuno-oncology is one of the biggest arenas)? Some market analysts are already worried about a bubble and more people should catch on soon enough. 

The other interesting byproduct of all these drugs targeting the same receptors is the maturation of the concept of me-too-biologics. There is already a lot of confusion regarding biosimilars and their legality. Adding more shades of grey into the mix is not to going to make it easy to establish future drug development guidelines. It will be interesting to see how this additional complexity pans out in the near future. 

Footnotes

[1] Clonal Expansion Versus Functional Clonal Inactivation: A Costimulatory Signalling Pathway Determines the Outcome of T Cell Antigen Receptor Occupancy - Annual Review of Immunology, 7(1):445

[2] A new member of the immunoglobulin superfamily--CTLA-4.

[3] Immunosuppression in vivo by a soluble form of the CTLA-4 T cell activation molecule

[4] Enhancement of Antitumor Immunity by CTLA-4 Blockade

[5] Improved Survival with Ipilimumab in Patients with Metastatic Melanoma — NEJM

The boorish nature of the stock market is not a secret to anyone, especially with biotech stocks during this (rather extended) boom/bubble phase. I usually don’t cover (mostly due to the fact that I am illiterate of the inner workings of investing/trading) the biotech industry stock market news, but something interesting that has rather overpowered my twitter feed recently. It is the Relypsa story. Frankly, I wasn’t familiar with either the company or its (freshly FDA-approved) drug Veltassa until recently. Some background for readers, who like me aren’t familiar with the drug - it is a novel polymer molecule that binds to potassium and hence can be used for the treatment of hyperkalemia (a condition seen in patients with chronic kidney disease (CKD)). It is given as an oral suspension that sequesters potassium in the gut thereby lowering its overall levels. As you might expect this drug has been shown to cause some drug interactions and therefore comes with black box warning. 

Well, this sounds like a run-of-the-mill new therapeutic with some minor concerns that requires a careful watch by the physicians. But, this is where the story gets rather interesting. The presence of the black-box warning obviously spooks the traders and Relypsa’s stock ($rlyp) takes a tumble to below $20. To add fuel to the fire, at around the same time AstraZeneca acquired ZS Pharma, which is also studying a potassium binder (Zirconium silicate to be exact) (ZS-09) in a clinical trial, for an absurd $2.7 billion (at $90 a share, a 42% premium at its stock price at the time!). Well it turns out (the trials are not complete yet but this is based on compelling predictions) that ZS-09 might actually have a higher (compared to Veltassa) incidence of side effects, especially edema and cardiovascular problems. Furthermore, Veltassa can begin treating patients by early 2016, while ZS-09 awaits completion of the trial and FDA-approval. 

Cannabis produces most of its effects by acting on CB1 and CB1 receptors; CB1 in the brain and CB2 in the periphery. That sounds reasonable, right? 

Wrong. There have been rumours in the past few years that CB2 might be expressed in microglial cells under inflammatory conditions and we might have missed them as previous efforts were looking at healthy brains. Well, that explains all the neuroprotective and anti-neuroinflammatory effects of CB2-selective ligands in animal models. 

Figure: CB2 involvement in neuroinflammation.

But apparently that is not the end to this caveat. 

A number of posters presented at SfN15 conference show unpublished data that CB2 is infact found in neurons in a number of brain regions including prefrontal cortex (in a cocaine model), hypothalamus, ventrolateral thalamus and striatum in normal rodent brains! Moreover, it is involved, to a significant extent, in some of the tetrad behaviours (catalepsy, analgesia, hypothermia and locomotor dysfunction) upon THC administration commonly associated to CB1. A CB1 and CB2 individual knockout study by Z. Xi from Dr. Gardner’s group at the NIH found that CB2 is also involved in centrally-mediated analgesia and catalepsy behaviours! 

Other unpublished data from Jeff Conn’s lab shows the involvement of CB2 in anti-schizophrenia effects of their positive-allosteric modulator (VU0467154) of M4, by preventing dopamine release to D1-medial spiny neurons! 

Over the past decade, there has been growing evidence that the strength of these synaptic interactions (also known as synaptic plasticity) are dynamic. Moreover, it turns out that the plasticity of these connections is critical for neuronal development and cognitive functions of the brain. I had the opportunity to hear Nobel laureate Dr. Sudhof, at this year’s Society of Neuroscience conference, speak about their research highlighting the importance of synaptic proteins with neurological disorders such as Autism and Schizophrenia. In 1992, Dr. Sudhof’s group in the quest to identify the membrane receptor of alpha-latrotoxin (the toxin from Black-widow spider venom), first cloned Neurexin. Further work by Dr. Sudhof and others has proven that most of the neuronal synaptic plasticity is mediated by intersynaptic proteins such as neurexins.

Source: http://www.bradleymonk.com/wiki

A number of synaptic proteins such as cerebellin, neurolignin, LRRTM2 and latrophillin bind and activate neurexin. But it was discovered that both alpha and beta neurexins are highly polymorphic due to extensive alternative splicing. Interestingly, these polymorphisms are highly regulated and show cell-type selective splicing and also change ligand binding characteristics. 

Over fifty different neuroligin gene mutations have been identified to be related to autism. But, the genetic background of individuals and not individual mutations is what determines physical signs and symptoms in patients. But sadly we are still a long way away from truly understanding this phenomenon. Sudhof himself said the following (I’m paraphrasing him here) regarding the increasing frustration in lack of progress in neuroscience among general public - 

Autism research field hasn't failed, we have only failed to communicate the magnitude of the challenge.

Progress in cancer research has required >$1 trillion, it should not surprise us if understanding brain disorders would take at least as much or even more?

Here is further reading, if you are interested about synaptic plasticity and autism - 

Synaptic proteins and receptors defects in autism spectrum disorders

I’m going to resuscitate this blog for some blogging while I’m here at the Society of Neuroscience conference 2015 in (cold) Chicago. 

If you are into cannabinoid research or just mass spectrometry, I’ll have my poster (Hall A 53.20/H17) up later this afternoon. Drop by and say hi! I will unfortunately miss the Alzheimer’s therapeutics mini symposium (that I was really looking forward to attend) because of my poster manning duties. 

In the meantime, here is some fun reading for the morning - 

Emerging Functional Divergence of β-Arrestin Isoforms in GPCR Function

GPCR dimerization is a tricky area of drug discovery with many ambiguous details yet to be elucidated. This week, I came across an interesting paper in Nature Chemical biology on the signaling mechanisms of angiotensin 1 receptor (AT1-R) and alpha adrenergic 2c receptor (​α2C-AR) following dimer formation. Using a series of BRET-based signaling assays, the authors identified that upon forced dimerization and dual activation of both receptors using native ligands, the heterodimer complex signals through Gs pathway, thereby leading to increase in cAMP! The authors conclude that prior knowledge of signaling pathways of monomers may not necessarily provide a complete picture of their signaling activity once they dimerize.

A couple of caveats that we need to keep in mind is that this data was obtained in a recombinant overexpression system (HEK293 cells) and brings up again the age-old question - Do these dimers really existing in native cells? and if they do, how relevant are they for human physiology? The other caveat is that this Gs signaling is only observed once the (larger) Gi signaling is blocked! So, not all dimers work through this alternate signaling pathway. 

My condolences to all researchers who work in GPCR-dimerization, their work just got more complicated with needing to watch out for another 'little' proviso.

An interesting study that was published earlier this month in PLosOne, whose major goal was to develop a murine model for the study the effects of e-cigarette smoke. Using this (shown in the picture below) new study model, they found that repeated exposure to e-cig smoke resulted in inhibition of pro-inflammatory processes (leading to decreased concentration of pro-inflammatory cytokines) thereby making them more susceptible to bacterial and viral infections. They attribute these effects to the high levels of free radicals (although much lower than in regular cigarettes) present in e-cigs. 

Yes, this work is on mice and we need to take all conclusions from the study with a grain of salt. But, we may also find it beneficial to put in more effort into understanding the long term effects of e-cig usage, before it really catches on with teens. (we may be already too late - http://www.nbcnews.com/health/kids-health/u-s-kids-favor-e-cigarettes-over-traditional-ones-n269241) 

I am going to cheat a little by posting a bunch of papers about B-cell immune system and antibodies (focussing on the bispecific kind), as I had done a lot of reading, about them, for my journal club presentation this week. 

Lets just get to it then and try to fit in all (most) remaining papers of January in one post -

Antibodies as such have become a hallmark strategy for therapy, especially in the fields of cancer and inflammatory diseases. Traditionally, therapeutic antibodies have been designed to bind efficiently with target surface (or in solution) antigens through the Fab domains of the molecule and illicit an effector function through the Fc domain of the molecule. We have made tremendous progress in the optimization of antibodies for higher specificity, selectivity, durability, better pharmacokinetic and pharmacological profile by modified/mutating these domains. 

Unfortunately, most Fc receptors (Fcgamma subtypes) that bind to the Fc region of the antibody are present on most phagocytic cells and not on T cells. In order to involve the highly effective cytotoxic T cells in the equation in cancer therapy, introduction of CD3 specificity to the second arm of the antibody was envisioned in the late 90s. Unfortunately, developing/synthesizing such heterodimeric antibody by combining four individual antibody components results in low yields and products of poor quality. Novel antibody engineering methods have now enabled us to create bispecific antibodies without extensive optimization steps. 

We know that such a strategy could work in theory, but what about in practice? The trifunctional bispecific antibody Catumaxomab targeting EpCAM and CD3 was the first bispecific antibody approved by EMEA in 2009. In Dec, 2014, the US-FDA approved its first bispecific antibody drug – Blinatumomab (from Amgen) which targets CD19 (B cells) and CD3 (T cells). As of 2014, a number of bispecific antibodies are in clinical trials targeting various receptors - 

More recently #9, the group from Roche reports the development of a novel glycoengineered bispecific antibody (XGFR) that targets EGFR and IGF-1R simultaneously by combining two previously known antibodies targeting these receptors, using the key-in-hole strategy. They report a strategy, that involves 'minimal' optimization steps, to combine two monoclonal antibodies to create a bispecific anitbody with no loss of affinity and better ADCC-mediated biological effect. Although the field of bioengineered antibodies is heading towards CART therapies, there is still room for improvement in design and development of multi-antigen targeting antibodies.

If you want a folder of papers for further reading on bispecific antibodies, I would certainly recommend the following (in no particular order) - 

#8 - Kontermann, R. (2012, March). Dual targeting strategies with bispecific antibodies. In MAbs (Vol. 4, No. 2, pp. 182-197). Taylor & Francis.

#9 - Schanzer, Juergen M., et al. "A novel glycoengineered bispecific antibody format for targeted inhibition of epidermal growth factor receptor (EGFR) and insulin-like growth factor receptor type I (IGF-1R) demonstrating unique molecular properties." Journal of Biological Chemistry 289.27 (2014): 18693-18706.

#10 - Kalia, V., Sarkar, S., Gourley, T. S., Rouse, B. T., & Ahmed, R. (2006). Differentiation of memory B and T cells. Current opinion in immunology, 18(3), 255-264.

#11 - Guilliams, M., Bruhns, P., Saeys, Y., Hammad, H., & Lambrecht, B. N. (2014). The function of Fc [gamma] receptors in dendritic cells and macrophages.Nature Reviews Immunology, 14(2), 94-108.

#12 - Lambris, J. D., Ricklin, D., & Geisbrecht, B. V. (2008). Complement evasion by human pathogens. Nature Reviews Microbiology, 6(2), 132-142.

#13 - Jiang, X. R., Song, A., Bergelson, S., Arroll, T., Parekh, B., May, K., ... & Schenerman, M. (2011). Advances in the assessment and control of the effector functions of therapeutic antibodies. Nature reviews Drug discovery,10(2), 101-111.

#14 - 1.Kontermann, R. E. in Bispecific Antibodies (ed. Kontermann, R. E.) 1–28 (Springer Berlin Heidelberg, 2011). at <http://link.springer.com/chapter/10.1007/978-3-642-20910-9_1>

#15 - Elgert, K. D. (2009). Immunology: understanding the immune system. Chapter 4 - Antibody structure and function. John Wiley & Sons.

#16 - Morrison, S. L. (2007). Two heads are better than one. Nature biotechnology,25(11), 1233-1234.

#17 - Spiess, C., Merchant, M., Huang, A., Zheng, Z., Yang, N. Y., Peng, J., ... & Scheer, J. M. (2013). Bispecific antibodies with natural architecture produced by co-culture of bacteria expressing two distinct half-antibodies. Nature biotechnology, 31(8), 753-758.

#18 - Garber, K. (2014). Bispecific antibodies rise again. Nature Reviews Drug Discovery, 13(11), 799-801.

#19 - John B.B. Ridgway, Leonard G. Presta, and Paul Carter‘Knobs-into-holes’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Eng. (1996) 9 (7): 617-621 

#20 - Lewis, S. M., Wu, X., Pustilnik, A., Sereno, A., Huang, F., Rick, H. L., ... & Demarest, S. J. (2014). Generation of bispecific IgG antibodies by structure-based design of an orthogonal Fab interface. Nature biotechnology, 32(2), 191-198.

#21 - Merchant, A. M., Zhu, Z., Yuan, J. Q., Goddard, A., Adams, C. W., Presta, L. G., & Carter, P. (1998). An efficient route to human bispecific IgG. Nature biotechnology, 16(7), 677-681.

#22 - Dudgeon, K., Rouet, R., Kokmeijer, I., Schofield, P., Stolp, J., Langley, D., ... & Christ, D. (2012). General strategy for the generation of human antibody variable domains with increased aggregation resistance. Proceedings of the National Academy of Sciences, 109(27), 10879-10884.

#24 - Mack, M., Riethmüller, G., & Kufer, P. (1995). A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proceedings of the National Academy of Sciences, 92(15), 7021-7025.

Lets get straight into it - 

#4 E. Ghosh, P. Kumari, D. Jaiman, and A. K. Shukla, “Methodological advances: the unsung heroes of the GPCR structural revolution,” Nat. Rev. Mol. Cell Biol., Jan. 2015.

An excellent review of all the technological advances needed to solve the crystal structures of these complex membrane proteins. Although we are at the crossroads of being able to solve the structures of receptors (from different classes) in their modified/engineered state, we are a long way from solving their structures in their native state (if that is even possible). But I do agree with the authors that it is time we put to use some of these tech upgrades into structural biology efforts of other membrane proteins.

#5 Bowles, Nicole P., Ilia N. Karatsoreos, Xiaosong Li, V. Kiran Vemuri, Jodi-Anne Wood, Zhiying Li, Kellie LK Tamashiro et al. "A peripheral endocannabinoid mechanism contributes to glucocorticoid-mediated metabolic syndrome."Proceedings of the National Academy of Sciences 112, no. 1 (2015): 285-290.

As some of you may know, chronic use of cortisone results in a metabolic syndrome including obesity (especially fat accumulation in the face and torso). Now for the first time, this study provides some clues into the involvement of the cannabinoid system in this process. The authors found that upon either CB1 knock-out or administration of CB1 antagonists prevents the precipitation of cortisone-mediated metabolic syndrome in rats. Further, only the peripheral CB1 receptors were involved in this cannabinergic effect. So why is this interesting? Well, CB1 antagonists (or rather inverse agonists) were highly touted to be the next best class of anti-obesity drugs (The drug Rimonabant was approved for such an indication) in the 90s. The severe CNS side effects of CB1 antagonists/inverse agonists led to the abandonment of further drug discovery efforts in this research area. This new study adds to growing evidence that a peripherally-restricted CB1 receptor antagonist/inverse agonist could still be a valuable tool in the treatment of obesity and other metabolic diseases. 

#6 Konermann, Silvana, Mark D. Brigham, Alexandro E. Trevino, Julia Joung, Omar O. Abudayyeh, Clea Barcena, Patrick D. Hsu et al. "Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex." Nature(2014).

The CRISPR-CAS9 story has captured the attention of the media in the past couple of years, especially after the inventors/researchers having won the flashy, $3 million Breakthrough Prize. Adding to the fuel, there is this critical (and very expensive) IP fight brewing up in this area of research. But advances in this area have kept with the interest (especially from the lab of Dr. Zhang). In this study, they engineered the (previously developed) crispr-cas9 activator complex to improve its efficiency and study its application in a genome-scale transcriptional activation. They created an incredible 70,290 RNA guide library targeting every gene in the human genome and studied the changes of transcription in these genes, in a BRAF inhibitor-resistance cell model. This is obviously a very exciting advance in the field especially as a medical research tool and for disease diagnostics. I am still unsure of its applicability as a strategy as a gene-therapy tool in humans. I might be wrong here but we are still a long way from solving the targeted gene delivery problem. 

#7 C. M. Bishop, R. J. Spivey, L. A. Hawkes, N. Batbayar, B. Chua, P. B. Frappell, W. K. Milsom, T. Natsagdorj, S. H. Newman, G. R. Scott, J. Y. Takekawa, M. Wikelski, and P. J. Butler, “The roller coaster flight strategy of bar-headed geese conserves energy during Himalayan migrations,” Science, vol. 347, no. 6219, pp. 250–254, Jan. 2015.

This paper was definitely not part of my daily biochemistry/pharmacology diet, but did catch my attention (as it should yours). I heard about bar-headed geese for the first time in 2014, in one of the episodes of Wildest India documentary (which you can watch on Netflix and I highly recommend it), and was fascinated by their remarkable ability to fly in such high altitudes. The authors managed to identify that these birds do not necessarily fly at very high altitudes throughout their yearly over-himalayan migration trip, but rather use a "roller coaster" strategy to ascertain the terrain below and fly at the most energy-efficient altitude! 

PS. NPR has a wonderful podcast covering this research - Highflying Geese Save Energy By Swooping Like A Roller Coaster. 

I have decided to join the twitter trend #365papers and blog about one paper that I (will) have read for each day of 2015. I doubt I would be able to blog each paper in detail, but I promised myself to write a short paragraph noting the most interesting technique/method/discovery/insight from each paper. It looks like I am already behind (its the 13th already!!), lets see how long I keep up with this task...

So here are first three -

#1 Gamage, Thomas F., et al. "In-vivo pharmacological evaluation of the CB1-receptor allosteric modulator Org-27569." Behavioural pharmacology 25.2 (2014): 182.

The recent discovery of allosteric modulators of cannabinoid receptor 1 (CB1) has sparked a lot of interest in this area. Interestingly, one of the two most promising compounds, Org-27569 (the other being PSNCBAM-1) was shown using in-vitro experiments to bind to the extracellular surface of the CB1 receptor and increasing the binding affinity of cannabinoid agonists. But it was found to antagonize/inhibit the functional (g-protein mediated cAMP) signal of these agonists! Now the authors, have performed an in-vivo pharmacological evaluation of the ligand only to find that it does not show any in-vivo effect! So what now? Hopefully in the coming months/years there will more studies (published) on the in-vivo efficacy of other novel allosteric modulators, to understand this paradoxical result.

PS. Since its publication, additional studies have also confirmed the lack of in-vivo efficacy of Org-27569. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4186448/

#2 Yin, Jie, Juan Carlos Mobarec, Peter Kolb, and Daniel M. Rosenbaum. "Crystal structure of the human OX2 orexin receptor bound to the insomnia drug suvorexant." Nature (2014).

Another GPCR crystal structure has been solved! This time it is from the lab of Dr. Rosenbaum at the University of Texas Southwestern Medical Center (after having moved there from Kobilka's lab). The structure itself would be of interest for researchers working towards developing drugs targeting this receptor, but what interested me more is the use of fusion protein P. abysii glycogen synthase (instead of the more popular T4lysozyme). They also used a double affinity purification protocol (His-tag followed by flag-tag based) stabilized the receptor in 0.05% lauryl maltose neopentyl glycol (LMNG) detergent. It would be interesting to compare the overall structure of the unpublished crystal structure from Heptares (most likely stabilized by mutations) with this fusion protein structure.

#3 Ling, Losee L., et al. "A new antibiotic kills pathogens without detectable resistance." Nature (2015).