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Pharmtastic

The fantastic world of drug discovery

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Further data on GPCR-arrestin interaction

Another interesting (and well written) study that ‘needs’ to be shared is the nature paper (“Visualization of arrestin recruitment by a G-protein-coupled receptor”) on the interactions between g-protein coupled receptors and arrestin from Lefkowitz and collaborators. This time they turned to hydrogen deuterium exchange and electron microscopy combined with some complex 3D reconstructions to understand the protein dynamics of receptor-arrestin interactions.

A couple of things (with valid reasons) that I found really interesting was the use of Vasopressin receptor with some structural modifications including a N-terminal T4Lysozyme as the token GPCR for this study. The other being the use of a nanobody to stabilize the ligand-receptor-arrestin complex. This study not only confirmed some of the previous EPR studies but also reveals other changes in the N- and C-terminus of beta-arrestin. They move on to suggest a biphasic mechanism of arrestin recruitment, which sounds very plausible considering it is very similar to the g-protein mechanism. 

On a secondary note, the number of failed techniques/constructs to study this collossus protein complex, pile on as you continue reading down the paper, which makes this study all the more impressive. Looking forward to more (crystal structure of the complex?) from these guys in the near future.

Filed under gpcr Leftkowitz arrestin nature

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prichugh asked: So I have an assignment for class to come up with a hypothetical assay for how to determine what olfactory GPCRs are associated with a certain scent (while given the cDNA of all olfactory GPCRs, the cDNA of various G-proteins and all downstream effector/signaling molecules. I also have the specific scent extracted in a soluble aqueous form) and then after that I must identify a SINGLE agonist that will stimulate the found GPCRs. I'm having difficulty figuring out where to start. Can you help?

Hi,

I am sorry to be getting back to you so late. Clearly, I need to pay more attention to my inbox! I doubt you still need an answer for your assignment. But I will give it a go anyway.

Frankly, this is a very difficult question to answer even if it is just hypothetical. The following are the problems that makes it a complicated study - 

1) How many different ligands are there in this “scent extract”? How closely related are they to each other? Is there anyway to even separate them? If there are highly chiral compounds that would mess up their purification further!! Are they are mix of agonist and antagonists? How stable are they? Most scent molecules are usually volatile. 

2) Regarding olfactory receptors - There are over 1000 human olfactory receptors! Trying to work out which one from all those would be very tricky to say the least).

One way I can think of performing this mammoth, hypothetical study would be to overexpress these receptors, individually, in mammalian expression systems (using the cDNA). You can then perform, individual, functional assays such as cAMP/GTPyS assay (for Gi and Gs) or IP3 assay (for Gq) with the scent extract to get an idea for which receptors get activated (lets assume there are no antagonists/inverse agonists in the extract). Then purify (using chromatographic methods) the scent extract into individual compounds and repeat the functional assays against previously identified compounds. 

Hope this makes sense. Let me know if you have more questions.

Cheers.

~Krishna

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Are Antibody-drug conjugates really “magic bullets”?

People in my lab (Mark and Jay, I’m talking about you guys) have finally found my blog and were wondering why I haven’t posted in a while. Well the only excuse I could come up with was to blame it on my controlled addiction to twitter. Typing out <150 characters is a lot easier than long(ish) blog posts. The wider (sciencier) audience makes it all more the more attractive. Then again, I am not going to completely give up on this blog yet. So here it is -

Recently, I had the pleasure to listen to John Lambert, CSO of Immunogen, a biotech leader in Antibody-drug conjugate (ADC) development. They already have a breast cancer, ADC drug (Kadcyla) in the market with the help of Genentech. I am going to let the following video explain the basic concept of why we want drugs conjugated to antibodies -

I wanted to point out a couple of not-so-eye-popping yet crucial factors that were not really clear to me, someone not working in this field.

Simple logic suggests that localized delivery of a cytotoxic drug to cancerous cells/tumors by conjugating it to a target-specific antibody (that may or may not already have intrinsic cytotoxic effect) is the pinnacle of drug therapy. But adding a drug molecule into the equation brings us face-to-face with a whole other monster - Pharmacokinetics!

Typical antibodies have the advantage of being made up of aminoacids just like any other protein in our body and their metabolism is therefore not a major concern during drug development. ADCs, on the other hand, have a cytotoxic drug that gets metabolized into products which requires developers to extensively study their toxicity and their kinetics.

You might wonder, Why is this so important? If these molecules are at the tumor site, and if the metabolic products are toxic, shouldn’t they be all the more effective in killing the cancerous cells?

Well, the important thing to keep in mind is that almost all anti-cancer antibodies target specific proteins (HER2, EGFR, etc.) that are highly expressed on the cell-surface of cancer cells. But, these proteins are also expressed (in lower concentrations) on normal cells. Although these ADCs are considered to be “magic bullets”, when administered, they move throughout the body (in our bloodstream), attach to both normal and cancer (predominantly) cells and hence can get metabolized throughout the body. 

But it is not all gloom and doom for ADC therapies. Due to the targeting effect of the antibodies, the concentration of drug molecules required to have a viable effect is much lower than typical radiation/chemo therapies using the drug by itself. Therefore, they have a similar toxicity profile to that of the chemotoxic drugs, but generally better tolerated by patients.

This to me points us towards the next generation of antibodies that target cancer-exclusive proteins. Although, this is easier said than done. I am also not sure, if any truly exclusive proteins exist in tumour cells. The proteins with altered glycosylation and other post-translational modifications (of which there are quite a few of) might be a better target for developing cancer exclusive antibodies (or even lectins).  

Filed under antibodies drugs adc conjugates magic bullet immunogen

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The $100M Club: Where the Major League Life Sciences Companies Are | Xconomy

Great list by Luke Timmerman. Please do check it out. Pretty impressive growth by the Boston biotech scene, but I’m not sure if they can keep this up for many more years to come.

I hope I am wrong. I will hopefully be graduating in a few years and would like many more options while looking for a job. 

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Drug discovery: Excersized rats live longer and stapled peptides last longer!!

Had a chance to listen to another couple of interesting talks this week.

The first one was by Dr. Konstantin Khrapko from the Harvard medical school, talking about the somatic mutations of mitochondrial DNA. I have to admit that I have almost no experience in his field of mitochondrial DNA research. Apparently, the mitochondrial DNA (mDNA) present in each mitochondria, in each cell, undergoes a number of mutations over our lifetime and they influence a number of physiological functions. The mDNA have almost no DNA repair mechanisms unlike the nuclear genomic DNA. Hence, large number of mutations get accumulated over time and cause age-related diseases. One of the interesting phenomenon that Khrapko and others in the field have observed is that these mutations are identical in all mitochondria in the same cell (or bunch of cells risen from the same mother cell) indicating a common source and a quick spread. They are not sure about how the initial mutation/deletion gets transmitted to the others, but this can be measured pretty accurately now. 

Logic would dictate that mtDNA mutations, when present at levels lower than in phenotypically normal Polgmut/+ mice (dashed green line), are irrelevant for aging. However, in aged human colon, the typical histological pattern of mitochondrial defects (blue crypts in the inset) associated with increased mtDNA mutant fractions in individual crypts suggests otherwise15. Fractions in colon include clonally expanded mutants only. Error bars represent estimated variation of the data. Figure provided by L. Greaves and D. Turnbull (University of Newcastle).(Source: Mitochondrial DNA mutations and aging: a case closed?Konstantin Khrapko & Jan Vijg Nature Genetics 39, 445 - 446 (2007))

So how does this affect the rest of us not working in this field? Well, when they developed a rat model by causing similar mutations/deletions in the mDNA of fertilized eggs, the offsprings grow old and diseased rather quickly!! This rapid aging with features including, muscle loss, har loss, greying of hair, CNS disorders, poor metabolism etc is rather clear from the pictures of these rats. The rather more interesting observation was that when these rats were made to exercise (since birth), they avoided this prognosis completely and were indistinguishable from the regular rats!!

So what does all this mean? Well they are still trying to figure it out. I don’t know about you but In the meantime, I would help myself to a healthy serving of exercise in the hope that it will extend my lifespan!

The second talk was by Dr. Tomi Sawyer from Aileron Therapeutics on the development of stapled peptides and how this work led to a $1.1 billion aggrement with Roche! I am sure most of you readers are familiar with the fact that peptides are an excellent alternative to small-molecule drugs due to their specificity and ‘easy’ development. But, one of the major drawbacks since their develpoment a couple of decades ago has been their poor stability in the gut and in the plasma. One of the novel methods that Aileron have used to stabilize these peptides is by “stapling” these peptides using chemical linkers. This not only improves their stability as a peptide but is also able to hold the peptide in the form of a alpha helix thus enabling their use in protein-protein interaction modulations.

One of the targets they have been working on for Roche is the MDM2 and MDMX dual-targeting alpha-helical peptides as an anti-cancer therapy. These stabilized peptides can interact with both MDM2 and MDMX proteins, with good affinity, and prevent their interaction with p53. This allows p53, a pro-apoptotic protein, to kill the cancerous cells. Although I was familiar with this strategy from the FierceBiotech’s coverage of their recent Phase I trial success, I did not realise that some of these peptides have a plasma half-life of around 24 hours!! This obviously makes it a very interesting drug design strategy to watch out in the near future. 

Filed under stapled peptides mitochondria mDNA drug discovery seminar

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Big pharma at its best and worst!

This week, I had the opportunity to listen to Dr. Doug Johnson, a medicinal chemist from Pfizer in Cambridge, Boston. He spoke about his group’s decade old project of developing FAAH inhibitors and beta-secretase inhibitors using click chemistry to study their interactions with the respective enzymes/proteins.

I happen to work in a lab that also works on developing selective ligands for FAAH and other endocannabinoid targets. It obviously got my interest that they had developed and optimized a drug molecule which is a highly selective FAAH inhibitor. This work could be one of those classic examples of how target-based drug design is such an asset that is used in the industry (and in academia) in drug discovery and yet how lucky you have to be to stumble upon the right bunch of steps (among a myriad of possibilities) to improve the pharmacological profile of the hit.

But the interesting bit (or rather a head-desk moment) of the talk came up during the Q&A session at the end of the talk. He was asked about why this compound was studied in a osteoporosis pain model rather than the more traditional endocannabinoid target models. And if you are familiar with how big pharma works, it would not come as a surprise that this was because of a major reorganization at Pfizer that led to the use of this highly selective FAAH covalent inhibitor for a “wrong” indication rather than in a neuropathic pain or MS model. The “pain group” in the company had apparently turned its focus onto ion channels and away from the endocannabinoid system during the reorganization. The cannabinoid drug obviously failed the Phase II trial and is now sitting in some cupboard waiting for the company to decide on which new (appropriate) indication it should be tested for.

Lets hope they get their act together and push this molecule to the right trial at the earliest.

Filed under Pfizer Drug Design FAAH cannabinoid system drug discovery

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Ligand structure based protein design!

Gone are the days when we just try (and hope) to design/synthesize/discover specific and selective ligands that fit to a protein of interest and elicit the effect that is required to treat/cure diseases. Although this quarter century-old drug discovery approach is the hallmark of most current drug discovery operations around the world, it has not been the most efficient in generating “winning” drug molecules.

Now a couple of groups at UW (Barry Stoddard & David Baker and their colleagueshave tried to reverse this age-old approach and have combined computational prediction models with some heavy biochemistry, to design new protein molecules with binding pockets that would best fit a ligand of interest! They published these results in Nature this week - Computational design of ligand-binding proteins with high affinity and selectivity and is definitely worth a read.

In summary, they developed specific proteins that bind to Digoxigenin (DIG), a steriod molecule. They begin their computational search by using over 400 protein scaffolds from known proteins which are then modified/mutated/refolded to improve the binding affinity with DIG to sub-picomolar levels. Yes, such computational studies have been published in the past and have been partly successful (refer 9-14 of the references in the above paper). But, this study follows up these predictions by actually expressing these novel proteins in-vitro and then studying the effects of mutations in the binding pocket and how it affects the binding affinity of DIG and other steroidal molecules. Remarkably, the results are very convincing and the newly designed proteins exhibit amazing selectivity towards DIG over other ligands.

This is a remarkable achievement and hats off to these guys for this wonderful and comprehensive work. I am looking forward to more from the groups involved in this study, in the near future, with some practical applications of such designed proteins.

Filed under protein protein design drug discovery biotechnology

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Llama-derived single variable domains (Nanobodie... [J Biol Chem. 2013] - PubMed - NCBI

Nanobodies, if you remember, are single-variable domain antibodies generated from llamas, which were used as one of the strategies for the crystallization of a number of GPCRs including the beta-adrenergic receptors and then the CXCR7 receptors. These nanobodies can stabilize a specific conformation of the GPCR and effectively mimic a small molecule or peptide agonist, antagonist or inverse agonist ligand.


It is great to see that apart from those experimental applications, they also have some very interesting clinical applications. This is the first paper with some in-vivo data that I have come across that show some very promising results for these antibodies. I hope to see more in the near future on other receptors as well.

Filed under GPCR Nanobodies science biochemistry drugdiscovery antibodies

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Chaperonin-mediated Protein Folding. [J Biol Chem. 2013] - PubMed - NCBI

Very interesting review of the history of the study of chaperonins by a legend in the field - Dr. Arthur Horwich. Protein folding is without question one of the most interesting topics in the field of molecular and cell biology, yet one of the least well understood.

We are still far far away from efficiently folding a particular amino acid chain into a functional protein by just adding a bunch of chaperonins, other ancillary agents along with the right environmental conditions. Lets hope that this “utopia” is too far away in the future.

Filed under chaperonins proteinfolding proteins biochemistry biologicalchemistry

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Chemical reporters for biological discovery

A very interesting review of some of the amazing bioorthogonal reporter molecules and their chemistry for biochemical discovery with all kinds of biomolecules.

My own experience (although only second hand. They are studied by other grad students in my lab) has been with activity-based protein profiling (ABPP) of serine hydrolase enzymes. . Although ABPP was touched upon briefly, it was not discussed in depth in this review. If interested, you might want to check this paper by Ben Cravatt from Scripps - Activity-based protein profiling for biochemical pathway discovery in cancer.

Filed under ABPP chemical reporters science chemistry