I had the pleasure of attending the last month’s two day GPCR seminar series held in Boston as part of the Experimental Biology 2013 conference. I won’t go into some elaborate description of all the talks or the posters that were presented. But I would like to point out some of the rather extraordinary tit-bits that I have picked up over the two days regarding how GPCR research is being done in some of the leading labs in the world.
- Atleast 3 biased ligands from Trevena (a spin-off from Bob Leftkowitz’s lab) are in the clinical trials and have shown some very promising results so far! (Yay! first biased ligand on the way people.)
- Michel Bouvier’s group are trying to apply some of the techniques we have learnt in the past decade of genome sequencing techniques for GPCR ligand screening. They have started screening compounds using a BRET-based ultra-high throughput screening of compounds in relation to over 20 different secondary signaling proteins/parameters in a single plate setup! The mind-blowing pictures of a 1536-well plates with different colored intensities brings back memories of DNA microarrays.
- Another interesting aspect of converging in-vitro data from these biased ligands to in-vivo mechanisms was covered by Dr. Tobin. One interesting detail about how they started this work was that one of his graduate students went ahead to prove that there is correlation between them, inspite of his previous objections to not waste money on this! Hope all the PIs in the room were listening and another story for the “Hey look, Grad students are right too!!” book that I am writing. (Unfortunately, it is still in its infancy :p)
- Jeff Conn’s group are trying to improve the effects of current ligands by adding allosteric modulators that are designed to favor one conformation over the other depending on the indication of use. (Nice to finally hear and meet one of the PIs I was looking to work for through grad school!)
- Jefe Aube’ provided some med chem humor when he showed some data that went against the popular jokes about medicinal chemists - “SAR to biologists usually means methyl, ethyl, propyl, butyl…futile” when talking about how simple SAR changes improved potency and efficacy of their novel scaffolds.
- Mark von Zastrow went over his recently published work (Comformational biosensors reveals GPCR signaling from endosomes) in Nature, with some extraordinary FRET images and videos that convincingly and (for the first time) directly prove that GPCRs signal even from the endosomes.
- Roger Sunahara compiled all the structures of GPCRs obtained so far and a few other studies added to them to prediced that G-proteins bind weakly to the ligand-free receptor. Then following ligand binding, they form stronger interactions and also change the extracellular conformation of the receptor preventing the release of the ligand.
- Another company I have been hearing a lot about over the last few years is Heptares (a spin-off of Christopher Tate’s mutation-based GPCR stabilization work at MRC, Cambridge University). Fiona Marshall showcased some of the details of how they carry out GPCR structural characterization. One of the coolest bits that blew my (and I am sure quite a few others) is that they solve a different crystal structure every 2 weeks!! Also that they prepare about 1000 mutations for each receptor to pick out the right mutations that stabilize the receptor for crystallization!! (How exactly are we in academia to compete with this?)
I unfortunately did not get a chance to attend the two Kobilka talks earlier in the week, but made it to the Lefkowitz’s talk on Wednesday. It was very similar to his Nobel talk but one bit that stuck in my head was that how proud he is of Kobilka’s endurance during some tough financial times in his lab. He is certainly an excellent scientist and an even better mentor (if that is even possible).
In all, it was a great couple of days to meet and talk to some of the people in the field of GPCR research. I only managed to take a couple of pictures of Dr. Lefkowtiz from afar and here they are -
Hopefully, I will be back with another post soon. As always, comments, shares and likes are welcome.
Umbrella project @MIT (at Jack barry field, cambridge MA)
I’m spending the night at the lab to finish up some writing work but having done with it earlier than expected I thought why not add a new post. Yes, its been a while but hey I still remember the password to my tumblr account!
I never got around to writing about the Nobel prize in chemistry this year which was awarded to the grandfather of GPCRs, Bob Leftkowitz sharing it with his protege Brian Kobilka. I will not dwell too much into whether they deserved it or what exactly won them the award, as a number of fellow bloggers and science journalists have dealt into this in great detail over the past few months. But I would like to make just one point that if you can spare about 50 mins, I would recommend watching their Nobel interview -
The stark contrast in their demeanor during the interview is very interesting and is rather intriguing as to how they managed to get along over the years with such a massive difference in personalities (as pointed our by Bob himself). It is my belief that ‘it’ (it being this combined, massive undertaking of GPCRs that took over 40 years) would never have been such as success if Kobilka was the predecessor with Bob being his student. This is no offense to Kobilka’s mentorship skills.
Anyway, lets move on to another monetary prize that has raised some eyebrows in the recent weeks - The Life science breakthrough prize!! The $3 million award to a select few scientists focusing on ‘breakthrough’ therapeutic strategies mainly targeting cancers and some neuro-degenerative diseases has elicited a rather bitter and unwarranted antipathy. I couldn’t disagree more with this aversion towards awarding such large sums of money to a select few.
First and foremost, it is a privately funded award trying to encourage rapid development of therapeutic strategies for some diseases that could potentially wipe out the human race in the coming years unless they are conquered soon. Secondly, the wide (and rightly so) distribution of federal money although has given numerous breakthroughs over the years, have come along in far too few numbers and too far apart. This monetary push from these tech magnates with stuffed wallets may turn out be the push that has been missing in this field.
I recently came across this paper- The Very Large G Protein Coupled Receptor (Vlgr1) in Hair Cells, by a group from Shangdong University about the Vlgr1 receptor in hair cells. Any guesses on how big this “very large” receptor really is?
Well it is 6,309 amino acids long!! Atleast the full length splice variant in that long and this is not the end of the mindboggling nature of this receptor. The N-terminal domain is 5,800 amino acids long and the C-terminal end is 150 amino acid long!
For someone who works with (read as struggles with) Class A GPCRs who range from 350-450 amino acid length, this number is blood chilling. Even with these class A GPCRs we usually truncate the terminal ends if longer than 100 aminoacids as they cause severe problems during recombinant protein expression. I completely sympathize with groups working on these Adhesion class of GPCRs and is quite an achievement just cloning such genes from their gigantic mRNA (17kb in this case!!).
Disclaimer: I stole the above title from the talk by Dr. Ben Bahr from the University of North Carolina, Pembroke.
So, I am finally back to blogging after listening to another interesting talk I had the opportunity to attend today. This one as the title says, talks about the chemotherapeutic interventions possible for treating Alzheimer’s disease. So the answer to the above question is simple - A single drug.
Dr. Bahr works on Alzheimer’s disease with a focus on the lysosomal pathways of cells and how their modulation can relieve some of the characteristic patho-physiological changes seen in Alzheimer’s disease. We know from recent research that a plethora of proteins and pathways are involved in causing the complex neurological disorder known as Alzheimer’s disease. Long gone are the days that based the entire source of the disease on the formation of Amyloid plaques in the brain. It is now well accepted in the Alzheimer’s field that proteins - alpha-beta peptides, tau, etc. and their related enzymes and signaling pathways are all effected in numerous ways and combinations.
Of course, Dr. Bahr is biased to the notion that the most obvious method of treatment would be to “boost” the endogenous lysosomal pathways that clear mis-folded proteins like the alphabeta and tau dimers (and multimers). He alluded to a aminoacid-based therapeutic agent that has shown some signs of improving just that and also showed data from small molecule analogues that were developed to mimic the chemically-modified dipeptide.
But the most interesting take-away message he wanted to deliver was this - no single magic bullet exists nor may ever be discovered to treat such a complex and idiosyncratic disease. We have to target this using a combination of different agents acting through different mechanisms but all with low efficacies and hence not perturb the body system too much.
This brings back up the fact of drug discovery that I’ve learnt in the few years I have been in touch with this field - IT IS NOT EASY TO TREAT AN AILMENT WITHOUT AFFECTING ANYTHING ELSE IN YOUR BODY. This is also probably one of the critical points that most non-biologists (for that matter most non-drug-discoverers) do not completely appreciate. If something is changed, there is always something unwanted that will also be changed. The trick is to minimize this as much as possible and when it comes to such complex diseases, localized in one of the most complex anatomic locations in the human body, IT IS NOT EASY!
It is not impossible, but it not easy.
Today, I had the wonderful opportunity to listen to the talks from some of the eminent personalities in the field of drug discovery and development, both from the academia and industry. Northeastern University, and the Center I work for, hosted this event today consisting of a series of 11 talks on the topic of “Drug discovery and development in the 21st century”.
The talk I was most interested in was from Dr. George Whitesides on “New Drugs: Why Are They so Hard to Develop?”. I am sure everyone in the fields of chemistry, nanomedicine and drug discovery are all familiar with his work and the accolades this man has been honored with. The talk dealt with one of the biggest questions in drug discovery currently. During the talk, he touched upon some interesting aspects about the thermodynamics of ligand-protein binding and the involvement of water molecules. He pointed out the fact that the mechanisms are extremely trivial and yet completely unexpected. These were results from their studies, on carbonic anhydrase and other enzymes, showing the balance between the enthalpy and entropy of the ligand binding reaction.
But the point that I found very interesting was his take on the current health care system. He raised the following question -
Is a “disease” a “profit opportunity” or
is “health care” a “public obligation”?
This might be another critical question that needs our attention in the quest to develop better drugs and provide better and cheaper patient care.
He alluded to an even more riveting issue later on when asked about his thoughts on about other important mysteries, that he believes, we need to be address. He put forward the following (I am paraphrasing here) -
We know that all these chemicals (proteins, etc) are not “alive”. We know that the cells, made up of these chemicals, are alive. So how did these chemicals become “alive”?
This in my opinion is the biggest mysteries of biology and life in general from a philosophical point of view. This might be one of those questions that we have very little hope of answering but it still deserves our attention and a thorough discussion. Such discussions can help people (even ones without a biology or science background) understand the complexity of life at even the most basic, single-cell system. This could obviously lead to more open-minded discussions about other public health issues such as the abortion laws and planned-parenthood.
Do you have other such profound questions in biology that you think we need to answer or in the least acknowledge? Feel free to leave a comment and we could have a discussion.
Everyone who works on receptor-ligand binding experiments will agree that the traditional radioactive binding assays are very annoying and not the least bit user-friendly. Unlike most of the enzyme-substrate fluorescent assays, ligand screening on most of the receptors (GPCRs) are still being performed using the age-old radioligand binding assays . Why is this?
A very limited number of fluorescent ligands have been developed for most receptors and ones that do bind have very poor affinities. Based on my experience with Cannabinoid receptors, I can confirm that there are hardly 2-3 fluorescent CB ligands thus far developed that can bind to the receptor and can exhibit good fluorescence. The main problem is obviously the presence of a large fluorescent group attached to known ligands through a linker chain. Considering how specific the ligand binding is in GPCRs, the presence of such a large molecule how much ever separated by a linker still causes lots of problems. I would imagine other problems of solubility, stability, etc also creep in and add to more worries for the chemists.
So what is the solution? This - Microscale Thermophoresis (MST) technology from NanoTemper technologies. This method involves the detection of microscale changes in hydration interactions in the unbound receptors and the ligand-bound receptors. It is known that a small change in the orientation of the hydration shell of a molecule causes a change in its thermophoretic movement. A powerful laser is used to generate small microscopic temperature gradients in the sample solutions of different ligand concentration. The sample also contains a fluorescent agent and based on the amount of the fluorescence in each sample, the change in molecules’ thermophoretic movement is determined. By comparing the fluorescent readings of different ligand concentrations, the precise binding affinity of the ligand can be determined. You can read more about it on their website - MST Background. They confirmed the use technology in GPCRs by determining the ligand binding affinities of specific ligands of 4 olfactory receptors expressed in a cell-free expression system and stabilized in peptide surfactants. (here). They then went on to perform ligand binding studies of non-gpcrs as well.
This method similar to the other label-free cell-based technologies is completely constrained to their instrument and their reagents. The instrument as you would have guessed is crazy expensive and so will be the reagents. This places it beyond the reach of most academic labs. And so we are back to fluorescent ligands. I feel that there is still a lot of scope for the development of non-radioactive ligands for binding assays.
Even if we cross the bridge of developing a good fluorescent ligand, we still have a difficult assay to develop. The removal of unbound, excess fluorescent ligand will cause a lot of problems for the use of receptor-membrane preparations. So, we will need to use whole cells and use multiple wash steps which will cause significant increase in downtime especially when using a 384-well setup and with no liquid-handling systems available (Yes, we are that impecunious).
DTect-All™ is a interesting technology developed by Domain Therapeutics. They use a FRET-based approach to quantify the receptor-bound fluorescent-ligand. The receptors are N-terminal truncated and added with a donor fluorescent probe like GFP and this setup might not need a wash step. Although I am not sure how the signal to noise ratio is in these experiments. (P.S. I have not be able to find any papers using this technique yet nor have I been able to take a look at their patent.) This approach might be more within reach of an academic laboratory and I look forward to more labs switching over to such assays.
I would love to hear you thoughts on these and other ligand binding technologies that you may have heard of or in use of in your lab.
This month’s Nature Reviews: Drug Discovery has this interesting article discussing the prospects of the next five years of drug innovation (by analysts from McKinsey & Company). Oncology drugs has been the winner these past years with respect to both growth in drug sales and the number of drugs released. According to their projections, this trend is expected to continue, and will only to be followed by a mile by CNS drugs.
Also, as expected, biologics will beat small molecules in terms of growth and will be the focus of the pharmaceutical industry. An increase in new drugs approvals is also expected and could raise to about 30 drugs in 5 years. But all is not fun and sunshine, the gloomy fact remains that the current drug POS (Probabilities of success) of 8.3% will stay put (or worse, could drop to as low as 4.7%) in the next five years. Further, since these new drugs, most of which are acting on the same old targets, will have a tough task to compete with existing alternatives their annual net sales will drop dramatically from (currently) 900m to 600m by 2016.
Essentially, all Big Pharma will have managed to do is cut R&D, increase layoffs, protect their asses by increasing partnerships with other Big Pharma companies and finally somehow manage to release the same number of drugs for existing targets over the next few years.
EvaluatePharma Ltd has released another projection study of drug sales up until 2018 and believe it or not Pfizer is expected to lose its No. 1 spot to Novartis by as early as 2014 and then drop further down to third below Sanofi. This was long coming since their business strategy was essentially to remove any kind of R&D spending and that overreaching Wyeth merger did not help things either.
The star of the biotech industry, Gilead is expected to have the highest growth rate (9%) with their robust retro-viral pipeline and new combinations of existing anti-viral drugs. AstraZeneca is expected to have the worst growth rate at -5%! This could be partly attributed to their new plan of lets-layoff-our-employees-and-do-R&D-elsewhere and their now dried out pipeline. I might be a bit biased here, but it is a great idea to fund drug discovery research in an academic setting and I hope it is a great success and they prove everyone wrong.
Recently, I started a Quora board to discuss all cool things (and not so cool things) about human irrationality. Please follow and share your experiences.