It has been widely assumed that the production of the ubiquitous second messenger cyclic AMP, which is mediated by cell surface G protein–coupled receptors (GPCRs), and its termination take place exclusively at the plasma membrane. Recent studies reveal that diverse GPCRs do not always follow this conventional paradigm. In the new model, GPCRs mediate G-protein signaling not only from the plasma membrane but also from endosomal membranes. This model proposes that following ligand binding and activation, cell surface GPCRs internalize and redistribute into early endosomes, where trimeric G protein signaling can be maintained for an extended period of time. This Perspective discusses the molecular and cellular mechanistic subtleties as well as the physiological consequences of this unexpected process, which is considerably changing how we think about GPCR signaling and regulation and how we study drugs that target this receptor family.
This perspective article in the latest Nature chemical biology journal, summarizes some of the recent work on the complicated world of endosomal signaling of GPCRs. Although it looks obvious in hindsight, it is quite mind boggling to realize that the different receptor conformations (stabilized by different ligands) dictates the activations of g-protein signaling pathways not just on the cell surface but also inside the cells!
Figure: (a) Studies in cells led to the discovery that PTH, as opposed to PTHrP, sustains G-protein activity and cAMP production after PTHR internalization into early endosomes. (b) This observation is changing how we think about cellular signaling of the PTHR and is motivating the development of PTH analogs able to promote the endosomal cAMP signaling. One of them, LA-PTH, mediates a markedly prolonged cAMP signaling response in cells and prolonged hypercalcemic responses when injected into mice. (c) LA-PTH is now in preclinical development via the US National Institutes of Health BrIDGs program63 for eventual testing as a future treatment for hypoparathyroidism.
It will not surprise me if further evidence comes to light, in the near future, that other signaling pathways (beta-arrestin, PKA, PKC, etc) are also directly or indirectly activated from endosomal membranes.
This rather interesting study by the California Department of Motor Vehicles shines some light on the impact of driving under the influence of marijuana since the time of its legalization in some of the american states.
Increased driver cannabinoid prevalence associated with implementing medical marijuana laws was detected in only three states:
California (2.1%), Hawaii (6.0%) and Washington (3.4%). Therefore, changes in prevalence were not associated with the ease of marijuana access afforded by the laws. They conclude by saying that -
Increased prevalence of cannabinoids among drivers involved in fatal crashes was only detected in a minority of the states that implemented medical marijuana laws. The observed increases were one-time changes in the prevalence levels, rather than upward trends, suggesting that these laws may indeed provide marijuana access to a stable population of patients as intended, without increasing the numbers of new users over time. Although this study provides some insight into the potential impact of these laws on public safety, differences between states in drug testing practices and regularity, along with the fairly recent implementation of most medical marijuana laws, suggest that the long-term impact of these laws may not yet be known.
AVI-7537 (GCC +ATG GT+T TT+T TC+T C+AG G) is an antisense RNA oligomer (19mer), with 5 “PMOplus” linkages (positive charges to enable better cell penetration), that targets the viral protein 24 (VP24) transcript RNA.
The VP24 protein is one of the 7 structural proteins of Ebola virus particles (EBOV) and has been known to inhibit host’s type I interferon responses. Therefore, inhibition of the translation of VP24 may lead to a more efficient host response to infection. VP24 also forms homodimers and binds to viral protein (VP35) and nucleoprotein (NP) and may play a role in the switch from viral replication to transcription, a function critical to the viral life cycle.
Lets start with some background about the structure and composition of Ebola virus (EBOV). EBOV particles are made up of a negative-sense RNA strand held in a cylindrical/tubular structure made of viral envelop, matrix and nucleocapsid components. The non-infectious RNA genome is roughly 19 kb long and encodes for 7 structural proteins (Nucleoprotein, virion protein 35 (VP35), virion protein 40 (VP40), glycoprotein, virion protein 30 (VP30), virion protein (24) and polymerase L protein) and 1 non-structural protein. Although great progress (Questions and Answers on Experimental Treatments and Vaccines for Ebola) has been made in the development of vaccines/antibodydrugs targeting these proteins, due to the lack of clear understanding of EBOV’s pathogenesis and the difficulty of working with the virus under biosafety level conditions, progress towards antiviral drugs has been gradual at best.
However, we do know that the L protein provides the RNA polymerase activity and therefore involved in the transcription and replication of the viral genome. Also, VP24 and VP35 have been found to show inhibitory effects on the host’s interferon response (anti-viral response). This makes these three proteins the most promising antiviral drug targets.
One of the obvious strategies for the development of an anti-EBOV drug would be to prevent of the replication of the viral particles by interfering with the translation of 1 or more of the above mentioned viral proteins. Sarepta Therapeutics (previously known as AVI BioPharma) have tried to use a (Modified)
RNA interference strategy, which involves the use of small modified RNA strands (oligomers) to target the viral mRNA and prevent the expression of key viral proteins.
But simple, small RNA oligomers are rather easily degraded by nucleases. Therefore, phosphorodiamidate morpholino oligomers were developed by the modification of the phosphate linkages. These oligomers are
highly negatively (Edit: un-)charged and therefore yet cannot easily cross the cell membrane. To aid cell penetration, these modified oligomers were conjugated to known cell-penetrating peptides (shown as PPMO in the figure below). Alternately, the phosphate modifications were made such that they provide a positive charge hence neutralizing the oligomer and, which improves their stability and cell penetration (shown as PMOplus in the figure below). They used the above three strategies, using multiple variations of sequences, targeting 4 different proteins (VP35, VP24, NP and L protein) and found that AVI-7537, a PMOplus oligomer targeting VP24, resulted in the highest* and longest efficacy (and survival rate) with the simplest dosage regimen in murine and non-human primate studies.
*The combination of PMOplus siRNA targeting both VP24 and VP35 resulted in higher efficacy (survival) but required a complex dosage regimen and therefore is not being used in human trials.
Edits: Following Jon’s comments below, I have made changes to the article to rectify some of the errors in the article.
Iversen, Patrick L., et al. “Discovery and early development of AVI-7537 and AVI-7288 for the treatment of Ebola virus and Marburg virus infections.”Viruses 4.11 (2012): 2806-2830.
Warfield, Kelly L., et al. “Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers.” PLoS pathogens 2.1 (2006): e1.
Geisbert, Thomas W., et al. “Postexposure protection of non-human primates against a lethal Ebola virus challenge with RNA interference: a proof-of-concept study.” The Lancet 375.9729 (2010): 1896-1905.
This interesting study of the endocannabinoid and fatty acid signaling systems and their effect on cognition, in the brain. FABP5 seems to modulate the levels of the free fatty acid in neurons thus modulating the activity of PPARgamma and CB1 receptors. The increased FABP5 activity results in increased transport of free arachidonic acid (released by the enzymatic hydrolysis of anadamide by fatty acid amide hydrolase (FAAH)) into the nucleus where it activates PPARgamma receptors. This results in the modulation of the activation of a number of genes associated with cognition and learning.
Interestingly, this further confirms that these binding proteins are a critical part of the missing link between these two signaling systems. This further validates FABP5 as a potential drug target.
Besides, this finding could partly explain the phenomenon of temporary cognitive loss associated with cannabinoid use.
As part of the Pfizer’s Collaborative Innovation Speaker Series, I had the opportunity to hear Dr. George Church speak about their work.
Here is the list of some of the interesting things I managed to note down -
- They are working to overcome the most wide spread disease on this earth (and maybe elsewhere!) - Aging! (Wow that sounds depressing). The interesting fact that he eluded to was that there are (only?) 71 centenarians on this earth. One interesting solution to this aging problem would be to identify that single gene/allele that is unique to these individuals and maybe manipulate it further. Unfortunately or maybe fortunately (Church is very optimistic) they haven’t been able to identify any single “aging gene”. Rather, it is most likely through a combination of multiple alleles that contribute to their extended life spans. This (weirdly) excites Dr. Church even more, as this provides them with multiple genes to manipulate and brings up the possibility of additive effects and even synergism.
- Moving on to the next problem which is the shortage of human organs for transplant. Their group has managed to manipulate the genome of pigs (removal of antigenic proteins and glycosylation modifications) to prepare them as potential organ donors!
- Next problem - New drug clinical testing. Apart from the ethical issues of animal and human testing of drugs, trials in different species seem to have very poor correlation with each other in a number of new compounds. Solution - Organ-in-a-chip! Which is essentially organs (or rather clusters of cells) that are mechanically and functionally active and interconnected through microfluidics.
If those projects haven’t blown your mind yet, lets finish it off with some of his genome sequencing wisdom - (Not exact quotes) “Genome remains rather similar between cells of the same organism but epigenetics differs a lot between cells. Studying epigenetics with every cell at every stage of life would be the ideal case.” Obviously this not possible (yet), but we are clearly moving in this direction.
There are n number of other projects, just as exciting, from his lab and I am really looking forward to seeing more such projects achieving mainstream success in the near future.
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.
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).
People love to rank U.S. biotech clusters. Most of these reports are full of data on venture financing, patents, jobs, and NIH funding. But many are riddle
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.
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.