November 4, 2011 § 2 Comments
One of the things we wonder about a lot in biology is what is going on inside a cell. We have many ways to get at partial answers — Western blots, GFP fusions, transcriptional profiling, various proteomic techniques — and the number and power of these approaches is increasing. Here’s a new window on the internal state of a cell that makes use of a fundamental process of biology: the presentation of peptides by the class I MHC complex (Caron et al. 2011. The MHC I immunopeptidome conveys to the cell surface an integrative view of cellular regulation. Mol. Syst. Biol. 7 533-547, doi:10.1038msb.2011.68).
To understand what’s going on here you need to know a little about the amazing mechanisms that underlie an immune response. One of the problems the immune system has to solve is that viruses live inside cells, so in the early stages of a viral infection there may be little to see, and little for the immune system to respond to, in the extracellular environment. And so, conveniently, we have evolved a system to allow the immune system to look inside the cell. For historical reasons it’s called the Major Histocompatibility Complex class I, or MHC class I — it was discovered as a genetic locus that controlled the rejection of skin grafts in mice (hence histocompatibility), by George Snell. Basically what MHC class I molecules do is to go fishing inside the cell for peptides of a certain length, which are required to be from proteins that are made within the cell. These peptides are then captured in a “bear trap”-like structure at the top of the MHC molecule (which I have sketched here), transported to the cell surface, and offered up for recognition by T lymphocytes.
You don’t really need to know about the other parts of this system for the purposes of discussing this paper, but thanks to a mechanism called “tolerance”, briefly touched on here, T lymphocytes generally manage not to respond to peptides that come from proteins made by the host — that’s you. Instead, they focus on the foreign peptides, which are presumed to originate from viral proteins. The point to remember is this: the MHC itself isn’t selective for viral peptides, but brings a broad sampling of what’s inside the cell to the cell surface. It’s not an unbiased sample; peptides from some proteins are over-represented, others under-represented, and specific arrangements of amino acids are preferred for binding. But it offers a view of what’s going on inside the cell that is hard to get any other way. The question is, what is this view telling us? Caron et al. set out to answer this question by using a drug to manipulate the internal state of the cell, and looking with mass spec to see what happens to the peptides presented on MHC as a result.
October 17, 2011 § Leave a comment
Here is a letter from HMS Dean Jeffrey Flier that came out today. Many of us have been working on this for months, so it’s exciting to see it go public! The Boston Globe also has a nice article, here.
Dear Members of the Harvard Medical School Community:
I am excited to announce that Harvard Medical School is launching an Initiative in Systems Pharmacology, a comprehensive strategy to transform drug discovery by convening researchers from an unprecedented range of disciplines to explore together how drugs work in complex systems.
The initiative will be led by Marc Kirschner, the John Franklin Enders University Professor of Systems Biology and chairman of the HMS Department of Systems Biology; Peter Sorger, professor of systems biology; and Tim Mitchison, Hasib Sabbagh Professor of Systems Biology and deputy chairman of the Department of Systems Biology. It will comprise of faculty from a broad array of disciplines, including systems biology, cell biology, genetics, immunology, neurobiology, pharmacology, medicine, physics, computer science and mathematics, drawing on expertise from the Quad and our distinguished affiliated hospitals and research institutions. The initiative will be fueled by a strong and diverse group of existing faculty and new recruits who will be based in several departments, and will be supported by an ambitious fundraising effort.
The Initiative in Systems Pharmacology is a signature component of an HMS Program in Translational Science and Therapeutics. Led by William Chin, the Bertarelli Professor of Translational Medical Science and executive dean for research at HMS, Translational Science and Therapeutics is being created with two broad goals: first, to increase significantly our knowledge of human disease mechanisms, the nature of heterogeneity of disease expression in different individuals, and how therapeutics act in the human system; and second—based on this knowledge—to provide more effective translation of ideas to our patients by improving the quality of drug candidates as they enter the clinical testing and regulatory approval process, aiming to increase the number of efficacious diagnostics and therapies reaching patients.
With this Initiative in Systems Pharmacology, Harvard Medical School is reframing classical pharmacology and marshaling its unparalleled intellectual resources to take a novel approach to an urgent problem: The alarming slowdown in development of new and lifesaving drugs.
A better understanding of the whole system of biological molecules that controls medically important biological behavior, and the effects of drugs on that system, will help to identify the best drug targets and biomarkers. This will help to select earlier the most promising drug candidates, ultimately making drug discovery and development faster, cheaper and more effective. A deeper understanding will also help clinicians personalize drug therapies, making better use of medicine we already have.
The initiative will support both new approaches in translational science, such as failure analysis on unsuccessful drugs and use of chemical biology to develop probes of biological pathways. It will also include a new educational program, one that develops a new generation of students, postdoctoral fellows and physician-scientists, the future leaders in academic and industrial efforts in systems pharmacology and therapeutic discovery.
Transcending disciplines, departments and institutions, the systems pharmacology initiative will present new opportunities for collaboration throughout the HMS community. To learn more about this important initiative and its potential to transform drug discovery and patient care, please visit isp.hms.harvard.edu. You can also see a video on the initiative at http://www.youtube.com/watch?v=l1p_uFI4BCE.
I hope you share our excitement about the potential of this promising initiative, and I welcome your ideas as we move forward.
Jeffrey S. Flier
Dean, Faculty of Medicine
July 29, 2011 § Leave a comment
When you’re sick — whether you just have a mild headache or you’re at risk of a heart attack — it’s likely that the drug that will be used to treat you is either a natural product or a human-made copy of a molecule originally found in nature. About half of the drugs on the market today were discovered by screening collections of small molecules made by bacteria, fungi, snails, leeches and other such creatures. Though the pharmaceutical industry has made serious efforts to get away from this reliance on the natural world, attempting to create rationally designed drugs that are perfectly crafted to fit the structure of the target — or even to make their own collections of random molecules through combinatorial chemistry — natural products still represent an important fraction of the new drugs that are being discovered and approved. And so it’s interesting to ask where these drugs come from, phylogenetically speaking. The answers, reported in a recent paper (Zhu et al. 2011 Clustered patterns of species origins of nature-derived drugs and clues for future bioprospecting Proc. Natl. Acad. Sci. USA 10.1073/pnas.1107336108), are surprising.
First, why would it occur to anyone to look for phylogenetic patterns of drug production? Every species produces biologically active molecules (otherwise the species itself would hardly be biological or active) and surely any biologically active molecule has some chance of turning out to be useful as a clinically approved drug? Well, perhaps not. Pharmacologists have come to the empirical conclusion that chemical molecules built on a set of specific patterns — called privileged drug-like scaffolds — have a much better chance of becoming actual approved drugs than other compounds. It turns out that the enzymes required to produce a specific scaffold run rather strongly in families, and many of these privileged drug-like scaffolds are produced by only one, or at most a few, families of species. So, for example, the 12 natural-product-derived drugs approved by the FDA as kinase inhibitors turn out to be built on just three scaffolds, and each of these scaffolds is made by only a few species. There is a similar pattern when you look at ligands for the nicotinic acetylcholine receptors: the 53 ligands known are built on 29 (wildly varying) scaffolds, and each of these scaffolds is made by a different family, or small set of families. There are 5 approved drugs within this set, each of which is made by only a single family.
July 15, 2011 § 1 Comment
I talk a lot about drug-resistant bacteria and why we should worry about their inexorable rise — the most recent example of which is chronicled here. Now I want to offer you another thing to worry about: drug-resistant fungi. It’s the same general problem — when you use a drug that inhibits the growth of some organism, and you use it a lot, that organism has a real incentive to evolve around the drug. The special worry with fungi, though, is that we never had a huge array of useful drugs in the first place. The best broad-spectrum antifungals are the azole derivatives, such as fluconazole; these inhibit an essential enzyme that is the product of the gene ERG11. But — same old story — they’re gradually losing their effectiveness.
Why are effective, broad-spectrum antifungals so rare? The problem is not so much that fungi are hard to kill, it’s that they’re hard to kill without killing us as well. Fungi are eukaryotes, and the pathways they use to thrive and survive are awfully similar to the analogous pathways in us. Even fluconazole suffers from this problem: the ERG11 gene encodes a cytochrome P450 enzyme, and we humans have many similar enzymes; cross-reaction of fluconazole with the human enzymes causes significant toxicity. The fact that fungi and humans have so much in common has made it hard to identify single agents that reliably kill (or inhibit the growth of) one, while sparing the other.
A new study (Spitzer et al. 2011. Cross-species discovery of syncretic drug combinations that potentiate the antifungal fluconazole, Mol. Syst. Biol. 7 499) now offers hope that combinations of drugs will do better. Spitzer et al. started with the observation that although there are only 1100 genes in the yeast Saccharomyces cerevisiae (biologists’ favorite model fungus) that are essential under normal lab conditions, many more genes can become essential under other conditions. In particular, if you knock out one non-essential gene, you often find that you can no longer knock out certain other non-essential genes without killing the yeast. This is called synthetic lethality. Roy Kishony likes to use the following analogy to explain it: suppose you put an eyepatch over your right eye. This may make you look as if you’re auditioning for Pirates of the Caribbean 5 (or are we up to 6 now?), but it doesn’t completely prevent you from seeing. The same is true if you put the patch over your left eye. It’s only if you wear two patches, one over each eye, that you get the “synthetic lethal” effect on your vision. Similarly, knocking out two non-essential genes — or, in this case, inhibiting their products with drugs — may have a lethal effect even though targeting just one of the two genes doesn’t do much.
To expand the universe of useful antifungal drugs, Spitzer et al. wanted to look for synthetic lethal combinations that involve drugs not currently used as antifungals. They took a library of bioactive drugs, including a number of clinically approved drugs that are no longer covered by patents, and screened them in combination with fluconazole at a concentration where fluconazole isn’t effective on its own. They used four different fungi: our old friend S. cerevisiae, and the human pathogens Candida albicans, Cryptococcus neoformans, and Cryptococcus gattii. Almost 150 compounds — over 10% of the library — showed activity against one or more of the fungi, including examples of some surprising drug classes including antidepressants, antibiotics, and antipsychotics. (I guess even fungi get depressed.) These drugs were not active against fungi on their own, but in combination with fluconazole they had an effect. You could call them conditional antifungals.
June 10, 2011 § Leave a comment
We’ve talked before about microbes playing dead to avoid the effects of antibiotics. A recent paper (Baek et al. 2011. Metabolic regulation of mycobacterial growth and antibiotic sensitivity, PLoS Biol. 9 e1001065) identifies a new mechanism that Mycobacterium tuberculosis uses for switching into a low-metabolism, drug-tolerant state.
M. tuberculosis, as you undoubtedly know, is the bacterium that causes tuberculosis (TB). It’s a nasty pathogen, made worse by the fact that it’s really hard to kill. Treating tuberculosis involves a 6-month-long course of antibiotics — anything shorter, and not only does the infection come back, it’s now drug-resistant. Multi-drug resistant TB (MDR-TB) is increasingly a nasty public health problem. People just aren’t very good at taking pills for 6 months, without fail, even after they’ve started feeling better.
Why does M. tuberculosis take so long to kill? The way these bacteria survive is rather bold: they live inside macrophages, the cells that normally help get rid of bacteria, and indeed inside the vesicles (phagosomes) that are intended to chew them up. Here they grow, but very slowly: they divide maybe once every 100 hours. Many studies have shown that antibiotics generally do better at killing bacteria that are growing rapidly. Maybe this slow growth has something to do with the poor killing.
It’s a stressful environment inside a phagosome. If you’re a bacterium, this is an environment that’s designed to kill you. There’s not much oxygen, the pH is low, and important nutrients, including iron, are lacking. Simply restricting the oxygen supply in vitro causes the bacterium to become slow-growing and antibiotic-tolerant. Baek et al. used these hypoxic bacteria in a transposon-based genetic screen for mutants that don’t slow down their growth when oxygen is limited, to look for genes involved in the pathway that controls the growth shutdown. The mutants they find are… in genes to do with the production of fat. Triacylglyceride, to be precise. Huh?
March 29, 2011 § 2 Comments
You’ve probably seen NMR machines at some point during your career. They usually have their own room, often with an extra-high ceiling to allow the operator to insert the sample without bumping his or her head. So it may surprise you to know that a miniaturized NMR machine that you can literally hold in the palm of your hand has now been developed by Ralph Weissleder’s group. And yes, there’s an app for that: the instrument is operated via a smartphone, making it possible to use NMR analysis of clinical samples literally at the bedside (Haun et al. 2011. Micro-NMR for rapid molecular analysis of human tumor samples. Sci Transl Med 3, 71ra16, doi:10.1126/scitranslmed.3002048).
The Weissleder lab and their collaborators have been working on this miniaturized NMR machine and the imaging reagents required to use it for several years now; Ralph brought a prototype to our faculty lunch meeting about a year ago. The goal is to use NMR as a sensitive way of detecting specific markers on very small samples of cells from patients who may or may not have a malignant tumor. The device uses a miniaturized magnet to create the field inducing the magnetic resonance, solenoidal microcoils to detect the signal with high sensitivity, and tiny fluid channels, embedded in polydimethylsiloxane beside the microcoils, into which the sample is injected. The whole device has a footprint of 10cm square. The other part of the magic is in the imaging reagents. These are magnetic nanoparticles that can be linked to a variety of monoclonal antibodies via some clever chemistry that allows the linking to be done in the presence of whole blood (for more details, come to Neal Devaraj’s Pizza Talk at 12.30 today). Using this system, it’s possible to get a reliable measurement of the level of a specific marker from just ~200 cells. And the measurement is amazingly quick: it takes under an hour.
March 8, 2011 § Leave a comment
As regular readers of this blog know, I am not looking forward to living in a world without effective antibiotics at all. (Well, I’m not insane.) I was therefore interested in a recent paper (Wei et al. 2011. Depletion of antibiotic targets has widely varying effects on growth. PNAS doi:10.1073/pnas.1018301108) that takes a small step in the direction of making the discovery of new antibiotics more rational.
There’s a general feeling out there that the best antibiotic targets to go after are probably the ones that are most needed by the cell. If a bacterium needs 99% of the activity of a protein to grow, then maybe reducing the activity of that protein by just a little bit would be enough to kill the bacterium, or at least make it grow much slower. To make it easier to find a small molecule lead that inhibits a particular target, you can reduce the expression of the target, for example by using bacteria in which mRNA production from your gene of interest is reduced. Wei et al. use a conceptually similar but practically different approach: they knock down the levels of the target protein using a clever strategy for inducing protein degradation. Their strategy goes like this.