Molecular gymnastics worth 10 billion dollars

Specific covalent inhibitors have long been of great interest to many people, despite the fact that irreversible inhibition has not really been on the very top of pharma companies’ wish lists until recently. I do find this somewhat ironic given the fact that a great many useful medications owe their efficiency to the covalent mode of action. We do not have to go very far for examples – take aspirin for starters…

Today I want to talk about epoxomycin, a molecule that has been associated with the name of Professor Crews (Yale). The company that emerged from this technology, Onyx, was recently bought by Amgen for 10+ billion dollars. Kyprolis is the name of their drug, which is a close cousin of epoxomycin. The difference between the two is just a couple of modifications to improve solubility and other drug-like properties. 10 billion is a lot of value for a fairly simple epoxy peptide, which means that there’s more that meets the eye. Indeed, epoxomycin (an irreversible inhibitor of proteasome 20S) displays a marvelous mechanism of action. The molecular gymnastics that take place during its interaction with the terminal threonine of proteasome 20S are shown below. Obviously, this is not your typical epoxide that reacts by a classic Sn2 mechanism with some active site nucleophile. If you look closely, the interaction has two distinct steps: hydroxyl of Thr1 attacking the carbonyl group followed by amine reacting with the epoxide ring. Thus, the specificity is defined by a multi-center engagement, something that I think is worth emulating in other contexts. Below is a link to the Crews’ seminal paper.


Proving vs disproving

I went for a walk with Jovana, my wife, on this crisp October night. There are leaves and all, which is truly poetic and makes one want to listen to the “Four Seasons” by Tchaikovsky. I may not have mentioned this before, but Jovana is a hematologist. She often tells me stories about her work at the hospital. Some of our discourses are more interesting than others, but the bottom line is that the daily decisions Jovana has to make are on a different level in terms of responsibility compared to my line of work (some people might even posit that I am somewhat irresponsible in anything I do, but that’s ok…). I will admit that earlier today Jovana had told me something that really resonated with me and made me think. Do you know what is the most commonly missed fracture in an emergency room? Here is the answer: “the second one”. This suggests that even professionals can have a tendency to stop searching for abnormalities or problems after ONE is identified. This also speaks to the challenges they face in trying to correctly process the available evidence with the righteous intent to come up with a correct diagnosis. It is common to selectively seek evidence that confirms his/her instinct and then stop without considering whatever’s left over. This problem underscores a deeper fault in our reasoning, namely our inclination to reach conclusions that are not based on reconciling ALL pieces of evidence. As long as the resulting picture accounts for our prejudices, we tend to make our judgment, which is in haste…

This discussion made me think of a parallel with a dangerous way we might reach conclusions in science. I suppose this is a reminder to all students. After all, they are the gatekeepers to the most precious part of the research enterprise: collection and analysis of the primary data. I recall Olah and his memorable injunction some 20 years ago when he said “you can never prove a mechanism of a reaction, you can only disprove it”. This is a profound statement (it is also fundamentally correct as it reflects the fact that our techniques constantly evolve and we will only see and appreciate more details, not less) because it highlights that scientists are naturally good at disproving things. We really are. To take this mantra one step further, I submit that our preferred way of doing science should consist of rigorously disproving our gut reactions and conclusions. Why? It’s because we are good at proving things wrong and also because we will not fall into the dangerous trap of having the conclusion ready before the available evidence supports it.

The honour roll: Oyo Mitsunobu

Prior to 1967, people in the Haight-Ashbury neighborhood of San Francisco thought that they had seen it all. Little did they know that a social earthquake was about to befall them, which later became known as the Summer of Love. The music of Jefferson Airplane was in the air and I just wish I was around then, but I was not even conceived at that time. There is another memorable event that hails from that eventful year. Across the Pacific Ocean in Japan, the late Professor Oyo Mitsunobu (1934-2003) developed one of the most remarkable and useful reactions in the history of organic chemistry, the Mitsunobu process. There are a myriad of reasons to love this reaction, which epitomizes the so-called oxidation-reduction condensations. In the Mitsunobu reaction, the cost of installing a new bond comes from a combination of oxidation (phosphine into phosphine oxide) and reduction (DEAD into the corresponding hydrazine) processes. There are people who would say that this is not atom-economical, blah blah. No, it ain’t. But go tell this to Ed Grabowski, the retired process chemistry guru from Merck. He will tell you a thing or two about the immense utility of this “ungreen” reaction. I wish we had more ungreen processes like that, I tell you this much! Any chemistry that you use without worrying about referring to the original journal reference is in the lore of chemical synthesis. The Mitsunobu reaction is certainly there.


The business we’ve chosen

It is important to keep trying to find ways of guiding our students through rough times in research. As advisors, we have been there before and it is useful to ensure that our students know that things are cyclical by nature and good times tend to follow the bad ones if you give it 100% of your effort. Some people go to prominent figures from the past for motivational language. But there are too many cliché quotes out there and I am not particularly fond of them because they state the obvious and sound dogmatic. For instance, we know that Churchill said: “Success consists of going from failure to failure without loss of enthusiasm.” It is true, but this statement tends to rub some people the wrong way.

So here’s a quote that’s rarely mentioned, yet it is a good one. It comes from one of the best movies ever made (Godfather, part II). Earlier today I was reminded about it when I heard that some of my students have not seen this film (really?). In the second part of the Godfather trilogy, Michael Corleone visits Hyman Roth and hints at his shady involvement behind the scenes, which triggers Roth’s jab back at Michael that ends by a CLASSIC “…This is the business we’ve chosen”. Check this out (he says it at about 2:24):

Those who decide on a career in science do it for reasons other than money. Their commitment to pursue knowledge creation is admirable. Yet, once in a while when we ruminate, there is a tendency to forget that we have voluntarily chosen this difficult, yet intellectually rewarding, path… Indeed, this is the business we’ve chosen.


Because of my lab’s current interests in co-crystals I have been trying to read the literature in this area. One recent paper I came across describes a well-executed study that has some… well, I am not kidding, magic (almost) in it. The paper hails from Bucar and co-workers in Cambridge. According to the authors, efforts to generate a co-crystal between caffeine and benzoic acid have consistently failed over the past six (!) decades. Bucar’s team was no exception to this ill fate, but not for their lack of trying. It is hypothesized that, despite the anticipated stability of the resulting material, a high kinetic barrier to co-crystal formation accounts for this lack of success. For decades, this kinetic barrier has hindered the formation of what should be a very thermodynamically stable co-crystal form. When the authors tried to improve their chances using a heteronuclear seed (a caffeine/substituted benzoic acid was used in this capacity), they finally succeeded in growing the long-awaited caffeine/benzoic acid co-crystals. This achievement aside, it is what happened afterwards that caught my attention. Apparently, ever since the first successful crystallization, the crystals would always form after numerous attempts to rigorously clean the glassware by scrubbing it with all sorts of potions (acids, bases, bleach, you name it) and deliberately NOT using the aforementioned seeds any longer. In other words, the authors could not replicate their NEGATIVE results from the past. Please read the paragraph starting with “One possible explanation for the unexpected…”. Imagine this: you try for a long time, you get nowhere, and then you finally reach your crystalline Nirvana. Form that point on, your lab gets its “mojo” in that those chemicals that used to never co-crystallize, would from now on ALWAYS co-crystallize. According to the authors, these results speak to the exceptionally strong seeding capacity of the complex they used in order to obtain co-crystals. I think we need some of this in our lab too! We need some of this pixie dust, guys…


Eeating my own words

We have been having some exciting results in heterocyclic fragment screening using X-ray crystallography. The initial hits made with the help of Aman and Elena now enable further design and we will hopefully nail some of our targets together with Shinya, Conor, Jeff, and Rebecca in my lab. By “targets” I mean a series of methyllysine-binding proteins for which we intend to identify specific and cell-permeable probes. It will take us a while (longer than the remaining 2 months of my sabbatical), but we are in this thing for the long haul.

I want to cover one particular line of reasoning that has been brewing in my head for a while now. The sheer number of new (from my lab’s perspective) tools that have emerged out of the SGC collaboration, is quite staggering. They range from sophisticated soaking methods to co-crystallization and docking studies. I have turned into a total fan of Schrodinger Glide software ( Dr. Conor Scully, a research associate in my lab, introduced me to this tool and I have continued learning on my own. I am having a lot of fun, although I sometimes feel bad about peppering the great Schrodinger team of developers with all sorts of questions. It is a full assault, I must admit, but these guys are real pros. Earlier today, Hege Beard, my contact there, informed me that she has developed an algorithm that will help us with covalent docking of aziridines to cysteine-containing protein targets. We have had some glitches with this, but the method is working really well now.

So where is my point for today, you might ask? Here it is. As I immerse into the elements of design and docking of ligands for our targets, I am forced to revise some of the dogmatic thinking I have been using in the past. For instance, one of the points I used to love chuckling about had to do with some curious publications that described the design of a molecule and experimental validation of its activity in biochemical and cellular assays. This would eventually lead to an atomic level view of the pivotal interaction, manifested in a co-crystal structure. And there came the moment of truth when scientists would humbly admit that their molecule, while active, potent, etc, engaged its target in a totally different way from their initial prediction. To back up my words with a concrete example, here is something form SGC. In this nice work, a kinase inhibitor (in red below) of Pim1 kinase was developed. I made this picture using PyMol. The co-crystal structure shows that the molecule does NOT bind in the anticipated “Type 1” mode (it is when the so-called hinge region is engaged in protein/ligand contacts).


Having said that… I was recently on a lecture tour in B.C. and talked to a biochemist who told me about the so-called two-stage binding in cytochromes. This is totally unrelated and I am not an expert in this at all, but it makes me wonder about my previous position I just described. Here is my proposal: when you see a binding mode that is not consistent with the projected (designed) one, it may be a simple manifestation of kinetic/thermodynamic control and the fact that there are different phases to an interaction. In other words, what eventually crystallizes corresponds to the most stable arrangement that, by its nature, does not invalidate the design that took a different part of the binding site into account!

Strain in pi-clouds

Of late, I have been having a number of discussions about pi-systems with my students. There are many reasons for this surge of interest, too many to mention in one post. This discussion of pushing the boundaries of unusual pi-reactivity reminded me of some really innovative work coming out of Prof. Neil Garg’s lab at UCLA. Neil has pioneered some cool ways of making (and using) indolynes, the distant cousins of benzynes. One of his earlier papers related to this technology appeared back in 2010. I really appreciate this work, which is due to my lab’s long-standing interest in medium sized rings, particularly that of indolactam V, a well-known PKC inhibitor. In Neil’s indolactam synthesis, indolyne intermediates are put to clever use. What you see below is the key step that enables this synthesis. The chemistry involves regioselective addition of an amine nucleophile to the strained indolyne pi-system.


Counting oxidation states

I want to talk about the concept of an oxidation state. According to IUPAC, it is “a measure of a degree of oxidation of an atom in a substance”. While inorganic chemistry has a fairly clear-cut set of rules about how to count oxidation states of metals in organometallic complexes, it is certainly not so simple in the case of organic compounds. There are vastly different ways of assigning oxidation states to carbon centres in organic molecules and things get unnecessarily confusing. I decided to take a look at some online resources… In the following video ( the oxidation state of carbon in methane is assigned to -4, while in ethane it is assigned to -3! Of course, everyone understands that this is sheer nonsense. If we follow these guidelines, an aldehyde and a ketone will have DIFFERENT oxidation states at the carbonyl centre, which is not consistent with their fairly similar properties.

I would say that we have to go back to the drawing board and ask a question: why bother with oxidation states at all? There is only one purpose: to understand structure and reactivity better. In inorganic chemistry, Pd in the oxidation state “0” does things that Pd in the oxidation state “+2” is not known for (or known to do differently). This knowledge really helps us. Why don’t we keep things simpler in organic chemistry? Well, hybridization states and C-C bonds (including multiple ones!) screw us up. To make things more useful in this oxidation state mess, I think it is important to first consider one-carbon molecules. I will just look at the following four and unambiguously assign oxidation states based on the number of carbon-heteroatom bonds.

ImageMore elaborate carbonaceous molecules are composed of fragments that correspond to the variations of the blocks above. I admit that carbon-carbon multiple bonds make counting tricky. If a carbon atom has one pi bond and no heteroatom partners, its oxidation state is “+1” (e.g. ethylene). If a carbon is BOTH part of a pi-system and is connected to a heteroatom, the oxidation level is defined as the number of pi-bonds plus the number of heteroatoms attached to the carbon under consideration. Take the following case as an example:


The reason I really like things this way is that they enable me to think about heterocycles with some clarity. If someone knows of a better way to easily count oxidation states, let me know.

Unleashing some powerful reactivity

The Passerini and Ugi processes are two of the best-known multicomponent reactions that are based on the isocyanide functional group. In each of these processes, the isocyanide reacts with two carbonyl components. A carboxylic acid and an aldehyde are engaged in the Passerini process, while in the Ugi reaction it is the imine that reacts with the isocyanide instead of an aldehyde. When we started looking at amphoteric aziridine aldehydes with Ryan Hili back in 2006, we thought of aziridine aldehydes as 1,3-molecules based on the distance between the nucleophilic and electrophilic nodes of reactivity. This was a nice way to differentiate from isocyanides.


While aziridine aldehyde reactivity was in its infancy back in 2006, the isocyanide’s innate ability to react with the donor (eg aldehyde) and acceptor (eg acid) components had ample precedent in the Ugi and Passerini reactions I just talked about. Ryan and I felt that something must happen if you simply mix an aziridine aldehyde and an isocyanide. However, this simplest mode of reactivity has been elusive thus far. While we had plenty of luck with other processes, it has been kind of frustrating not to be able to find a precedent for what appears logical: mix isocyanide and aziridine aldehyde and get something cool. We are still trying to reduce this idea to practice. Unless my students correct me, aziridine aldehydes and isocyanides just stare at each other in solution. I still don’t understand why. Most likely Mother Nature is trying to teach me a lesson in kinetics here…

Along these lines, here is an interesting example that I really enjoyed reading about. The chemistry comes courtesy of Prof. Wang in Tsinghua University, China, and details the chemistry of enamide aldehydes that use their enamide portion in order to attack the isonitrilium ion, which is in turn created during the well-established isocyanide attack at the aldehyde (just take a page from the Passerini reaction!). The elements of amphoteric reactivity are on full display here. As a result of this well-orchestrated sequence, one gets access to polysubstituted pyridines. In the graphic below I am not attempting to illustrate all the gory details. While a couple of steps (oxidation and acylation) are left behind, the core of the process is present. Enamido aldehydes seem to be more than adequate to unleash the synthetic prowess of the isocyanide functionality…


Lab revelations

I am going to talk about some of my own work today. My sabbatical is going well, but I can sense that the Winter semester is around the corner. As of January 1 I am back to my regular job at the Chemistry Department. Thus far, my experience at SGC ( has been a blessing. I have learned a lot of new things about structural biology. More importantly, some of my students are now trained in protein chemistry and crystallization (thanks to Elena). I also have to thank my good friend Al Edwards, who was at the beginning of it all. I refer to his role in the foundation of SGC together with Cheryl Arrowsmith as well as to his shaping of my interests in protein crystallography. Below is Al, by the way, along with a drawing he made at Starbucks some 6 years ago when we discussed standard conditions for crystal growth by slow diffusion. It all looks deceptively simple, but the devil is in the details. The trick is to get into the right “zone” that is conducive to crystal growth. Passing that zone is easy and it is all about conditions, conditions, and conditions (to paraphrase the real estate dictum).


As far as me doing lab work, here is the man lesson thus far: it is really good for an academic to go back and get calibrated in the lab from time to time. Besides its educational value, this experience reminds me about how rarely things actually work in research. Ironically, this is something that is special about research experience because we feel good when things do work. At the same time, if one’s heart is not 100% in research, failures can be a real drag. I have always enjoyed the challenge, so it is ok for me. But I will tell you this: if you are a professor and are sitting in your ivory tower overlooking a range of projects, it is easy to forget that each of your graduate students is in fact focusing on a specific and often narrow area of research. There is a significant ramification here: it is more difficult to get frustrated in the ivory tower. If there are no results in a particular project, there is always another, parallel, area that does generate results. In time, things fluctuate in terms of success between the areas. It certainly is tougher to be in the students’ shoes. It just ain’t the same when your job is fixed to one problem. Now… there is a practical and obvious lesson here for the students: always (yes, always) balance several different projects on your plate. In this case, the chance of getting discouraged about lack of results will be smaller.

For example, we evaluated a battery of new conditions aimed at understanding macrocycles and their folding in the crystalline state. I hope we’ll get some new hits but things have been difficult thus far. I set up 480 experiments and one of them worked, giving me crystals that unfortunately did not diffract today. But that’s ok – a couple of hours later I went back to my chemistry lab and talked to Piera, Adam, Shinya, Frank, and Victoria about their plans in boron chemistry. See: there was a total “switch”. We failed with our crystals earlier in the day, yet I got rejuvenated after a switch to something totally different. We bounced ideas and things are looking up… It’s all about balance.