Questions for the kinase biologists

I am working on a kinase inhibitor design project and I realised that there are some key questions that we need to get answered from the biologists before we can rationalize the selectivity of various kinase inhibitors for a given binding site. I also realised that these questions need to be answered for many other kinds of protein-inhibitor interactions.

1. Whenever we get different IC50 data for two inhibitors, we immediately try to look at binding interactions that may be different for the two moelcules to rationalize this observation. But as I have alluded before, it's not the IC50 but the Ki that's really to do with different binding interactions. The Ki and IC50 are related by an equation that includes both the Km value of ATP and the concentration of ATP in the two experiments, or in general, these two parameters for the natural binding substrate for the protein. Only if these two are the same for both inhibitor experiments is the IC50=Ki. So make sure you confirm this. Otherwise, extrapolate and calculate the new IC50s based on identical values for these parameters. Then rationalize the IC50s based on binding interactions.

2. For many kinases, three events are absolutely essential for activation:
a. Phosphorylation of one Ser, Thr or Tyr residue,
b. Binding of ATP (duh), and
c. Dephosphorylation of another Ser, Thr or Tyr residue.
Think of it like a logic gate. IF the answers to all a. b. and c. are YES, THEN the kinase will be activated and proceed to perform its function. (I got this from Alberts et al.'s Molecular Biology of the Cell)
In the assays that are run, it is important to know (and not very easy to always determine as I have been told) whether the necessary residue is phosphorylated or not. For one thing, inclusion of this knowledge in your docking and modeling can naturally make a big difference. And secondly, depending on the state of phosphorylation, you can think of different modes of inhibition for your inhibitor (eg. ATP blocking + substrate blocking).

As usual, it's important to know what the biologists are doing. They don't know the nuances of modeling/crystallography and you don't know the nuances of their assays. But it's important for both camps to think of questions which the other camp should answer that will affect their own work.

Inside the "graduate kitchen"...and what I found there

The "graduate kitchen", just like the "graduate lounge" and the "rabbit hole" of "Alice", is a non-existent entity. But you fantasize about it, especially in light of the dismal thing down the hall that tries to approximate it.

Everyday that I trot down the hall into it to heat my lunch or get my daily coffee, I am assailed by familiar smells, that are yet so unfamiliar that even the Flying Spaghetti Monster would not be able to unravel their composition and sources. The dingy place consists of a table with unidentified food scraps on it, a giant something in a corner approximating a fridge, and stodgy white appliance right next to it, apparently the kitchen microwave.

Each one of these objects has its own story. If you open the microwave oven door, you see what looks like an extra layer of insulation, which turns out to be a 1 inch thick coating of food that has been spilled and splattered about it for the last couple of years. But this is anything but disgusting, at least not so if you don't find finely carbonized ash disgusting. That's what that layer looks like, carbon...or something that resembles it. It's not exactly carbon though, because it still has an 'organic' smell, neother pleasant nor repulsive. One dreams of what molecules could be in there...the cure for cancer of AIDS perhaps. Looking at the specks and layers, I had an idea; scientists could use this system as a model system for studying what happens to organisms and organic molecules when they are cast aloft into space and transported over long distances, bombarded by the less than ambient ultraviolet and other kinds of radiation rampant in the galaxy. The specks in the microwave bear witness to this cruel assault; having been fried countless times over by microwaves, they could perhaps be similar to the molecules that arrived after a perilous journey on early earth to sow the seeds of life. Obviously, Fred Hoyle and Francis Crick must have anticipated the modern grad student lifestyle.

Then there's the fridge. Even my home fridge was a great experimental system; the Chinese roommate who vacated the apartment when I came had a crab in there that was at least six months old. Immense benefits of bleach immediately dawned upon me. A similar, but much more exalted, scenario exists in the "graduate kitchen" fridge. There is some black goo on the fridge floor, long since petrified, that reminds me of a liquid alien form in an X-Files episode. Dig deeper if you dare to, and you find foods of different shapes and sizes, that used to actually form a part of international cuisine. It was two years ago that I stuck a notice on the fridge; "Please do not leave food inside for more than a month...or until it starts to stink". I should have realised; especially when it comes to international cuisine, the stink is in the nose of the beholder. Needless to say, I did not want my own gallon of milk to suffer such a fate. That's why I gulp it down within a week, and post a sign saying "Poison, do not ingest" on it to prevent theft. Then there's the top freezer compartment, which tests the resilience and shelf life of frozen dinners more than their manufacturers could have imagined in their wildest dreams.

In the corner of the "kitchen" sit two transparent lunchboxes. I vaguely remember two students long since graduated, once eating lunch from those lunchboxes. I want to inform them that their fungal growth experiment has worked.

But of course, it's the microwave that really interests me. I wish I could scrape off some of those particles of unknown composition and submit them to chemical analysis and NMR. Then at least, my daily visits to the "graduate kitchen" would have become a little colourful.

Having said all this, just like an unpleasant relative, the kitchen becomes part of your daily life, and so actually manages to somewhat endear itself to you. The random journal issues and magazines tossed on the table entice you to no end to want to stay. Some brave souls, including our department chair in the lead, take on occasional tasks of heading a search-and-rescue operation into the kitchen. At least I wipe something when the source of the spill is my lunchbox or mug. But I am not one of those brave souls.