Back in India

It took me exactly 45 minutes to get to the taxi stand from the time I got out of the plane.

The flight landed at 8.30pm at the Delhi airport. I mentally prepared myself for the endless queues and delayed baggage. If I got out before 10pm, I told myself, I was lucky. I was little apprehensive because I dislike dark and Delhi Taxi drivers are notorious. My cell phone was in safe custody and I had no access to any phone.

But surprise!

There were 4 medical booths were we had to deposit a form stating whether we had any fever etc etc. Precaution of swine flu epidemic. There were bunch of people with face masks sitting in these booths collecting the forms. I do not know what purpose all this serves other than giving employment to bunch of people.

The immigration lane was short and sweet. There was literally no wait time. The fellow at the immigration booth actually smiled and said Good Evening. I was ready to faint from the shock of it when he reverted back to type and wanted to know why I had gone to US and what I was doing etc etc. Typical nosiness.

Then came the baggage claim. It took about 5-10 minutes for the bags to come to the conveyor belt. But by 9.15 pm I had my bags. There was no wait at the customs. They asked me where I was coming from and took the customs slip and I was through.

There was a pre-paid Taxi booth who made out the chit for a pre-paid taxi. And I was home by 9.45pm.

Of course lest we think that travel is a breeze the taxi driver was the perfect Delhi wallah. He wanted minimum Rs 100 as tip. We haggled back and forth. He told me that he did not have change for Rs 100 and it had been a long day and that no taxi driver worth his salt would come for such a short distance as he had done. finally we settled down for Rs 50.00.

Welcome home.

Deepavali with Bella

My housemate has gone to a one day conference in North Carolina so I am dogsitting Bella.

Yesterday night she got miffed with me because I refused to let her sleep on my bed. She scratched on the door, cried, barked, and tried all sorts of tricks but I refused to let her inside my room.

This morning, I tried to tell her that it is Deepavali and she should take Ganga snanam. But she paid no heed to it. She has been insistent that I should play with her. The play consists of my throwing her favourite toy- a bone shaped cotton cloth- as far as possible, and she running to catch it. Occasionally, as dictated by her, we play a tug of war with that toy. If I work or refuse to play with her, she growls and drums her feet impatiently.

This morning, I took her for a walk and then locked her up in the house while I went for my jog. She did not take too kindly to that treatment. When I came back home, she had managed to express her anger by tearing up a catalog and some roses my room mate had left in the living room.

At present as I type the blog, she has been throwing her toy at my computer, angry that I am paying more attention to the computer than to her.

Happy Deepavali!

Nobel Prizes and We

Every newspaper is full of V. Ramakrishnan. After all he is of Indian Origin. So we have a cause to celebrate. (Okay, I am proud too. After all he too studied at M.S. University, Baroda.).

That apart, now will begin the usual question: Why can't Indians do well in India and win Nobel Prize.

I am not going to get into the merits of Nobel Prize. As all prizes go, this also involves lots of pushes and pulls. What I want to focus on is why it is difficult to do good research in India, especially in the Universities.

1. Faculty recruitment- The Universities, which abroad are the places where research is done, has been relegated to a second place. They are teaching workshops. We do not bother about the kind of faculty we hire at these places. Forget research, they do not even need to be good teachers! The research has been pushed into institutes but no one is asking where the students for these research institutes is coming from. The worst of it is that once a faculty has been hired there is no way of getting rid of that faculty short of retirement. So there is no motivation to perform. Actually, if you underperform you will be promoted and given all sorts of awards. There is a crying need to bring in the sort of tenure system into our Universities. You are booted out unless you perform both as a teacher and as a researcher. The UGC did propose a sort of evaluation for promotion but the teachers are against it. They do not want their teaching program to be evaluated because it can be manipulated. That is another problem- suggest anything and everyone will be ready to point out why it will not work. So unless faculties are expected to adhere to rigor, we can forget about good teaching as well as good research.
2. Faculty salaries- This is the bone of contention. Since salaries are decided by UGC, all of us get the same salary. There is no incentive to do good teaching or research. Am I going to get extra for putting in that much of work? Why can't the universities be allowed to decided who gets paid how much? Of course, there will be people who will tell you that this will lead to mismanagement but why can't we put a procedure in place to decide how much salaries will be paid?
3. Promotion- thanks to UGC an Assistant professor has to put in 9 years of work before he or she will be promoted regardless of how well they teach or how good they are as researchers. They have to, regardless of their expertise, do one orientation course and two refersher courses. No, don't ask me what good they are. I slept through them. I still have one refresher course to attend. Now, tell me what motivation will I have to be a good faculty? I do it because I have this inner urge to do something great but it is so frustrating at times that I feel like chucking the whole thing off. It is different that I have nothing else to fall back on and therefore, I plod along.
4. Money- Thanks to this policy of Universities as teaching workshops, money is scarce. Money is needed to buy chemicals as well as equipments. Science has moved away from using simple things to do research. The technology changes every day and we need to buy the sophisticated equipments to good research. We need expensive chemicals but where is the money to buy them? Invariably, every faculty is expected to write a grant. Which is a good thing. The bad thing is that how much money I am going to get from my grant is decided by a bunch of lunatic financial officers with no appreciation of science or research. I, for example, wrote a grant and asked for 20 lakh to buy equipments. I need a shaker incubator and a centrifuge very badly. I was given 10 lakhs to buy two items. Neither of them is a shaker incubator and a centrifuge. Even the equipment I am allowed to buy will not be the sophisticated version that I need but a simple one that will just help me to do some basic analysis. Now, tell me why should a financial officer decide what I should purchase and not purchase?
5. Procedural delays- To buy any equipment above 50,000 rupees (which invariably all equipments are) I have to get permission from the purchase committee headed by a finance officer. I have to get 4-5 quotes and am allowed to buy only from the person who quotes the least. Needless to say I have to spend time getting these quotes, presenting before the purchase committee and then only I can buy. It takes 6 months to get an equipment. This is not unique to Universities. Institutes too have to follow this procedure. This is ostensibly to reduce corruption but who ever wants to make money can still find loop holes to make money.
6. Infrastructure- The labs are invariably constructed by the great CPWD with more than 150 years of experience. They know how to get seepages correct, how to make sure that the drain does not work, how to do shoddy work, how to create a workplace that is generally not workable. Can we please get good lab spaces that are temperature controlled so that I do not have to figure out a process for summer that is different from of winter? The infrastructure is terrible. We still do not have good internet facility. We do not have access to library books and journals. Our libraries are the pits. Less said about them the better.

There is a good article in the rediff that talks about recruiting good faculty globally amongst other things.

The problem with us is that we are fond of talking and complaining. At some point we need to stop talking and start overhauling the system. Not so that we can get a Nobel Prize winner but because we need to put up a good education system. It is essential to remember that people like V. Ramakrishnan were taught by teachers who are increasingly difficult to find in our University systems.

From Targets to drugs -III

All hits do not convert into a drug molecule.

Suppose one finds a small organic molecule that seems to inhibit the activity of the protein of interest, what does it really mean? It means nothing because we have to prove that it is effective in vitro as well as in vivo.

The first thing that any chemist or biologist will ask is what is the binding constant. Binding constants are nothing but the concentration of the inhibitor that is required to bind to the protein and inhibits its activity by say 50% (This is a gross exaggeration but this is the simplest I can explain Michealis-Menten equation.). The lower this number the better the inhibitor. Typically, we look for molecule that will have a binding constant in the order of sub micromolar or nanomolar range. That is a tall order. Not all molecules that we discover will bind in that range.

There are other consideration. The molecule or drug should be such that it can enter into the cell easily. This is again a tall order because each cell has a bilayer membrane made of lipid molecules. So essentially you are looking at non-polar molecules or molecules whose charges are not exposed.

The molecule should be small in size so that it can diffuse into the cell. If it is large molecule then it will require special channels or pores to enter into the cell.

The molecule should be specific to the protein of your interest. In other words, look at the protein I work with. As I said it hydrolyzes ATP. Now, there are thousands of proteins inside the cell that can breakdown ATP. If I want to create a drug that inhibits the ATP activity of my protein, I need to make it absolutely specific so that only my protein is inhibited. This becomes absolutely important when you consider that a drug has no specificity with regards to the cell it enters. It can be a diseased cell or a normal cell. What you look for when you create drug molecules is that in the diseased cell, the protein of your interest is present and if you target it then the diseased cell will be killed. However, you cannot prevent your drug molecule from entering into normal cell and therefore, if you have a non-specific drug it will kill the normal cell too, creating havoc.

So you see getting a drug molecule is a tall order. It is has to be small molecule, specific to one protein, and binding to it very tightly.

If you succeed in finding such a molecule, then comes the next part. You have to show that it is not toxic. So each drug has to pass through pharmocokinetics studies. You have to show that the drug molecule is not creating unwanted side effects, it is not carcinogenic and it is not going to alter the DNA make up your cell. You have to also show how it is metabolized and excreated from the system. These studies are done in animals.

Once it has passed this stage, the human trials begin. Only then the FDA gives approval for a drug to be marketed.

These studies take time. The average success rate is between 1-2% and it can take up to 20-30 years of intense studies to market a drug.

Can this time be cut down? Yes, if we know how to make proteins in large quantities and solve their structure. If we have better programming tools for in silico studies. In fact, many pharmaceuticals are also looking at alternative models for pharmocokinetics studies. If we are successful in eliminating or reducing even one of the bottlenecks, we can drastically cut down the time.

From Targets to Drug-II

I meant to post this in the morning but I got side-tracked with the structure of the inhibitor. I think I have it but, boy, I have no clue how it can be produced enzymatically. Oh well, most probably it will be wrong. Many times in the past I thought I had it only to find out that I was wrong.

So let us go to part II of targets to drugs.

I was talking about using the bacteria to produce protein. Sort of like a factory. The only problem is that the biological systems are entirely unpredictable. So when we put in a gene into the bacteria and ask it to make the protein, it entirely depends upon the bacteria. If it likes the gene and the protein, it will make tons of it. If it does not like it, it will not make it. Sometimes, it will make the protein but the protein might be inactive. There are numerous reasons for this. The bacterial proteins and the eukaryotic proteins differ in many respects. One aspect is what is known as post-translational modifications. Proteins are made of amino acids linked together by peptide bonds. After this is made, the cell can add little pieces of decoration like phosphorylation, glycosylation, ADP-ribosylation, sumoylation etc. etc. The bacterial cell hardly ever does this. However, all eukaryotic cells do this bit of extra decoration. Sometimes these decorations are essential for function, and at other times they are not. The only way to know what is essential and what is not essential is to do the experiment. So one takes the gene and puts it into bacteria and ask whether the protein is produced or not. And whether the protein is active or not. And whether the protein can be crystallized or not.

When I was working for a pharmaceutical company, I came across such a case. The protein could be produced in bacteria but could not be crystallized. We had to move to another system to produce the proteins.

Bacteria are the simplest and the easiest. But if the bacteria does not work, we move through the chain- yeast, baculovirus, and mammalian cells. Each one is tougher and more expensive than the previous one- Mammalian cells are the most expensive, and require lots of care, and really not worth it unless one has no options.

Once we have figured what which of the system works the best for us in terms of protein production and activity, we have to move to the next thing.

Discovery of small molecule inhibitors for the protein target can be done in two ways. One can use them for high throughput screening or one can crystallize the protein and do in silico protein-drug modeling.

All proteins are not really amenable for high throughput screening. Basically in this type of screening, you have a protein whose function can be estimated easily. For example, I work with an enzyme that hydrolyzes ATP, the energy currency in a cell. When ATP breaksdown it releases inorganic phosphate which can be estimated by a method known as Fiske-Subbarao method. Therefore, if you have a protein that can be purified in large amounts and its function can be easily estimated then you can put it through a high throughput screening. In such a screening, you test a variety of chemical compounds and see which compound inhibits the activity of the protein. When you find such a compound, you have a hit.

Instead of doing this laboratory thing, you can do in silico method too. For this you need to solve the structure of the protein. This is another bottleneck. As a colleague of mine put it succinctly, proteins do not like to be crystallized. They were not evolved to do that. Therefore, you have to find conditions and coax the protein to crystallize. This can take time. There are some proteins that you cannot crystallize at all. But assuming you can crystallize and have the structure, you can use in silico programs to model small molecule-protein interactions and see which one of them will bind to the protein. When you find such a molecule, you ask the chemists to synthesize it and test it. The problem with in silico modeling is that you are trying to mimic a 3-D molecule in 2-D space. Does not work! There was much hype about this but slowly the realization has sunk in that the protein in space can not be mimicked on a computer screen.

Assuming everything works and we have a hit, we have to move through steps to make sure that the hit can indeed be developed as a drug molecule.

Next lecture!

From Targets to Drugs-Part I

It takes anywhere from 20 to 30 years of intense research to get a drug to the market.

This is a long discourse on biological principles and I will try to make it as simple as possible.

There are two types of cells on this planet: bacterial and eukaryotic. The eukaryotic cells encompass protozoan cells, mammalian cells, yeast cells, and plant cells.

Though there are lots of differences between the various kinds of cells, most of the biological processes are common. For example, the way glucose is utilized inside a cell to generate energy is more or less uniform across the vast span of different cells. What biologists try to do is to exploit subtle differences between various organisms. A case point is an enzyme called thymidine kinase. It is made both by human cells and herpes virus cells. The enzyme performs the same catalytic reaction. However, there is a difference in the structure of the two enzymes and this could be neatly exploited to generate drugs that will inhibit only Herpes virus Thymidine kinase but not the mammalian cells.

This is the Holy Grail that biologists search for in their life times while trying to discover drugs. Small subtle differences that can be used as drug target.

Identification of drug target involves understanding the biology of an organism. It means that we have to study the proteins that are involved in doing the biochemical reaction. This is the first bottleneck.

To understand a reaction, where do we start? With the advent of Genome sequencing, we know the sequences of many organisms. But all we really know is the way ATGC is organized. We do not know what it means. We do not know which of these particular organization of ATGC encodes for a protein. That is where computational biology comes into picture. We use computer programs to predict the coding sequences. Even if we assume we can accurately predict the protein encoding genes, we do not know what the protein does. Even for something that has been well studied like E.coli, there is a substantial genome data that we do not understand. For human sequences, we do not know the function of more than 80% of the proteins. For pathogenic organisms we know even less.

Therefore, our first goal becomes to understand the biology of an organism, to find our favourite protein and to study it.

Let me put this in a little bit perspective. The protein I work with was initially purified from calf thymus tissues. I would take 10 kg of calf thymus tissues and process it through various stages to get less than a microgram of pure protein. This amount is useless for anything but basic reactions.

What researchers do is to take the DNA encoding the protein into E.coli and ask the bacteria to produce it. As E.coli is easier to grow, theoretically we should be able to produce humongous amount. Ah! this is the second bottleneck.

As my E.coli cells are thawing and I have to start a protein preparation, I am going to stop here and continue with this exposition tomorrow.