Methods for drug discovery

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Forward approach and Reverse approach are the two widely used methods lying under the drug-discovery process. The final steps (testing) for both are similar, the only difference is that Reverse Approach establishes the target site for the drug to attack first while the Forward Approach follows a hit and trial method for finding the exact target before actually testing the drug.

The Reverse or Target-Based Approach

This method is like having a complete blueprint before you commence the plan: you know where to fire and what path to take. Target based pharmacology has been emerging lately due to drastic developments in the way we understand chemicals and how they react with each other and with our body. This process has been accelerated by the genomic revolution. The sequencing of the human genome coupled with advances in automation and parallelization technologies have afforded a fundamental transformation in the drug target discovery paradigm.

There are many places in the human body where drugs can bind but the most common ones are proteins, and more specifically, the G-Protein Coupled Receptors. But why do most drugs treat diseases by targeting the G-proteins in the first place?

https://youtu.be/WORIhbaRABg

Different types of GPCRs are involved in different processes. All these processes are regular to most diseases and functioning of the body:  vision, taste, immune system regulation, behavioral changes, water balancing, smell, and countless others. This makes GPCRs the target site for many treatment-drugs.

High Throughput Screening – To Speed Things Up

HTS is used to both find target sites and test the drugs on target sites.

https://youtu.be/WRSJhZYq7UM

It runs more than a million tests in a go, and requires four elements:
(1) suitably arrayed compound libraries;
(2) an assay method configured for automation;
(3) a robotics workstation; and
(4) a computerized system for handling the data. (See here)

When the chemical library to be used for testing on the assays is resolved, the assays, chemicals, and the algorithms to be used for the analysis are all integrated at one place.

In the HTS:

  • The cells and tissues to be tested are filled in small plates with around 96 well-like holes.
  • A chemical library is then selected out of thousands of chemical classes which is assumed to work for the related disease. (We’ll discuss how in a bit.)
  • The different chemicals in the library are added to the cells and tissues in the wells in a planned pattern and many times more than one compound is added to a single test cell to check the interaction between different compounds more accurately and see if they yield better results or how do they affect each other.
  • Intermittent incubation between almost every step is needed to make sure the samples get enough time to show results.

How are the results determined?

In HTS methods, all this is done using automated machines, with the sole purpose to save time, decrease error and help with cures for diseases requiring quick action.  

https://youtu.be/ra0e97Wiqds

Computer-Aided Drug Design aka Virtual HTS

The genomic revolution has made it possible to group similarly behaving gene classes and other cells in a computer database. Now, whenever a new drug development plan is made it can be tested by running millions of virtual assays using processes like docking and structural analysis, in which structures of various chemicals are made and they are put (docked) on the targeted gene sites (virtually on a computer) to notice how they interact. Saving us from running all those tests for real, it saves a lot of time and money, and helps avoiding repetition during more complex discoveries.

https://youtu.be/xGRdOEWE9QE

The Forward Approach – Starting Without Prior Target Knowledge

Choosing a chemical library

A chemical library is used in HTS methods where various chemicals are kept classified for their similar structures or effects but differing by intricate traits (yet not so intricate that they can’t be grouped together). These various groups are selected in different combos depending upon the type of disease under research.

Given a thought it seems very difficult as to what compounds should be preferred for testing if we don’t know the target first. Not really difficult, this can rather be called more time-consuming (which can actually be future-productive, we’ll tell you why).

Mostly, compounds bind with the chemicals in the body and show effects. But under phenotypic screening, we are actually running tests to look for variations in the phenotypes (physical expressions of genes) of specific cells (chosen arbitrarily). When we are dealing with such changes, it is less known if the compounds will react or not, and if some do, they are further verified for the desired result and the target is reached using target deconvolution. As compared to which, there’s more surety of how the reaction will proceed if a chemical-chemical interaction is undertaken from the starting, i.e. we already know the target.

Yet Phenotypic Screening is better for the long run because it involves testing a broader spectrum of compounds over the same types of cells: This gives us cues about how various chemicals interact with the body to a still higher extent whenever unexpected reactions are encountered. This makes this process best for research purposes and updating the existing chemical and drug libraries.

The whole process for drug discovery works on the principle of Reductionism. As the understanding of small molecule interactions keeps broadening the target sites become more precise and the assays to be performed can be reduced to look for more specific results.

Apparently, one method of discovery supports the other. With every research done for a new disease, we always end up with an updated database which can be utilized by both the methods.

Penicillin – Discovery By Accident

https://www.youtube.com/watch?v=0ZWjzcsTd5M

In the era where Antibiotics failed to cure the very thing they swore to destroy, penicillin still stands unravaged by the evolution of the bacteria. The time Fleming was studying over various cultures to look for an antibiotic, there was no means to use high throughput methods for screening numerous molds for one successful antidote. So, our first encounter with Penicillin, the first ever antibiotic, was by chance. 

Isolation

For the first time, when Fleming noticed the invasive fungus (which he later called Penicillin) he tried to observe it by isolating it from the culture. This was very unstable as the techniques he used were preliminary and not very reliable. Yet the instability left him with enough time to observe the antibacterial effects that the fungus had on various bacteria.

Penicillin is a secondary metabolite of certain species of Penicillium and is produced when the growth of the fungus is inhibited by stress. It is not produced during active growth. (Wikipedia)

The first major isolation and mass production of Penicillin was done by Howard Florey and Ernst Chain using deep-tank fermentation.

Penicillin trials have been almost successful since the early times. Till date, different classes are purified further to increase the efficacy of the drug.

Discovering Diuril – A Case Study

https://www.youtube.com/watch?v=Ab9OZsDECZw

Diuril was a drug whose path of discovery was well-planned but the results diverged significantly from the expectations. Diuril is the brand name for Chlorothiazide. Its discovery was led by a search for a treatment for edema; it was seen that both edema and hypertension can be handled in the same way, by decreasing the fluid volume of the body.

Now, specifically for hypertension, this could be done in two ways. Either by decreasing the blood volume or somehow the fluid volume. Reduction of fluids was clearly preferred over that of blood. The one part of the body which controls the fluid excretion is the kidney. So, the target was almost clear. Initially, when the chemical libraries were being chosen to run the assays, Sulphonamide chemistry was picked up as the basis just to start somewhere.

Apparently, the compounds prepared from this research were acting as Carbonic Anhydrase Inhibitors (CAIs). The Carbonic Anhydrase enzyme acts as a catalyst in ionization of carbon dioxide to bicarbonate ions in the body. The more the ions, the more is the blood pressure. CAIs reduced this enzyme’s activity in the body and therefore it was observed that it reduced the number of ions produced by its action as well. However, the trials suggested that the ion reduction rate in the body fluid was not enough because the carbonic anhydrase itself was not present in the body fluids in such high concentrations, that the reduction of ions caused by its blocking could suffice for the required results.

Finding Better Compounds

It was also seen that the “removal of sodium and chloride ions” only, lowered b.p. in a better way than inhibiting the Carbonic Anhydrase enzyme. It directly reduced the volume of vessel-fluid, and the process was faster. The compounds under research for better diuretics were thiazides contributing to the chemical class which was to be used here. Upon more research one such compound called Chlorothiazide was synthesized in the process. It was expected that this compound as well would only work as a CAI. However, it portrayed impressive removal of sodium and chloride ions without inhibiting Carbonic Anhydrase enzyme in noticeable amounts.

Clinical Trials and Production

Chlorothiazide became the ideal antihypertensive drug. Reason being, it had a fast rate of action the moment it encountered any abnormal rise in the salt content. It also adjusted its working as the concentration approached normal. Plus point was that its side effects were not severe and could be neglected. The first trials were conducted on dogs as well as rats, and then on hypertensive humans. The tolerance levels observed were satisfactory making it viable for sending to the markets.

Reference: bpspubs.onlinelibrary.wiley.com

How Are New Medicines Made? – Drug Discovery
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