The Discovery Of New Drugs

Published Date: 02 Nov 2017

Purdue University

GPCRs – A Platform for the Discovery of New Drugs and Human Diseases

Chintan Patel

ENGL 106

Mr. Paiz

01 April 2013

Introduction

G protein coupled receptors (GPCRs) are involved in virtually every physiological process you can think of, from sensing colors, flavors, and smells to the action of neurotransmitters and hormones (Marasani, Anil, Venu, & Jayapaul 370). GPCRs are considered one of the most important proteins in human genome because pharmaceutical industry has targeted them to a greater extent than any other protein class. Even though they represent less than 1% of the human genome, they are 50-60% of the current drug target (Ibegbu, Mullaney, Fyfe, & MacBean 43). GPCRs are the largest and most diverse group of membrane receptors in eukaryotes (Strasser, Andrea & Hans 5). These cell surface receptors act like an inbox for messages in the form of light energy, peptides, lipids, sugars, and proteins.

From a chemical standpoint, the GPCRs provide a superb example of a molecular machine whose subtle workings we have only started to understand. The drug design has been greatly hampered by the limited knowledge of high-resolution structures of GPCRs (Lundstrom 103). My hypothesis is that the limited technology and limited knowledge of GPCRs structure is halting new drug designs and that in turn is preventing from treating certain diseases. The more we know about GPCRs structure, the more structure-based drugs will be designed.

What do GPCRs look like?

GPCRs bind a tremendous variety of signaling molecules (ligand), yet they share a common architecture that has been conserved over the course of evolution. GPCRs consist of a single polypeptide that is folded into a globular shape and embedded in a cell's plasma membrane. Seven segments of this molecule span the entire width of the membrane — explaining why GPCRs are sometimes called seven-transmembrane receptors — and the intervening portions loop both inside and outside the cell (Kroeze, Douglas & Bryan 4867). The extracellular loops form part of the pockets at which signaling molecules (ligand) bind to the GPCR. http://www.nature.com/scitable/content/ne0000/ne0000/ne0000/ne0000/14673543/U4.cp2.1_nature01307-f1.2.jpg

What do GPCRs do?

As their name implies, GPCRs interact with G proteins in the plasma membrane. When an external signaling molecule (ligand) binds to a GPCR, it causes a conformational change in the GPCR. This change then triggers the interaction between the GPCR and a nearby G protein (Marasani, Anil, Venu, & Jayapaul 370).

G proteins are specialized proteins with the ability to bind the nucleotides guanosine triphosphate (GTP) and guanosine diphosphate (GDP). G proteins that associate with GPCRs are heterotrimeric, meaning they have three different subunits: an alpha subunit, a beta subunit, and a gamma subunit. A G protein alpha subunit binds either GTP or GDP depending on whether the protein is active (GTP) or inactive (GDP). In the absence of a signal, GDP attaches to the alpha subunit, and the entire G protein-GDP complex binds to a nearby GPCR and is inactive. However, when the signal (ligand) binds to the GPCRs, G protein is activated because GPCRs remove GDP from G protein and attaches GTP to it (Kroeze, Douglas & Bryan 4868).

Whenever G protein is active, they can relay messages in the cell by interacting with other membrane proteins involved in signal transduction. [1] G protein usually targets effector proteins [2] which produce second messengers [3] and those second messenger molecules lead to different responses in cell (Kolb, Peter and Klebe 11573).

The Problem & Focus

The Problem

GPCRs play a role in an incredible array of functions in the human body. Increased understanding of these receptors has greatly affected modern medicine. Unfortunately, it’s difficult to study the detailed structure of GPCRs because of the great difficulties in crystallizing [4] them (Attwooll). GPCRs are membrane bound proteins that span the cell membrane in the form of seven transmembrane helices which are connected by three loops; three on the intracellular side and three on the extracellular side.

Most importantly, GPCRs are membrane proteins, any attempt to take them out of the membrane would rapidly destroy their integrity; it’s like trying to study a delicate embryo by taking it out of the womb. This is the reason why GPCRs are notoriously hard to study. I also want to point out that technology limitation also plays a role. The methods used to study GPCRs are not effective. Due to the limited knowledge of the GPCRs structure, new drug design has been halted in recent years. In order to understand the functions of GPCRs, you first need to understand the structure since the structure determines the function (Kroeze, Douglas & Bryan 4867). Without the knowledge of most of the GPCRs structure, nothing can be done in preventing certain diseases such as halting cancer growth.

The Focus

This paper examines the importance of GPCRs structure and the impact of the limited knowledge of its structure on drug design. First it will begin with methodology section so others can replicate this process. It will then focus specifically on these areas:

The importance of GPCR functions in human health.

Their role for targeting certain diseases, such as halting cancer growth.

The impact of current discovery on drug design.

Major concern surrounding the continued development of GPCR-targeted therapies

Current assays (methods) used to arrive at the GPCR structure and its connection to the limited knowledge of GPCR structure.

This paper will explore the role of GPCRs, the current discovery, the methods used, and the drug design and connect these areas to the limited knowledge of GPCRs structure. This paper will provide reasons on why it’s important to know the structure of GPCRs. It will begin by highlighting some of the crucial roles played by GPCRs in certain diseases, such as cancer. It will briefly touch on the relation between cancer growth and GPCRs to reinforce the main themes: the importance of its structure and to prove that the researchers have limited knowledge of GPCRs structure. To reinforce the point of the GPCRs structure, this paper will describe the current discovery on the GPCRs structure and its impact on drug design. This discovery will prove how finding one structure of GPCRs can greatly influence drug design.

This paper will then switch gears and will mention the current assays (methods) that are used to find the structure of GPCRs. It will mention some reasons as to why researchers have limited knowledge of GPCRs structure in terms of technology or the assays (methods) used currently. This paper will clarify in the end on how everything discussed in this paper relates to GPCRs structure. This paper will then make a call for a change. It will conclude by providing limitations and directions for future research.

Methods

This section will report on the methods used to gather information for this report. This will include critical thinking behind when choosing appropriate sources for this report. In order to accomplish this, I began with a search for online articles. I began with a simple keyword search of glance at G protein coupled receptors in Google scholar. Out of 28,500 results, I decided to examine "G-protein-coupled receptors at a glance" article. After reading few paragraphs, I decided to keep it since it provided an overview on GPCRs. This article provided sufficient background information on GPCRs. To help me continue my research, I made nine categories based on my research questions: background, introduction to GPCRs, their functions, their importance, drug design and cancer, a new discovery of the structure, problems, future predictions and tools used to study GPCRs. My goal was to find each article that pertained to each category.

My first goal was to find the introductory information on GPCRs to see the big picture. I began my search in a Purdue library website. With nothing specific in my mind, I searched the keyword, "GPCRs," in the books & media search bar. From 41 results, I decided to closely examine, "Modeling of GPCRs a practical handbook." One of the main reasons I decided to choose this source was because the author spends few chapters just describing the GPCRs. This book provided a detailed introduction to GPCRs.

With no other books that related to my topic, I then began with my search for articles in Google scholar. To find a specific article, I began with a keyword, role of GPCR in cell signaling with quotation marks around it. I decided to examine the first and only article titled "Cell signaling: Role of GPCRs." After reading abstract, I decided to preserve it. This article listed some key words at the end of the first page. I used one of the key words Paracrine to find similar articles to make sure that the information presented in my article was accurate. In Google scholar search bar, I typed paracrine signaling and GPCR. From 5,010 results, I closely examined "G-protein-coupled receptors and signaling networks: emerging paradigms." From this article, I was able to confirm the information presented in my previous article.

To find the relation between GPCRs and cancer, I began with the phrase "G protein coupled receptors and cancer" in Google scholar and decided to examine the first article, "G-protein-coupled receptors and cancer." By reading this article, I was able to generate one category for my research and that was tools used for GPCRs research. This article repeatedly mentioned the phrase, "G-protein-dependent assay," so I typed this phrase in Google scholar and I received one result. I decided to examine "Tools for GPCR drug discovery" article. By examining the cancer article, I was able to find this article. From this, I thought that I should connect cancer and tools used for GPCRs research.

I was astonished that I was able to find tremendous information on GPCRs. However, one thing was missing. I did not find any concerns regarding GPCRs research. To accomplish this goal, I decided to search at Purdue library website. I began to search for articles only. In the articles search bar, I typed "the state of GPCR research." From 6629 results, I decided to examine the first article, "Twenty questions: the state of GPCR research in 2004," since it matched my phrase. However, the Purdue library did not have full text of this article. So I copied the article title and paste it in Google scholar. From there, I was able to accomplish my goal. This article was listed first out of 5,540 results.

To conclude my research, I decided to search for an article that summarized the problems and future predictions of GPCRs. I began to search for books, however I was unsuccessful. I then turned toward articles. To increase my chance, I began my search in Google search bar instead of Google scholar since I wanted every possibility that I could find. In Google search bar, I first began with the phrase "GPCR problems." From the results, I discovered about the Nobel Prize given to two biochemists for discovering the structure of GPCR. This gave me an idea, so I changed my phrase to "GPCR new discovery and any impact on drug design." From 239,000 results, I decided to closely examine, "The golden age of GPCR Structural Biology: Any impact on Drug Design?" I chose this article because the golden age means time of prosperity. I knew that this article will discuss about the new discovery. I was right, this article discussed about the new discovery, it mentioned about GPCR problems and future directions.

To further reinforce some of the points mentioned, I conducted an interview. I chose interview questions after I finished my research report because I wanted to know what I should ask and whether or not if I found enough information on certain areas. I based few of my questions on GPCRs problems and how to solve them since this was the core of my research. And other interview questions were based on GPCR roles, recent discovery on GPCRs structure and the methods used currently (see Appendix). I chose to interview biochemistry and biology professor since biochemistry professor would have more knowledge on GPCRs structure and biology professor would have more general knowledge. This combination would provide me with all the information that I need to know.

Results

Now that we have discussed the methodology section, this paper will now move on to reporting the results of the study. This section will answer research questions in order that they were presented in the introduction section.

Question 1: Is the function of GPCRs in cell membranes important in human health even though they represent less than 1% of genes in our genome?

The cells within the human body continually communicate with one another in order to fulfill their various tasks. For that purpose, they are equipped with sensors with which they receive signals from their environment. Sensors on cell surfaces are known as receptors. Numerous processes taking place within our body -- such as sight, smell or taste -- are performed by an important family of receptors known as G protein-coupled receptors (GPCR). G-protein-coupled receptors (GPCR) are involved in directly and indirectly controlling an extraordinary variety of physiological functions, (Schoneberg, Schulz, Biebermann, Hermsdorf, Rompler, & Sangkuhl 173).

As I mentioned earlier, the function of GPCR is to activate G protein, which in turn activated effector proteins which produces second messengers. Those second messengers lead to the different responses. Mutations in GPCR and/or G protein can cause many diseases, (Ibegbu, Mullaney, Fyfe, & MacBean 50). For example, mutation that alters G protein activation because of GPCR may cause disorders characterized by either insufficient or excessive transmission of signals, (Spiegel, Weinstein, & Shenker 1121).

Some of the most important roles played by GPCR are (Marasani, Anil, Venu, & Jayapaul 370):

Physiological Roles

Brief Descriptions

Regulation of immune system activity and inflammation

GPCR receptor bind to certain ligand which mediates intracellular communication between cells of immune system

Cell density sensing

A novel GPCR role in regulating cell density sensing

The sense of smell

Ligand binds to one type of GPCR known as olfactory (smell) receptor.

Autonomic Nervous system transmission

GPCR is responsible for controlling: blood pressure, heart rate and digestive processes.

The visual Sense

Electromagnetic radiation is converted into cellular signals via GPCR.

Behavioral and Mood Regulation

GPCR bind different neurotransmitters such as serotonin, dopamine and GABA.

Question 2: Have they been useful for targeting cancer cells and other diseases?

Malignant cells often hijack the normal physiological functions of GPCRs to survive, proliferate autonomously, evade the immune system, increase their blood supply, and disseminate to other organs (Dorsam, Robert & Gutkind 79). Many GPCRs are over expressed in various cancer types, and contribute to tumor cell growth when activated by circulating or locally produced ligands by cancer cells. Many GPCRs can only be used to stimulate responses. However, one GPCR functions as a metastasis suppressor, KISS1. The KISS1 metastasis suppressor gene has emerged as a promising molecular target for the management of metastatic disease, such as cancer (Nash, & Welch 647).

Question 3: Does the recent discovery of the structure of GPCR impacts drug design?

The new discovery showed the crystal structure of --- the beta 1-adrenergic receptor --- that does not have a chemical signal or a ligand bound to it. The researchers say the finding will likely to offer a major boost to drug development because designers can use information gleaned from the crystal structure to learn how to build new, more effective drugs.

"Now by understanding the native structure of these receptors- which are likely very similar to each other—drug designers may be able to create therapies that are exquisitely targeted says Dr. Xin-Yun Huang, a professor of physiology and biophysics at Weill Cornell Medical School (DISCOVERY OF THE ATOMIC STRUCTURE). In fact most crystal structures of the GPCRs come with drugs attached to them and therefore they are invaluable starting points for designing more potent drugs with better safety profiles. However, the new GPCR discovered has no drug (ligand) bound to it.

Question 4: What are current assays used to arrive at GPCRs structure?

The quest for new G protein coupled receptor targets is challenging the limits of today’s cell-based assays. There are many different assays that can be used to find certain GPCR functions as well as its structures. They are: Receptor Binding Assay, G protein dependent functional assay, and receptor dimerization assay. Here is the summary of each assay used: (Zhang & Xin 372-382)

Assays

Description

Receptor Binding Assay

This assay is used mostly to find the interaction between the GPCRs receptors and their ligands such as affinity of ligand to receptors. In this assay radioactive toxin is used as the ligand. And when that radio labeled toxin binds to the receptor, the products formed are trapped on filter. And this allows researchers to study products more closely.

G protein dependent functional assay

This assay is useful to identify new compounds that target GPCRs. One way to do this is to monitor cAMP [5] concentration inside the cell. The cAMP is regulated by GPCRs so if any molecule/ion affects GPCR then that can be seen because the cAMP concentration will change inside the cell.

Dimerization Assay

Dimerization is just interaction of two GPCRs receptors. GPCRs interact with each other at the plasma membrane to carry out certain responses that cannot be done by one GPCR. In this assay, researchers genetically make plasmids that attach to the GPCRs and that results in the dimerization of GPCRs and this will allow researchers to study the dimerization process.

Question5: What are the major concerns surrounding the continued development of GPCR-targeted therapies?

The number of GPCRs for which structural information is available has increased dramatically in recent years, providing valuable insights into ligand recognition and mechanisms of activation and structure-based drug discovery is now a reality for this family, (GPCR-Based Drug Design Conference). And there is still much to be learned about how GPCRs work and how they can be selectively modulated. Fortuitously, technologies designed specifically to tackle the GPCR challenge are blossoming (Filmore 25).

Many researchers are concerned about the structure of GPCRs and their impact on drug design. GPCR signaling is incredibly complex, and attempting to integrate this complexity into disease processes and the identification of useful drugs is a major hurdle (Ellis 619). Moreover,

based on predictions from the human genome, a large number (>200!) of GPCRs remain to be discovered and their functions characterized (Ellis 619). Faced with a plethora of potential therapeutic targets and the inability to decide which ones to target, some will wait until a particular GPCR has been assigned to particular function before launching a drug discovery programme. We need to develop specific concepts about the function of GPCRs first.

Interview

The information that was collected from both interviews matched. Both professors agreed that the current problem regarding GPCRs is that researchers have limited knowledge of GPCRs structure. And both also agreed that this was due to GPCRs location and limited technology. Biology professor went deep for the technology limitation and biochemistry professor talked more about the GPCRs location since it satisfies their knowledge. When it came to discussing whether roles or structure is important for determining GPCRs structure, there was disagreement. Both agreed that both roles and structure was important, however they took one side. Biology professor chose role as more important than structure and vice versa for biochemistry professor. "It will many years for researchers are able to find almost all the GPCR structures (Broyles interview).

Discussion (include interview here  especially for call for a change section)

With result section being completed, this paper will now move onto a discussion of what results might mean for the continued search for GPCRs structure.

GPCR roles

From the result section, we can see that the GPCRs play many roles. And this roles played by GPCR has made researchers astonished. This had interested researchers for over thirty years in search for their structures. Due to the limited knowledge of their structures, many roles have not been discovered yet. The roles listed in results section is only five percent of the total roles yet to be discovered. When one considers that the human genome expresses gene for between 800 and 1000 different GPCRs and marketed drug target less than 50 GPCRs, it is evident that the field of GPCR drug discovery and development is still growing (Gilchrist 191).

Understanding their roles will greatly influence drug design since most GPCRs share common roles. And understanding those common roles will greatly enhance researcher’s understanding of GPCR. Structure without function is a corpse and function without structure is a ghost. They are interconnected so as a result limited knowledge of structure is preventing researchers from gaining GPCRs roles. The future of drug development relies heavily on the understanding how these GPCRs work. Since a protein’s function is dependent on its structure. Without knowing what roles GPCRs play, companies can’t design drug because drugs target each GPCR based on the role that GPCR play.

GPCRs assays and recent discovery

Abnormal function, expression, and regulation of G-protein-coupled receptors (GPCRs) have been implicated in many diseases and, as a result, GPCRs represent one of the largest receptor classes targeted by drug discovery programs. Most GPCR-modulating drugs on the market weren’t initially targeted to a specific protein but were developed on the basis of functional activity observed in an assay. Historically, ligand-binding assays were used to identity compounds that target GPCRs. While these assays measure a compound’s affinity to a receptor, they cannot measure its activity, are not amenable to high throughout discovery, and some use radioactivity, which is difficult and expensive to use on a large scale.

Today researchers use assays that monitor the accumulation of a second messenger such as cAMP (G protein dependent functional assay) instead of problematic ligand binding assay. Even these current methods have many limitations. One limitation that this assay holds is that it’s not very sensitive to cells that express low levels of GPCRs (Zhang & Xin 376). Due to this, researchers cannot detect GPCRs activation in certain cells. GPCRs are the single largest target of current drug discovery programs yet, of the approximately 200 GPCR with known functions, only a very small fraction of them are currently utilized in drug discovery due to technology limitations, leaving many of them as putative new targets for the development of new therapeutics (Diwu 1). This assays discussed can be helpful to find GPCRs structures, however they are not good enough.

The recent discovery by two biochemists proves the importance of GPCRs structures. Even one discovery is likely to boost the drug design. Their research on proteins central to cell communication has aided the discovery of many pharmaceuticals and may open up ways to design more-selective drugs (Noorden). This discovery also shows that the technology is improving which can open up many opportunities in future. This discovery also proves that there is more to know about GPCRs which brings us back to the limited knowledge of their structure.

GPCRs role in cancer growth

Interfering with GPCRs might provide unique opportunities for cancer prevention and treatment (Dorsam, Robert & Gutkind 79). Overexpression of GPCR is one path that cancer cells take to proliferate their growth. And most GPCR known right now are stimulators meaning they only stimulate processes. There is only one GPCR that is able to under stimulate the process and that is KISS1. Only this GPCR is interest to researchers when it comes to halting cancer growth. Increasing the function of KISS1 can greatly suppress the growth of cancer.

However, not much is known about this GPCR and not only that, researchers don’t know if other GPCR exist just like KISS1 that can suppress metastasis (Dorsam, Robert & Gutkind 89). The discovery of one of the GPCR: CXCR1 which is involved in cancer growth has astonished researchers. Targeting CXCR1 can also greatly reduce cancer cells (Singh, Farnie, Bundred, Simões, Shergill, Landberg, & Clarke 644). However, researchers don’t know everything about this GPCR just like KISS1. This goes back to designing drugs to target cancer cells. Because of the limited knowledge of this GPCRs structure, researchers can’t target cancer cells and this can be due to technology limitation.

Call for a change

Researcher’s goal right now should be to improve assays or invent new assays for GPCR detection instead of spending time on old assays. "They should also find a way to take out GPCRs out of the membrane to study them (Bos interview). They should develop assay for GPCR arrays. Limited knowledge of the structure of GPCR and halting of drug design is all due to the limitation of technology. Things can be done to improve this by just improving the assays. When I discussed about the cancer growth, this proved that the researchers have minimal knowledge of GPCRs structure since they cannot halt the cancer growth. I believe that the new technology will not only support drug discovery, but also allow researchers to examine receptor activation and signal transduction in a new way previously not possible. Drugs can only be designed if structure is known because drug act as ligand and can only bind to specific site on GPCRs and this can only work if we know the whole structure of GPCR.

Limitations and &Future Directions.

This paper will now switch gears and will discuss about the limitations and the future directions. Limitations

This study is subjected to limitations. Due to time limit, this report was more condensed. It may be beneficial to do this kind of research if ample amount of time is given. Since I can’t discuss one area without discussing the other areas, this report could have been longer.

Another limitation that this report holds is that it’s hard to perform an experiment to validate the information since it requires intricate technology. This kind of experiment cannot be done without expensive equipments.

My interview also had some limitations. Two professors that I interviewed graduated twenty years ago from college and at that time, none of the GPCRs were discovered. This means that this issue is current. Because of that, professors don’t have enough knowledge of GPCRs yet because I know that I asked other professors and they didn’t know much detail about these proteins. In other words, people who are specifically focusing on these proteins would have enough knowledge about them.

Future Directions

Since the 1990s, advances in the structural and biochemical characterization of GPCRs have led to a better understanding of these signaling molecules (Granier, Sébastien & Brian 673). However, the application of structural approaches to developing safer, more effective drugs for these important clinical targets is just beginning. The development of several technologies in future will allow elucidation of structure of virtually any GPCR encoded in the human genome. The more assays will be developed, the more we will know about GPCRs structures and more drugs will be design which in turn will help treat certain diseases such as cancer growth. Not only that, researchers hope that they will find a way to take those proteins out of the membrane to find structure easily, this is not impossible to do.