Therapeutic Drugs: A History of Discovery, A Future of Design

Zach Jarou PSL 480 Fall 2007 Michigan State University

Introduction By their most basic definition, drugs are exogenous chemical compounds that modify the way the body and mind work by acting upon biological targets. Throughout history and today, many different groups of people have used drugs for many different purposes, including medical, recreational and spiritual. My interest in this topic is due to the culmination of several different factors. I have always been fascinated by the idea that our physical and mental states could be reduced to a network of complicated, self-regulating chemical reactions within our bodies. As a child, I had no idea about the complexities of this system could function, but my experiences here at Michigan State have done wonders in explaining the details; to take a reference from Dr. Steve Heidemann of the Physiology Department, each of the trillion cells that make up our bodies are nothing more than miniature Rube Goldberg machines. By understanding the functions of different biochemical pathways present in each cell and the mechanisms by which they are regulated, researchers are able to attribute failures in these pathways to certain disease states. To date, researchers have been very successful in synthesizing various chemical compounds that can be administered to patients to correct deficiencies in these systems. Unfortunately, most of this research has been targeted towards

diseases that more commonly afflict people in Western cultures due to their ability to pay for such products. Worldwide, there are still many devastating diseases for which no cures exist either because the prevalence is considered too small to warrant the costly research required or their inabilities to pay for treatment if one was created. During my time at MSU, I have been lucky to have numerous opportunities to get involved with and be mentored by several talented faculty members. Dr. Donna Koslowsky, of the Microbiology and Molecular Genetics department, served as my Cell and Molecular Biology professor in the spring of 2007 and from my achievements in the course, encouraged me to apply for the National Science Foundation’s Undergraduate Research Fellowship in Mathematical and Biological Sciences. I was subsequently accepted and began working in Dr. Maria Zavodszky’s computational biology lab. Our lab is focused on correctly predicting the orientation that ligands, or other small molecules, take when they dock into the active or regulatory sites of their target proteins. The final reason for my interest would be my interest in pursing a career in the pharmaceutical industry, which is one of the largest and most profitable in the world.

The History of Drug Discovery Throughout history, drugs have played important roles in the

lives of the human race. As early as 10 million years ago, the accidental discovery of plants with healing powers probably occurred by our earliest nomadic ancestors. It was not until the formation of agricultural communities, 20,000 years ago, that individuals began to specialize in role of healing; ambitious leaders emerged from these communities and their authority often came from their ability to heal the sick. These early medicine men created a variety of medicines containing mainly botanical specimens but also could contain animal parts, which deepened their mysterious powers. Guided only by vague symptoms and no knowledge of anatomy or physiology, once a certain treatment was found to have an effect, it was often used for a variety of ailments. As time went on, valid explanations were sought for particular treatments; the first of these pharmacopoeias was compiled in 2735 BC by the Chinese scholar-emperor Shen Nung (Burger 1995). In the 4th century BC, Hippocrates of Greece, the “Father of Medicine,” became interested in using inorganic salts, rather than botanicals, as medications. His authority lasted through the Middle Ages and while his famous oath may have survived better than his preferences for inorganic salts, they are still used today under certain circumstances (Burger 1995). The next revolution in medicinal thought occurred in 2nd century AD Rome, when Galen prepared medicine by soaking or boiling plants. His insistence of carefully identifying the type and age of plants was

one of the earliest examples of controlling drug purity. His teaching had a lasting impact, as the herb gardens of medieval monasteries contained the necessities for his prescriptions (Burger 1995). Today, more than 200 major pharmaceutical firms, comprising a $602 billion industry as of 2006 and expected to reach $1.3 trillion by 2020, perform the majority of drug discovery. These large multinational corporations are often vertically integrated to discover, develop, manufacture, market, sell and distribute new drugs to treat human ailments. Annually, the FDA approves about only 25 truly novel drugs, along with several reformulations. As of 2003, each new drug costs about $1.7 billion to develop; to encourage drug companies to pursue this research, patents are granted to protect the drug discovering company’s exclusivity rights for 20 years to ensure that they are able to recoup their huge investments before other firms are able to introduce lower priced generic substitutes (DiMasi 2003).

From Mythology to Molecular Biology While these early pharmacists may have been somewhat successful in accurately prescribing the proper medication to treat a patient’s ailment, they were practically clueless about the mechanism of its action to restore health. The supernatural explanations for healing held by the earliest shamans and vitalistic separation of the organic life processes from the inorganic matter first suggested by

Aristotle have been replaced. Improved understanding of individual organ function and Pastuer’s 16th century germ theory of disease reduced the allusion to mystical forces, but it was not until 1828 when Wohler synthesized urea from inorganic components that vitalism was rejected by the majority of scientists. In 1674, Anton Van Leeuwenhoek became the first person to view a living cell using microscopy and by the mid-1800s the classical cell theory was introduced. But, the question still plaguing the minds of many biologists was how tiny structures visible under their microscopes were linked to the molecules being characterized by physiological chemists. The attempt to explain the phenomena of life starting on the molecular level first began with the convergence of several disciplines in the early 20th century to form a new field known as molecular biology. The results of these early experiments can best be summarized by Crick’s “Central Dogma” which states that genetic information coded in DNA is carried out of the nucleus by RNA to a ribosome where the code corresponds to amino acids required to create a specific proteins, which are the “molecular machines” of the cell.

Mechanisms of Drug Action Recognizing the hierarchical organization of the human body is

important to understanding the mechanisms by which drugs work. Each of our bodies is comprised of nearly 1.5 trillion cells, which are specialized into nearly 200 different types to form different tissues found in the various organs of our bodies. These organs perform individual functions within larger, interconnected organ systems that support the continued survival of the organism. Thus, changes at the cellular level are reflected upwards through the chain. In order to cause a change in cellular physiology, a drug must interact with a biological target, such as a receptor, enzyme, or other protein. Receptors come in many different varieties based on their location within the cell and their mechanism of action. Because the size and chemical nature of ligands determines their ability to move through the selectively permeable membrane of the cell, transmembrane receptors are located within the plasma membrane and serve to conduct the signals of hydrophilic ligands into the cell, while lipophilic signals are able to pass directly through the membrane to interact with intracellular receptors. There are two main classes of transmembrane receptors, including those that are metabotropic, which activated second messenger systems within the cell via a series of intracellular events, and ionotropic receptors, which directly open channels that allows the flow of ions down their respective concentration gradients. Intracellular receptors often act as transcription factors, which bind to specific DNA sequences to regulate

the activity of RNA polymerase. Drugs that bind to receptors can either be agonists, which activate the activity of the receptor, or antagonists, which bind to but do not activate the receptor, limiting the ability of agonists to bind. The other major class of biological targets, enzymes, are proteins that act to catalyze chemical reactions within the cell. Enzymes are extremely selective for their substrates and the products of one enzyme typically become the substrates for others to form metabolic pathways. Enzymes catalysis occurs in a region referred to as the active site, where the activation every of the reaction is lowered through the formation of multiple non-covalent interactions with the ligand. It is important to note that enzymes only increase the rate of the reaction, but do not alter its equilibrium. Other than the active site, enzymes may also have binding sites that allosterically or otherwise regulate the activity of the enzyme; this is important to efficiently allocate energy and required compounds within the cell. The majority of drugs used on enzyme targets work through reversible inhibition, which can competitively compete with biological substrate to bind into the active site or uncompetitively binding to an allosteric regulatory site. A few inhibitors are irreversible and react with the enzyme to form a covalent linkage between the pair.

Modern/Future Drug Discovery/Design Methods

In recent history, drugs have traditionally been discovered using a variety of different methods, including identifying and isolating active ingredients from traditional remedies, serendipity or by trial-and-error testing on animals. As scientists’ understanding of biological systems has advanced, accompanied by advances in automation and computational technologies, the methods used to identify potentially therapeutic drug compounds has, and continues to, increase in complexity. High-throughput screening combined with combinatorial chemistry and in silico methods are greatly increasing the rates at which novel drugs can be brought to market. Traditional methods based on bioprospecting have been highly successful by both using traditional and indigenous biomedical knowledge, and also by searching for previously unknown compounds in organisms. A 2007 estimates that between 1981 and 2006, up to 63% new chemical entities were derived from plants or other natural sources. Overall, this figure reaches as high as 75% including compounds such as morphine from opium and digoxin from digitalis (Newman 2007). Many notable drugs have also been discovered by accident, including penicillin, LSD and viagara. It is also interesting to note that research carried out at Michigan State led to the accidental discovery of the anti-cancer drug cis-platin. In the late 20th century, the use of synthetic compounds became increasingly favored as drug candidates. High-throughput screening is

a drug discovery method involving the usage robotic automation to rapidly observe the effect that millions of compounds on a particular biological target. Currently, there are HTS robots that can test up to 100,000 compounds per day. It is very unlikely that any of the compounds screened will act as a perfect drug, but there will instead be many compounds that cause differing degrees of response. By examining the similarities between these compounds, scientists are able to determine which the spatial arrangement of chemical interactions that are necessary between the target and ligand to alter its activity, known as a pharmacophore (Hann 2004). In order to increase the interaction specificity, the pharmacophore serves then as a lead compound whose structure is modified in various ways to create several new molecules with activities that must be determined by another round of highthroughput screening. A process known as combinatorial chemistry can also automate the generation of these compounds. This process can continually be repeated for several iterations and in a brute-force manner in which the exploration of chemical space function can be thought as the equivalent of picking a lock. Although experimental high-throughput screening may eventually generate good lead compounds, the process is very time and money intensive and is in essence based on nothing more than sophisticated trial and error. Newer computational techniques merging

biological and computer sciences have developed a technique known as virtual or in silico high-throughput screening; rather than discovering drugs, they can be rationally designed based on the structure of the desired target obtained from x-ray crystallography. By assigning discrete coordinates to each atom of the protein, it can be 3dimensionally visualized on a computer. During the overall process, several possible docked conformations are generated for each ligandprotein complex that must be subsequently to indicate their correlation to which most likely to occur in vivo. As with any type of computation there is a trade-off between time and accuracy, therefore research is currently being done to investigate which chemical models and scoring criteria are most efficient in generating good drug leads. From my research in the Zavodszky Lab, I have had the opportunity to work with a docking program known as SLIDE that was developed at MSU by Dr. Leslie Kuhn’s Protein Structural Analysis & Design Lab. SLIDE, which is an acronym for “Screen for Ligands by Induced-fit Docking, Efficiently,” operates on a fairly simple chemical model. First, the space containing the active site is permeated with a grid, to which specific favorable interaction types can be made with the protein are assigned. Then, the ligand, which is also represented by several interaction centers, first binds a rigid base fragment by matching triplets of these points onto complementary points of the protein; the flexible side-chains are then added in a way that avoid

steric clashes between the two. Finally, each complex is scored by the predicted free energy as a result of their hydrophobic and hydrogen bond interactions. Although x-ray crystallography has been successfully used to characterize many targets, the structures of several proteins still remain unknown. Protein folding, a process to predict structure given an amino acid sequence, is another area of research that if elucidated could have important implications to virtual screening. Since the completion of the human genome project, scientists have been scrambling to annotate the sequence into the individual genes responsible for coding particular proteins. Aside from possibly filling in the gaps left by x-ray crystallography, consideration of a patient’s individual genetic make up could allow for the development of increasingly sophisticated medications based upon a especially as the prices for personal genome sequencing continues to significantly decrease (Smith 2003).

Program: MMKIN (Michaelis-Menten Kinetics) As previously described, the mechanism of some drugs is to alter

V0 =

Vmax [S] K M + [S]

the rate at which enzymes are able to perform catalysis. In situations where the concentration of non-allosteric enzyme is much lower than its substrate, the kinetics can be modeled by the Michaelis-Menten equation: Where Vmax is equal to the maximum rate of catalysis that can occur due to saturation of the enzyme, therefore increased substrates will have no free active sites to interact with and KM is equal to the substrate concentration related to the rate ½ Vmax. Competitive inhibitors block the substrate at the active site and thus can be overcome by increasing the substrate concentration. Noncompetitive inhibitors act upon the enzyme-substrate complex and thus the rate will never reach Vmax. The HP50g program I have designed will calculate the initial rate, V0, for the enzyme and substrate alone, as well as under both types of inhibition.

MMKIN Flow Diagram

MMKIN Program Listing BEGIN << 2 FIX 1CF 2 CF TN "Km?" ":M:" I 'KM' STO TN "Vmax?" ":rxns/sec:" I 'VM' STO TN "[S]?" ":M:" I 'CS' STO TN CLLCD "Inhibition? 1. None 2. Competitive 3. Non-competitive” DUP 1. DISP 0. WAIT 'C' STO IF C 92.1 SAME THEN ‘(VM*CS)/(KM+CS)’  NUM ‘ANS’ STO ELSE IF C 93.1 SAME THEN 1 SF INHIB ELSE IF C 94.1 SAME THEN 2 SF INHIB END END END TN CLLCD "Initial Velocity, Vo =" 2 DISP ANS 3 DISP “rxns/sec” 4 DISP 3 WAIT { KM VM CS CI KI ANS } IF C 92.1 SAME THEN MMKIN ELSE CLEAR END >> I << INPUT OBJ>> TN << 800. 2.E-1 BEEP>> INHIB "[I]?" ":M:" I 'CI' STO TN "Ki?" ":M:" I 'KI' STO TN IF 1 FS? THEN ‘(VM*CS)/((1+(CI/KI))*KM+CS)’ NUM ‘ANS’ STO ELSE ‘(VM*CS)/(KM+(1+(CI/KI))*CS)’ NUM ‘ANS’ STO PURGE CLLCD "Again? 1. Yes 2. No -key 1 or 2-" DUP 1. DISP 0. WAIT 'C' STO

References

Burger, A. (1995). A Brief History of Drugs in Understanding Medications: What the Label Doesn't Tell You. American Chemical Society Publication. DiMasi, J. et al. (2003). The price of innovation: new estimates of drug development costs. Journal of Health Economics, 22(2): 151-185. Hann, M. and Oprea, T. (2004). Pursuing the leadlikeness concept in pharmaceutical research. Current Opinion in Chemical Biology. 8, 255-263. Newman, D. and Cragg, G. (2007). Natural products as drug over the past 25 years. Journal of Natural Products, 70(3): 461-477. Smith. C. (2003). Drug target validation: Hitting the target. Nature, 422: 341-7.