MULTIMEDIA - Disease Targets
Understanding disease at the level of individual molecules

Introduction and Overview

Interactions between proteins are at the root of all diseases, from bacterial infections to Alzheimer's disease to cancer. Understanding how proteins function and malfunction is the essential first step toward the design of drug therapies.

It’s easy to think of drugs as tiny encapsulated miracle workers.  From something as common as an anti-inflammatory such as Aspirin, to medications that help people cope with severe clinical depression, to the different chemical treatments for malaria, we depend on the pills in the bottle to fight the good fight against foreign invaders, deficiencies, and malfunctions within our bodies.  The term “drug” encompasses any substance used for the diagnosis, treatment, cure or prevention of disease.  Drugs are molecules that affect biological processes.  Their pharmacological activity can be correlated to their structure.  Therefore, drug development is a highly complicated, multi-step process that makes use of a wide array of chemical techniques and knowledge.   In the case of infectious diseases, the most cutting-edge drug development uses techniques that focus on the mechanism of the disease-causing agent, or pathogen—that is, the specific actions it takes to harm the body. 

Credit: Jason Miller, UCSD

Graphic shows the molecular structure of a few of the trillions of variants of a protein (colors reveal different amino acids) used by a type of virus called a phage.  Phage use these proteins to tether themselves to the bacteria they infect. 
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The most common disease-causing agents are viruses and bacteria.  Bacteria are living organisms, only some of which are harmful.  Some, like the normal inhabitants of our intestines, are vital to our health.  Viruses are clusters of genetic material with a protective protein shell.  Many are harmful, but some, such as the phage that infect bacteria, can be beneficial.  Because viruses require the machinery of the cell they infect in order to reproduce, scientists do not consider them to be living organisms.

Currently, symptoms of viral infections can be treated, but drugs available to combat the viruses themselves are very limited.  Bacteria, on the other hand, can be killed using antibiotics.  The downside of treatment through antibiotics is that many bacteria have mutated into drug-resistant strains.  For example, multidrug-resistant tuberculosis is an emerging public health threat.   To develop new drugs for viral and bacterial infections, researchers are investigating the mechanisms through which pathogens infect their host organisms. The objective is to understand the pathogen/target cell interaction, and then prevent the interaction from taking place by strengthening the body’s immunological defenses, or interfering with a process that is used by pathogens but not the cells of the body. 

Understanding the specific ways in which pathogens enter and then harm the healthy cells of their host is vital to designing effective drug treatments.  Viruses and bacteria must disguise themselves from their host’s immune defense system, often attaching to and multiplying within the same immune cells that normally destroy the invasive pathogens. The disease agents significantly alter the biochemical machinery of the host cell, either through interactions between the pathogen and the proteins making up the host cell’s structural and transport network—the  cytoskeleton, injection of harmful proteins through a needle-like structure on the pathogen, or even manipulation of typical immune defenses.  For example, certain immune cells engulf invading pathogens and self-destruct.  Some pathogens, however, manipulate the process so that the infected cell survives as a host to the disease agents.

Credit: Rebecca Phillips, UCSD Image of a mammalian cell (nucleus in blue) containing a bacterial toxin (green) that clusters in spots at the cell membrane.  More information >>

After the pathogens infect the host’s healthy cells, they further alter the normal biological processes within the body.  In order to bud off and reproduce, pathogens can demand more nutrients than normal cells. They can also trigger a healthy cell to slowly shut down and die. 

Pathogens introduce their genetic material into a host through a variety of mechanisms.  Likewise, the effect of the pathogen—the extent of the damage it can do—is host-specific.  In order to prevent the harmful effects of these pathogens, scientists must understand the cause of the disease at the level of its protein components.  In its most basic form, the biological interface between a pathogen and a cell it infects is merely a series of chemical reactions that involve interactions between proteins. 

Interactions between proteins are also critical in non-infectious diseases, such as Alzheimer’s, hemophilia, heart disease, arthritis and cancer.  For example, in arthritis, enzymes—protein catalysts—called matrix metalloproteases or MMPs are overactive.  MMPs have a zinc ion at their active site.  Because the zinc ion is essential for the MMPs to perform their catalytic function, blocking the zinc is a strategy for the development of arthritis treatments.

Credit: Seth Cohen, UCSD

Graphic showing inhibitor binding to metal in enzyme

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Given the central role of protein interactions in infectious and non-infectious diseases, the first step in drug development is to understand the structure of these proteins. Scientists use a combination of several techniques to do this—nuclear magnetic resonance (NMR),  x-ray crystallography and mass spectrometry.  NMR works by placing the protein within a magnetic field.  Once in the field, the magnetic atomic nuclei will “wobble” or resonate back and forth between a stable and unstable condition.  The frequency of radio waves required to achieve this state of nuclear magnetic resonance varies with the structure of the molecule.  This means that a series of frequency readings can offer some insight as to the 3-D shape of a molecule or protein.

NMR spectrometer

Credit: UCSD’s Center for NMR Spectroscopy and Imaging of Proteins

X-ray crystallography is considered the most accurate technique to determine the shape of a protein.  In this process, an uncontaminated crystalline sample of the protein is prepared.  After the often tedious process of sample preparation, the protein is bombarded with x-rays.  As the x-rays bounce off the crystal, they create a diffraction pattern that looks like a series of dots.  These dots are then analyzed using complex mathematical formulas. Because the density of the atoms in the protein dictates the way in which the rays scatter upon impact, the diffraction pattern offers enough information about the structure of the protein to create a highly accurate 3-D image.  However, some proteins are very difficult to crystallize and are not readily amenable to X-ray diffraction. 

Researchers also use mass spectrometry to reveal protein structure.  This process breaks down atoms and molecules into their ionic components.  Based on the mass to charge ratio of the ions, scientists can accurately guess the composition of the sample.

Because proteins are generally more delicate than other molecules, scientists use a technique called MALDI (Matrix Assisted Laser Desorption/Ionization)-Mapping to perform mass spectrometry on proteins.  

 See movie, “How a MALDI mass spectrometer works.” Click here >>

In this process, the protein sample is dissolved in a solution of purified water, a crystallized molecule and an organic compound.  The organic compound enables the non water soluble (hydrophobic) compounds to dissolve.  Similarly, the water allows for the water soluble (hydrophilic) compounds to dissolve.  Once the protein dissolves in solution, the solution is placed on a metallic slate.  The solvents evaporate, and crystals form.  The newly-formed matrix contains the protein throughout its structure.  Then, a laser beam is fired at the matrix in order to ionize the protein sample.  The matrix structure protects the protein from the damaging effects of the direct laser beam.      

To learn more about how researchers explore the mechanisms of disease, watch the video, “Understanding Disease at the Level of Individual Molecules.”  A supplemental video about protein folding, “How Do Proteins Fold?  Theory Meets Experiments” delves into the ways researchers from different disciplines explore protein folding.

Featuring Claude Benchimol, Senior Vice President of Research and Development at Invitrogen; Seth M. Cohen, Professor of Chemistry and Biochemistry, UCSD; Partho Ghosh, Professor of Chemistry and Biochemistry, UCSD; and Elizabeth A. Komives, Professor of Chemistry and Biochemistry, UCSD.

UCSD-TV (Real Player) click here >>