Design of Biometric Peptides for Antibiotic Use
Lenore M. Martin, Assistant Professor
Department of Biomedical Sciences
The University of Rhode Island (URI) has long
been noted for its strong research programs in both oceanography and pharmacy,
with the "Drugs from the Sea" initiative leading to the discovery
of many promising drug candidates and toxins in the laboratory of my colleague,
Yuzuru Shimizu. Shimizu has been a faculty member at URI for more than 20 years,
building an internationally recognized natural products research group. Four
years ago, as chair of the Pharmacognosy Department, Shimizu recruited me from
Rockefeller University in New York City where I was a research associate with
Nobel Laureate R.B. Merrifield. My research at that time focused on the chemical
synthesis of proteins and peptides (small proteins) found naturally in the human
immune system and on developing novel ways to analyze proteins found in biological
fluids. When I was a graduate student at UCLA, I developed an efficient method
for synthesizing an active fragment of a natural antitumor antibiotic secreted
by bacteria. The time seems ripe for bio-organic chemists like me to attempt
to replicate, and maybe even to improve on, the plethora of drug candidates
found in our environment.
One immediate goal of my research program is to use combinatorial peptide libraries to help ameliorate the public health crisis brought about by an alarming rise in the number of pathogenic (disease-causing) bacteria that are resistant to antibiotics. Indeed, in some cases, hospitals have documented the presence of bacteria that resist every known antibiotic, and children with severe bacterial infections are dying because no antibiotic therapy exists for them. It is heartbreaking to return to the pre-World War II days when bacterial infections killed large numbers of people worldwide. I remembered from my doctoral work on developing novel antitumor drugs that the main source of new anticancer drug leads was to test for the substances secreted by certain strains of bacteria in order to kill other bacteria. The first penicillins and other first generation antibiotics fall in the class of substances which chemists call "natural products." It seemed a logical next step for my graduate student, Bi-Huang Hu, and me to search the scientific literature for reports describing novel natural products obtained from a diverse group of bacteria and plants and for uninvestigated building blocks that repeatedly appeared in antibacterial substances.
In nature, many important substances such as proteins, DNA, and carbohydrates are termed biopolymers because they are made up of small repeating units attached end to end in long strings. The Merrifield Method for synthesizing proteins and peptides is based on an understanding of the nature of biopolymers. Living organisms make proteins by connecting individual amino acids, which I call "building blocks," like beads on a string. As each new building block is added, the string gets longer and the behavior of the string changes. In 1963, Merrifield suggested using insoluble polystyrene beads (like Styrofoam) and attaching one end of the growing string of building blocks to the beads (see Fig. 1). This type of anchor makes the string easier to work with and facilitates the automation of the chemical process whereby amino acids are attached to each other. Solid-phase synthesis, as the Merrifield Method has come to be known, has been extended to the synthesis of molecules other than peptides and is used to create a variety of biopolymers in the lab.
One recent development in our research is the observation that nature uses the same small number of building blocks over and over again to generate the tremendous diversity we see around us. Biopolymers are not the only molecules that can be constructed from a set of standard building blocks. Learning from this, we found that the solid phase technique enables us to devise novel building blocks and combine them in a virtually infinite number of ways to make replicas of natural antibiotics or to generate entities having totally new properties. A group of products generated simultaneously from the same set of building blocks is called a combinatorial library. Just as a conventional library might be made up of books in one subject area, combinatorial libraries are made up of compounds generated from a selected set of building blocks for a given purpose. Our selection process is driven by the effectiveness of compounds within a library, mimicking the process of biological evolution. We have found that although, in theory, we can successfully design a compound to have the desired properties, the library gives us some "wiggle room." Thus, while we seek to refine the methods by which we make our building blocks, we simultaneously seek to refine the process of selecting the best peptide out of each library.
Some of my former colleagues at Rockefeller were instumental in documenting the mechanisms that cause bacteria to become resistant to antibiotics. When I arrived at URI, we were just beginning to develop our synthesis of a novel type of building block, so we first tested the power of the combinatorial technique with a commercially available amino acid that is not found in nature. Combining just four building blocks in our peptide string, and using two types of building blocks, we prepared all 16 possible combinations in the first library (see Fig. 2). The components of the library were attached to polystyrene beads, so we could easily keep the strings separate from one another, yet prepare them simultaneously. Even though I had read about the tremendous diversity available through combinations, the prospect of having sixteen new compounds to test for useful activity was dramatic. Moreover, this first library contained only two types of building blocks! I was beginning to understand how overwhelmed my colleagues in industry felt when their companies jumped on the combinatorial bandwagon without considering the consequences. If one tries to produce too many compounds simultaneously, the result is usually an unwieldy mess---difficult or impossible to untangle. I felt that the answer to this problem lay in careful design of the building blocks and coupling the creation of libraries to a meaningful and efficient method of evaluation.
The rise of antibiotic resistance in bacteria may be circumvented by selecting compounds that target a vital biochemical process in bacteria. The biological process we targeted in our evaluation is the replication of the bacterial DNA. To divide and form progeny, each bacterium must first duplicate its DNA so that it can pass along genetic information from generation to generation. Bacteria use a slightly different process to duplicate their DNA than do humans and shellfish, so we looked for antibiotics which selectively prevent bacteria from reproducing and do not harm other types of organisms. I have already described, to some extent, the way we designed the building blocks based on naturally occurring antibiotics. Three building blocks and their method of preparation are now patented. In order to assess the activity of combinatorial products synthesized in our laboratories, we kept in mind the possible applications of antibacterial peptides. We have developed two methods for screening our libraries; one uses bacterial enzymes and DNA and is performed in my laboratory, the other uses live bacteria and is done in collaboration with Marta Gomez-Chiarri in the Department of Fisheries, Animal and Veterinary Sciences. Gomez-Chiarri and I are investigating the possible uses of peptides in aquaculture. She is concerned about preventing the spread of disease in fish and is addressing the problem of contaminated shellfish. Both issues pose serious economic and public health risks in the near future. We plan to collaborate with each other and with other researchers in environmental biotechnology. We plan to form a strong community of researchers and pool our expertise to address complex and urgent problems.
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