Structural Biochemistry/Translational Science

Translational science is a type of scientific research that has its foundations on helping and improving people’s lives. This term is used mostly in clinical science where it refers to things that improve people’s health such as advancements in medical technology or drug development.

Examples of Application

For a long time, pathologists have noticed the fact that cholesterol was present in unhealthy arteries. In the 1960s, epidemiological studies illustrated the correlation between serum cholesterol and coronary heart disease. In the 1980s, inhibitors of HMG-CoA reductase (statins) became available to the market. These drugs were created using the biochemical knowledge of the pathways for cholesterol synthesis and transport. Subsequent clinical trials were performed to collect safety and efficacy data about the drug. After the safety and effectiveness of the drugs were confirmed, physicians and the public were educated about the drugs, and the drugs became widely used. All of this contributed to a reduction of death caused by coronary heart disease. Considering that heart disease is the biggest cause of death in the world, this example demonstrates how scientific knowledge can be used to create drugs that can improve human health or reduce human mortality. Other examples of how scientific knowledge has affected the clinical environment are “inhibitors of angiotensin-converting enzyme, inhibitors of oncogenic tyrosine kinases, and insulin derivatives with more favorable pharmacokinetic profiles”.

Glutamine [1] as a Therapeutic Target in Cancer

Another example of translational science in action is the discovery that certain cancers show a considerably high rate of glutamine metabolism. In addition, glutamine has been shown to be an integral part of metabolic functions and protein functions in cancer cells. Therefore, by designing drugs that can reduce glutamine uptake in cancer cells can potentially provide new cancer therapeutics. In fact, there are several types of drugs that have been developed that have been shown to suppress glutamine uptake. L-γ-glutamyl-p-nitroanilide (GPNA) is an example of a designed drug that serves as a SLC1A5 inhibitor that can inhibit the uptake of glutamine in glutamine-dependent cancer cells and thereby inhibit the activation of the mammalian target of rapamycin complex (mTORC1) which regulates cell growth and protein synthesis. However, more research is being done to develop a drug that specifically targets and inhibits cancer cells without damaging normal cells. In addition, translational science has led to the development of an FDA approved drug, phenylbutyrate, [Buphenyl (Ucyclyd Pharma) or Ammonaps (Swedish Orphan] that lead to a major reduction of glutamine levels in blood plasma. Also, L-asparaginase [Elspar (Merck & Co.Inc)] has also been shown to decrease glutamine levels, but it is extremely toxic in adults. The fact that cancer is a major disease that has plaqued our society in recent years, this is a good example that illustrates the importance of how translational science has led to the development of new therapeutics to help patients win the battle against cancer. It also illustrates that scientific research is constantly conducted to better drug development and a necessary aspect of science to better out lives.

Medication to Arthritis Pain

Also, through advancement of biochemical knowledge and its complementary technology, a new way to fight arthritis pain is formulated. The pain resulting from arthritis is commonly treated with aspirin, Advil, and ibuprofen. However, most of these medications lead to gastrointestinal organ damages. Up to 100,000 hospitalization and 16,500 deaths is in fact lead by side effects from those non-steroidal anti-inflammatory drugs (NSAID). Fortunately, studying NSAID at its molecular level led to identification of the solution to this major problem. The scientist discovered that NSAIDs inhibits two enzymes called “cyclooxygenases,” or COX-1 and COX-2. Even though COX-1 and COX-2 have similar functions, COX-2 is formed as a reaction to an injury and infection. This leads to inflammation and immune response, which is the reason why NSAID blockade of COX-2 relives pain and inflammation from arthritis. On the other hand, COX-1 enzyme is responsible for the production of prostaglandins, which plays role in protecting the stomach linings from the acids. As NSAID inhibits COX-1, it may lead to a major problem, ulcers. With this knowledge, biochemist scientists are able to create a type of medication that not only reduces pain and inflammation, but also removes the side effects caused by the drug. Today, this drug is called Celebrex and it is a great example of how the advancement of biochemistry and drug development technology can open up new paths to better treatments for millions of people suffering with illness.

Imprinting

Imprinting is a process that is independent from Mendelian inheritance that takes place naturally in the animal cells, which is an example of epigenetics. In more cases, two copies of genes work the same way but in some mammalian genes, either the father’s or the mother’s copy is amplified rather than both being turned on. Imprinting does not occur selectively based on gender, but occur because the genes are imprinted (chemically marked) during creation of eggs and sperm. For instance, imprinted gene of insulin like growth factor 2 (Igf2) plays a role in growth in mammalian fetus. Considering Igf2, only the father’s copy is expressed while the mother’s copy remains silent and not expressed in the life of its offspring. Interestingly, this selective silencing of the imprinted genes seems to take place in all of the mammals except platypus, echidna, and marsupials.

The questions that scientist wondered regarding this was why would evolution tolerate this kind of process that risks an organism’s survival since only one of two of the gene is expressed? The answer to this is that the mother and the father have different interest and this resulted from a competition. For example, the father’s main interest for the offspring is for it becomes big and fast because it will increase the survivability, which will in turn give greater chance of passing on the genes to the next generation. On the other hand, the mother desires strong offspring like the father, but due to limited physical resources for pregnancy duration, it is wiser to divide the resources among the offspring instead of just one. Today, more than 200 imprinted genes in the mammals are identified and some of these imprinting genes regulate embryonic growth and allocation of resources. Furthermore, mutation in these genes leads to fatal growth disorders. There are scientists now trying to understand how Igf2 and other imprinted genes stay silent in the life of cells because it can be manipulated for treatment to various mutational diseases.

Medications in Nature

There are many substances that can be used as medications and drugs already existing in the nature. For example, in 1980s, Michael Zasloff was working with frogs in the lab at the National Institutes of Health in Bethesda. There was an interesting component to the skin of the frog that allowed prevention of infection even with surgical wounds. This observation led to isolation of a peptide called Magainin that was produced by the frog as the response to the injury. Furthermore, peptide made from frog skin possessed micro-bacteria killing properties and there are hundreds of other type of molecules called alkaloid on the amphibian skin. After careful study, the scientist realized that the compound responsible for the painkilling ability is called epibatidine. Epibatidine is however too toxic to humans for pain relieving medication. Knowing the chemical structure, the goal of making similar effect drug isn’t too far.

The Sea and Cancer Treatments

The ocean is a vast resource for anti cancer treatment and its success stems from the diversity it embodies. The earth is made up of around 70% of bodies of water and there are thousands of species living in the ocean, making it the most diverse marine ecosystem. Out of the 36 known phyla, the ocean contains 34. There are many drugs that have been founded based on natural substances and the ocean is a large provider of anti cancer treatments. Marine microorganisms are extremely important and can be difficult to utilize based on where they live. Many microbes live in very specific environments and it can be difficult to find an adequate supply.

File:Diazona violacea - Carantec.jpg (fichier)

Diazona violacea

One of the maritime organisms used in cancer treatment is the ascidian Diazona. The Diazona is a source for the peptide Diazonamide A. Diazonamide A has been known to be a growth inhibitor in the cells. Compared to others like Vinblastine and Paclitaxel, Diazonamide A has the best result in inhibiting cell growth after a 24 hour exposure to Human Ovarian Carcinoma. After testing down as a joint project in the UCSD Medical center, Diazonamide A proves to be a viable anti cancer treatment. The peptide has potency with in vitro cytotoxicity because it inhibits mitotic cell division. According to in vitro date, the reason that Diazonamide A is a good inhibitor of cell growth is its attack on the cell’s tubulin assembly. Structurally, the peptide is not similar to any known drug candidate but it is still considered an “active lead compound”. However, the lack of availability of the peptide ultimately renders it unviable due to lack of practicality. Without a ready source, it cannot advance to the preclinical stage and is not a practical option for drug making and distribution. However, in 2007, a synthesis for the peptide has been available and thus finally allowing it to go further in development. An example of how peptide synthesis is an important step in drug development.

Taxol structure

Another organism that can be used for anti-cancer treatment is the Soft-Coral Eleutherobia. Similar to the hard coral, but they are soft bodied and have no defenses against the environment. Its extract, the organic cytotoxin Eleutherobin, shows potency in cytotoxicity of 10nl/mL. Similar to the Diazonamide A, Eleutherobin is also a mitotic inhibitor. It stops the HCT116 colon carcinoma cell line by stabilizing the microtubules. When treated soluble tubulin with Eleutherobin, the microtubules are stabilized in a similar fashion to Taxol. In certain circumstances, Eleutherobin competes with the Taxol for target sites. Eleutherobin is, therefore, almost identical to Taxol and it is also a leading prospect for cancer research. However, similar to the Diazonamide A, it is not a practical lead to follow as there is no supply. There have been synthesis but none could yield practical results within reasonable costs. Because of those difficulties, it has not been advanced to preclinical stage.

HALIMEDA

A fungal strain extracted from the surface of the Halimeda. Like the two previous examples, this strain is also a potent cancer treatment. It works against HCT116 human colon carcinoma. Unlike Eleutherobin, this is not similar to Taxol but it is potent against cell lines resistant to Taxol. Halimide as a natural product, can be converted into the synthetic product NPI-2358. NPI-2358 differs from Halimide with a t-butyl group. The NPI-2358 has shown more progress than either Diazonamide A or Eleutherobin in its clinical stages. It has shown great in vivo results and since 2006, has been in phase 1 of clinical trials. In in vivo experiments with breast adenocarcinoma, the NPI-2358 has shown significant necrosis in tumor. The experiment includes a the breast adenocarninoma to be grafted onto the dorsal skin of a mouse with a viewing window. After 15 days of treatment, the group treated with the NPI-2358 shows a reduction in the tumor size in comparison to the control group. Upon closer inspection, the NPI-2358 seems to target tumor vasculature.

An organism that has been founded through the sampling methods is the Salinispora. They are special in that they require salt for growth and in cultivating the bacteria, grains of salt need to be added directly. They have unique colorings and 16s rDNA sequence. More than 2500 rDNA strains from the Salinispora have been studied. Their geographical location is along the earth’s equator, they have been discovered in Hawaii, Guam, Sea of Cortez, Bahamas, Virgin Islands, Red Sea, and Palau. The extract from the bacteria has shown cytotoxicity against HCT-116 colon carcinoma. To prepare, the Salinispora is cultured in shake flask for 15 days with XAD absorbent resin, the resin is then filtered with methanol. The cytotoxic effect of the Salinispora is very effective, it has a broad but very selective of cancer line cells. Above 2 microM, the bacteria is effective against several cancer types. The bacteria proves to be a wonderful inhibitor of cancer mechanism. Salinosporamide A is very effecting in inhibiting proteasome in vivo. It shows an average IC50 of less ca. 5nM against the 60 call line panel. It is also active against several other types of cancers. Currently as of 2006, it is in phase 1 of human trial and shows great promise as a future drug against some cancers.

Similar to the Salinispora, another marine stain of the Marinospora also requires salt. They are discovered along the Sea of Cortez by deep ocean sampling. Like the Salinispora they also have unique 16S rDNA sequences. They are also morphologically identical to the Streptomyces. Their extracts show good potency against drug-resistant pathogenic bacteria and cytotoxicity against certain cancer cell lines. The fractionation of the strain also leads to a new macrolide class. Marinomycin A from the strain is shown to be very potent against melanoma. It has been advanced to the hollow fiber assay.

The ocean is a diverse place and the many organism that it houses has great medicinal effects. Many of the populations were previously unknown but with strategies of deep sea sampling, in two years, 15 new genera of bacteria from 6 families have been cultured. More amazingly, of the 15 genera cultured, 11 have shown two be good inhibitors of cell growth and can be possible anticancer ingredients. That simply shows the wealth that the ocean has to offer in battling against dangerous illness.

Stem cell

There are cell in the human body that is completely generic and it has the ability to express extremely broad array of genes. Simply put, stem cells are able to becoming all kinds of cells in the body with an unlimited potential. Specifically speaking, this cell exists for few days after conception and is called embryonic stem cell. Once these embryonic stem cells differentiate, there are cells called adult stem that too have similar ability as the embryonic stem cell. These cells are located throughout the body, mostly in bone marrow, brain, muscle, skin, and liver. Then tissues are damaged by injury, disease, or age, it can be replaced by these stem cells. Adult stem cells however are dormant and remain undifferentiated until the body signals for its need. Adult stem cells have the capacity of self-renewal but different from the embryonic stem cell in a sense that it exists in small numbers and aren’t too flexible in differentiating. Adult stem cells plays role in therapies that treats lymphoma and leukemia. Scientists are able to isolate an individual’s stem cells from blood and grow them in the laboratory. After high dosage chemotherapy, the scientist can use the harvested stem cells to transplant and inject into replacing the cells destroyed by the chemotherapy. James A. Thomson of the University of Wisconsin was the first one to isolate stem cells from embryo into growing them in the lab. Stem cell research opens possibilities to treating Parkinson’s disease, heart disease, etc. diseases that involve irreplaceable cells.

AIDS Treatment

To understand strategies to combat HIV-1 infections, a study of its biology must be conducted. HIV-1 was found to interact with the host cells by means of their glycoproteins, gp120 and gp41. The receptors CD4, CCR5, and CXCR4 recognize these envelope proteins and together, they lead to the fusion of the virus and the host cell. In the beginning, how HIV-1 was treated was by preventing the protein from maturing and stop the RNA to replicate into the DNA. However, since both of these happen after the host genome as already been infected- a much more attractive strategy is to stop the virus from fusing with the cell. This line of research leads to the discovery of many HIV entry inhibitors.

HIV attachment

Since the glycoproteins gp120 and gp 41 play an important role in viral infection, their structures were studied. The gp120 interacts with the CD4 by undergoing a conformational transformation. This transformation exposes the gp120 to the receptor proteins CCR5 o CXCR4. gp41 also has a major conformational transformation that changes from a prefusion complex with the gp120 into a structure that is able to place the viral and host membranes side by side. Entry inhibitions that can prevent that step from happening can prevent the cell from being infected. The gp41 has a six-helix bundle (6-HB) made from N-and C- HR regions. The N-HR forms a core and the C-HR packs tigtly against it. The formation of stable crystals from these peptides aids in the search of finding a peptide inhibitor for the six-helix bundle. An HIV-1 entry inhibitor is the T-20, which is a homolog to the helical C-HR region of the gp41.

Experiments in the absence of high resolution structure of gp41 shows that the T-20 interacts with the N-HR helical region and acts as an entry inhibitor. When high resolutions of the gp41 structures were available, the T-20 was shown to form a heterocomplex that inhibits the formation of the 6-HB hairpin is required of the viral and host genome fusion. This result shows that either the C-HR or the N-HR could act as an entry inhibitor. The N-HR peptides are trihelical and they have deep hydrophobic pockets, these pockets have a complementary pocket-binding domain (PBD) present on the C-HR peptides. This stable helical structure shows that a point for the success of the T-20 is its ability to have a helical structure as well. In general, to improve inhibitions, w can increase the helicity of the C-peptides, increase the T-20 interactions with the N-peptide trihelices, or to make N-peptides that form soluble, stable triple helix core. A good relationship between the tendency to form a stable helix with inhibition activity was seen from many entry inhibitors.

Unlike the T-20, the C-34 has a PBD sequence that can interact with the hydrophobic pockets of the N-HR core. The C-34 is therefore effective against strains of HIV that are resistant against T-20. The C-34 works by preventing the formation of the 6-HB. On the other hand, T-20 works by interacting with the lipids that interrupts the membrane fusion pore from forming. If both characteristics of both the C-34 and the T-20 were incorporated into a single inhibitor, the result would be very potent.

Recent studies have shown that fatty acid and cholesterol may be used to act as peptide fusion inhibitors for gp41. During T-20 binding to liposomes the LBD domain plays a role in the fusion process. Fatty acids were shown to have similar binding characteristics which make them possible candidates to act as fusion inhibitors along the binding locus. Researchers speculate that the reason for this is because (C-16)-DP combats HIV-1 by increasing inhibition around the viral membrane. Similarly cholesterol helps by targeting C34 to lipid rafts which also increase inhibition activity around the membrane. These kinds of applications are extremely useful when combined with drug treatments that have limited local concentration.

N-peptides are another viable solution for inhibition because they show similar properties to that of N-HR on gp41. They contain the 5-helix design which inhibits HIV-1 fusion at nM concentrations. Studies show that stability of the triple helical core may be correlated to the effectiveness of HIV treatment. It has been shown that disulfide stabilizes the trimeric coiled-coil core which will increase inhibition properties. When combined with N-HR and C-HR inhibitors this virus has an increasingly difficult time surviving without mutating further. The downside to N-peptide treatment is the higher molecular weight which may lead to immunogenicity if not used through injections.

A final possible fusion inhibitor was found using D-amino acids. These D-amino acids mimic binding to the trimeric core. Additionally these amino acids are highly resistant to protease degradation which makes them more effective than T-20 which cannot be absorbed by paracellular passages in the intestine. This leads to the possibility of using D-amino acids in topical treatments which can be easily applied at a less expensive cost.

Protein and Drugs

Each individual human body has variations in the genetic makeup that leads to difference in general health. There are environmental and lifestyle factors involved, but the response that every individual have on medications is due to a variant gene called cytochrome P450 protein. This protein is in charge of processing any kind of drugs the body intakes. Due to uniqueness of the individual’s genes, the encodings for the cytochrome P450 is very exotic. This information was discovered in the 50s as certain patients had different side effects to anesthetic drug, which was fatal. Through experiments, the scientist realized that genetic variation can cause a dangerous side effect because cytochrome P450 protein wasn’t able to break down the medication in the normal way. Even medicines like Tylenol can sometimes give no relief to the body because of the genetic variation. Fortunately, with greater knowledge of this, pharmacogenetic scientists can now develop drugs that are customized based on individual’s genes.

Obstacles and Potential Solutions

To mediate between laboratory science and clinical science is not an easy task. It requires a vast amount of different types of complex and specialized knowledge, and this brings up a lot of problems and obstacles. One research team is not even nearly enough to create a bridge between basic and clinical science. One proposal is to create research teams that specialize in different steps to interconnect basic science into the clinical environment. It also seems very practical to train individuals that can mediate between these different steps so that if a research team does not exist at one step, these mediators or translators can try to find assistance from teams at adjacent steps of the process. These proposed solutions on how to implement translational science depend on the cooperation of various types of scientists. The idea is to allow translational scientists to have easy access to the wide array of intricate and specialized knowledge needed to bridge the gap between scientific research and clinical and medical advancements.

Over the years, an increasing emphasis have been placed Translational Science. National fundings and policies have greatly facilitated the growth of the field creating opportunities for the advancement of applied clinical research informatics (CRI) and translational bioinformatics (TBI). Examples includes The National Cancer Institute's caBIG program which engineered a variety of service oriented data-sharing, data-managing, and knowledge management systems, and the CTSA which aim focuses on informatics training, database design/hosting, and execution of complex data analysis. The issue however, is that such programs, and the fundings that accompany it are geared towards solving immediate problems while neglecting to focus on foundational CRI and TBI research that is crucial to the growth of biomedical informatics subdisciplines that ensures future innovations. Furthermore, the funds are usually allocated to service oriented research that provides the resolutions to immediate problems while the policies restrict the resources of data networks to specific universities or centers. Several resolutions have been proposed in response to the issue at hand:

1. Rigorous campaign advocacy to ensure that foundational CRI and TBI knowledge and practice is both recognized and supported as a core objective of translational change which will engage informaticians as equal partners in planning an execution as opposed to mere service providers.
2. Community effort to refine and promote national scale agenda that focuses on challenges and opportunities facing CRI and TBI allowing them to do more than just react to new fundings and policies.
3. Creation of a forum to ensure that establishment of policies and fundings affecting CRI and TBI are open to researched of the field and not limited to the few institutions, investigators, or lenders.

National Institutes of Health (NIH) and Clinical and Translational Science Awards (CTSA)

The National Center for Research Resources (NCRR) of the National Institutes of Health (NIH) has created the CTSA for the purpose of encouraging translational science and research. The NCRR has used the CTSA to give funding to infrastructure for translational science in areas such as “biostatistics, translational technologies, study design, community engagement, biomedical informatics, education, ethics and regulatory knowledge, and clinical research units”. The CTSA has six major goals with regards to translational science. The first goal is to train individuals in the field across the entire translational spectrum. This involves giving training to MD to allow them to act as clinical investigators, but it also involves teaching PhDs the fundamentals about the medical world. The expected result from this training would be that PhDs would know when they have come across something that would be of medical importance and so that MDs could understand what it is that the PhDs are trying to say to them. Secondly is the goal of trying to simply the translational process. This means trying to speed up the translational process as much as possible without losing regards to safety. This would include making expertise available to clinical researchers by the way of institutional review boards, FDA regulations and applications for investigating new drugs. The third goal of the CTSA is to take advantage of advances in informatics, imaging, and data analysis by applying these advances directly to research that clinical investigators are doing. By taking advantage of these resources, it is more likely that the investigators will come up with a meaningful study. The fourth goal is to find a way to encourage and protect the careers of translational researchers. An example of a path that propagates this career is MD/PhD programs. These types of programs could bridge the so-called translational divide by education people in aspects of both the medical and scientific fields. The success of these programs depends largely on tuition assistance provided, making it so that the graduates of these programs are not burdened with large loans to pay off. The fifth goal is to provide team mentoring, as well as support to junior clinical scientists. This goal can be achieved in part through programs like the K12 awards. The final goal of the CTSA is to catalogue all research resources in order to make these resources available to everyone possible that could need them.

References

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