Blegdamsvej 3B, 2200 København N, 22 Bygning 22, Bygning: 22-2-22
Good health is a key factor of our life and we live with an increasing life expectancy. Despite technological and medical progression, life threatening infections are returning as a serious threat even in well developed countries like Denmark and the societal costs of infectious diseases are considerable. Over the next decades our society will go through changes that greatly affect our sensitivity to microbial pathogens where the most pronounced will be the increase in elderly, immuno-compromised and hospitalized people. Such citizens are susceptible to chronic infections caused by well known bacteria such as Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. In Denmark, ~100.000 hospitalized patients acquire infections, often in connection with the use of medical devises such as arteriovenous shunts, pacemakers, urinary and other types of catheters, orthopedic devices and mechanical heart valves with an estimated 200-300 annual fatalities. Skin ulcers that develop into chronic wounds are also a significant and partly neglected problem, which affects patients with impaired blood circulation in particular diabetes patients. It is assumed that ~ 50.000 patients suffer from wounds that require special treatment and medical attention because of bacterial infections. This is an important socioeconomic burden, which includes patient suffering, lost employment and reduced life quality. The problem is growing in pace with the increasing emergence of multidrug-resistant bacteria.
In the golden age of antibiotics discovery, potent antibiotics such as penicillin, tetracycline, and vancomycin were discovered and brought to the clinic during the 1940s-1950s. These medicines have saved millions of lives but the backside of the coin is that they by their mode of action (killing the bacteria) strongly promote development of resistant bacterial variants, which rapidly outmatch the original sensitive strains. Infectious diseases that were once treatable with antibiotics will with time become incurable. In order to identify new antimicrobial drug targets, it is imperative to broaden our understanding of the molecular basis of infection, rather than just going in for the kill.
My working hypothesis is the following. The biofilm lifestyle dominates in the above mentioned chronic bacterial infections. Consequently, they share similar characteristics; they tolerate the highest deliverable doses of antibiotics and resist the action of the immune system as well as controlling the infectious process by cell-to-cell communication and internal signal transmission.
Biofilms are agglomerates of microorganisms surrounded by a self-produced extracellular matrix. For example, in the biofilm mode, P. aeruginosa uses "quorum sensing" communication to inform its fellow bacteria that it is time to launch a shield against attaching white blood cells. However, quorum sensing is only the tip of the iceberg of factors governing control over progression of the infectious processes which suggests multiple targets for efficient disease control. In the present project, I intend to generate information about genes at play during infection with particular emphasis on those that are differentially expressed in the biofilm state. Based on this, new anti-biofilm measures can be identified which efficiently decrease the biofilm forming capability or persistence of the infectious bacteria.
So far the majority of biofilm research has been carried out by means of in vitro systems. Much important information regarding gene expression, organization, structural development, antibiotic tolerance and interaction with neutrophiles (PMNs) has been generated this way (see for example refs 95*, 101*, 117*, 124*, 145*). Available data does not support the presence of a unique biofilm program i.e. a collection of genes that is expressed during development and maturation of biofilms (ref 124*). However, transcending traditional biofilm research into in vivo conditions for example by use of the implant model recently developed by my research team (ref 148*) in combination with transcriptomics will likely promote the discovery new bacterial genes and key proteins. Focus will be on those that are involved in the control mechanisms of the infectious processes. The classical approach is bacterial genetics which employs mutations to perturb the function of gene products. I will also follow a complementary approach, namely "chemical genetics", which uses libraries of natural and synthetic small molecules to perturb expression and function of gene products. My ongoing collaboration with organic and natural products chemists at DTU will provide a diversity of active small molecules, and thereby develop a deeper understanding of the complex biology of bacterial infections.
This in vivo experimental approach will make it possible to identify a collection of infection relevant genes; those that we already are familiar with (for example quorum sensing controlled genes) but importantly, those among the 50% whose function is presently unknown. Selected bacterial genes are then genetically engineered to construct screens for chemical genetics purposes. This will then in turn assists in the identification of bioactive small molecules capable of reducing expression of whole gene collections relevant to infection, in particular those involved in the temporal progression of infection and biofilm tolerance to the immune system. In other words, by controlling the multiple lines of command, disease-causing bacteria may be disarmed and the biofilms eradicated by the action of the host's defenses. It is important to emphasize that this approach seeks to target genes encoding non-essential phenotypes such as those involved in bacterial adhesion, intercellular signaling (c-di-GMP) and cell to cell signaling (quorum sensing). Blocking those functions does not hamper growth per se and consequently does not create a harsh selection pressure for development of insensitivity as we see with conventional antibiotics.
Chemical genetics approaches are inherently steps closer to drug development than classical genetics; however, it is not the my mission to develop new drugs per se. Small molecule hits may very well have the potential to be drug candidates, but it is naive (and an unrealistic success criterion for university based research funded by public grants) to expect that clinical candidates can be provided. The identification of novel small molecules as powerful tools for biological investigation and proof-of-concept of novel anti-microbial mechanisms (which transcend the past setbacks of resistance/tolerance) defines the meaning of success. At this stage industry must take over (based on shared intellectual properties) and cover the cost of further drug development and clinical trials. Many pharmaceutical companies no longer have antibiotic drugs in the pipeline or, even more worryingly, research activities in the field. The responsibility of uncovering novel antibacterial targets and potent small molecule probes therefore more than ever resides in the academic sector. In addition to creating new and exciting science, these initiatives will also educate a new, strong generation of young scientists, prepared for the multidisciplinary research efforts increasingly demanded by both academia and industry.
My aim is to continuously develop and combine research in molecular infectious biology with chemistry. My ultimate ambition is a paradigm shift in future anti-microbial treatment, fundamentally different from the traditional strategies of directly killing the bacteria with antibiotics, which have prevailed ever since the discovery of penicillin.
What are the structural features of small molecules most likely to yield specific modulation of protein functions involved in the control of infectious processes? It remains difficult to predict which small molecules will best modulate these biological processes and disease states, especially with limited prior knowledge, e.g. protein crystal structures, of the macromolecular targets. It is therefore necessary to systematically screen thousands of small molecules to find a successful match between a chemical and its target. As with the previous screens I have developed (refs 59*, 95*, 117*) for QS regulated gene expression, transcriptomic based analysis mentioned above will provide target genes for construction of those new screens.
Small molecules come in a variety of different shapes, e.g. they may be flat, rod-like, or spherical, and they may contain a variety of atoms with many functions. Natural products are small molecules that tend to be complex, highly three-dimensional in structure, and very different from one to another, whereas compounds made by traditional medicinal chemistry tend to be simple, flat, and alike. Natural products represent a prime source of "chemical diversity", and the hit rates of screening natural product libraries for novel antibiotics by far exceed those of prevalent compound libraries in the pharmaceutical industry. An example of this is my work with garlic (ref 129*). Garlic contains a variety of small molecule chemistry capable of blocking quorum sensing in P. aeruginosa. Previous support from the Strategic Research Council and the German Mukoviszidose e.V. has enabled us to identify and synthesize QS blocker chemistry and we have used this to successfully treat lung infection in our infectious, pulmonary mouse model.
Much of the medicinal chemistry practiced in industry has failed to provide compounds suitable for antimicrobial drug development, imparted by the strategic mistake of focusing on quantity rather than quality (or chemical diversity). However, given their track record in antimicrobial discovery, there is little reason to assume that natural products could not continue to play an important role in this process. Consequently, in collaboration I will engage in a two-pronged approach for providing novel small molecule libraries for drug discovery: natural products, and synthetic small molecules mimicking the structural features of potent natural products. "Diversity-oriented synthesis" (DOS) offers the potential to meet structural demands. Unlike traditional strategies for chemical synthesis, the DOS approach enables chemists to rapidly and efficiently synthesize libraries of complex and structurally diverse small molecules in a small number of synthetic steps. Rapid optimization of small molecule properties by structural modification in follow-up studies requires synthetic routes that are as short as possible. Keeping the number of synthetic steps to an absolute minimum ensures that all critical aspects of the small molecule discovery process are met, including optimization, and scale-up.