Professor Rudolf Allemann Project Titles
1. Dihydrofolate Reductase and the Physical Basis of Enzyme Catalysis
The hallmarks of catalysis by enzymes are selectivity, specificity, and speed. However, despite their central role, the physical basis of the enormous catalytic power of enzymes is not well understood. In the absence of catalysts, reactions in organisms occur with speeds that range from moderately fast to very slow with reaction times that often exceed the lifetime of our universe! It is therefore not suspiring that all biological reactions are catalysed and rate enhancements of up to 21 orders of magnitude can be observed.However, despite the importance of enzymes for biology and biotechnology and many decades of experimentation, the causes of these remarkable rate enhancements are not fully understood. Efforts to design biomimetic catalysts from first principles have often led to results that are both remarkable and disappointing at the same time, in that the rate enhancements always fall far short of what natural enzymes can do.
Proteins are not static but highly dynamic and the role that enzyme motions play in the physical steps in catalysis, i.e. binding of substrates and release of products is well established. However, the influence of such dynamic motions on the actual chemistry of an enzyme-catalysed reaction is less well defined. In particular, the influence of fast motions that actively promote the reaction is a current hot topic in mechanistic enzyme catalysis. Hydrogen transfer reactions are of fundamental importance in all biological processes and are particularly well suited to study dynamic effects because hydrogen has a low mass and hence is more susceptible to be affected by motions. The effect of motions on catalysis may be the key to understanding the nature of the enormous catalytic efficiency of enzymes. Here we will investigate the correlation between dynamics and enzyme activity using dihydrofolate reductase (DHFR), an enzyme required in many essential biochemical processes including the synthesis of DNA and amino acids. It is therefore a long established drug target and several DHFR inhibitors have been developed that act as antibacterial, antimalarial and anti-tumour drugs.
2. Chemical Wizardry: How nature handles carbocations
Terpene synthases catalyze intramolecular, electrophilic C–C bond formations from alicylic prenyl diphosphate substrates (i.e. geranyl, farnesyl and geranylgeranyl diphosphate) in reaction cascades that often generate several ring systems and double bonds with exquisite regio- and stereo-specificity. These reactions are initiated through initial heterolytic C-O diphosphate ionization leading to highly reactive carbocationic intermediates, followed by C-C bond formation via electrophilic additions of carbocations to -systems. This initial cyclization step is often followed by hydride-shifts and/or Wagner-Meerwein rearrangements and further rounds of cyclizations facilitated by movement of fleeting carbocations. This propagating “carbocation-guided” cascade of reactions is terminated by 1,2-proton elimination or water capture to produce cyclic hydrocarbons or alcohols, respectively. Enzyme-bound carbocationic species are stabilised by characteristic interactions with catalytic residues such as -stacking interactions with aromatic residues, ion pairing with carboxylate moieties or the enzyme-anchored diphosphate group, and dipole-dipole interactions with amide bonds of the protein backbone.
The transient existence of these highly reactive carbocationic intermediates is usually inferred indirectly from experiments with substrates analogues and/or terpene synthase mutants. In addition, the use of aza analogues of putative carbocations involved in biosynthetic processes are particulary useful as selective inhibitors and mechanistic probes to evaluate the stereochemistry, structure and charge delocalization of these presumably trigonal carbocationic intermediates proposed to occur in terpene synthase catalysis. This project will involve the chemical synthesis of analogues of reaction intermediates during terpene synthase reactions and the analysis of their properties when incubated with the enzymes.
3. Intracelluar Biphotonic Nanoswitches: control of cellular pathways with light
The actions of cells are governed by protein-protein interactions. Key decisions about whether a cell differentiates or undergoes programmed cell death are taken at a molecular level by the interactions of large proteins. These interactions consist of shallow contacts over large areas of their surfaces, interactions which traditional small molecule medicinal chemistry finds difficult to target. Instead we aim to replicate the most important sections of the interacting surfaces and tether them to photochromic dyes. These dyes change shape when they absorb light of a particular wavelength and by carefully placing dye molecules, this shape change can be used to control the shape of the molecule they are attached to.
Since shape is crucial to the molecules ability to fit the shallow groove on its binding partner, the affinity of the molecule for its target can be switched by irradiating it with light. We have demonstrated the ability of molecules of this type to control the protein-DNA interactions found in transcription factors and key apoptosis inducing protein-protein interactions in vitro. The project focuses on preparing new dyes, exploring the range of molecules we can target, and switching molecules and their subsequent biochemical pathways in live cells.
4. Combining synthetic chemistry and biology approaches to generate novel drugs
Sesquiterpene synthases catalyse some of the most complex reactions known. In one chemical step, the sesquiterpene precursor FPP (1) is specifically converted to a complex hydrocarbon that may contain several rings and stereocentres. Traditional synthesis of such compounds is expensive and time consuming requiring many chemical steps. Many of the sesquiterpenes produced have profound biological activity and with minor modifications may be transformed into powerful new drugs. For example (S)-germacrene D (2) is produced by the enzyme germacrene D synthase (GDS) from FPP and is a powerful alarm pheromone produced by the pests aphids. Analogues of germacrene D may be useful as crop protection agents by repelling aphids from the plants using a spray. This project aims to produce such compounds through harnessing of the enzyme’s catalytic capabilities thereby bypassing the usual requirement of a lengthy synthesis.
Traditional synthesis will be used to prepare analogues of 1 containing minor modifications. Turnover of these FPP analogues using GDS will produce germacrene D analogues that will be analysed for biological activity against the aphid insect model. Using the results obtained, further analogues will be designed and produced by the enzyme to produce compounds that are stable in the environment yet retain the required biological activity.
5. Medical Chemistry to Control Auto-immune Diseases like Arthritis
One of the first responses of a body to infection or trauma is the migration of neutrophiles (white-blood cells) from the blood stream to the site of damage. Over-activity of this defence mechanism causes auto-immune diseases such as osteo-arthritis. Hence a potential method for treating such diseases is prevention of neutrophil migration. In order to leave the blood stream neutrophils undergo a massive shape change involving the cellular membrane allowing them to squeeze through tissue barriers. This process is catalysed by a cysteine protease known as m-calpain. Aromatic mercapto-acrylates such as 2 are known inhibitors of -calpain and are therefore potential drugs for these diseases.
In collaboration with the neutrophil signalling group at the Heath Hospital this project will use a combination of synthetic chemistry to prepare new analogues of 2, biological testing to analyse the effect upon the neutrophil cell cycle and crystallization with the enzyme to produce new and improved calpain inhibitors as potential drugs for the treatment of auto-immune disease.