Bakterielle Ribonuklease P-Enzyme - Evaluierung als Drug Target und Analysen zur Substraterkennung

Ribonuclease P (RNase P) is an essential 5'-precursor-tRNA (pre-tRNA) processing enzyme in all domains of life. The architectural diversity of RNase P enzymes is unique. In Bacteria, Archaea, and in the nuclei and organelles of many Eukarya, RNase P is a complex consisting of a catalytic RNA subunit and a varying number of proteins (one protein in Bacteria, at least four in Archaea, and up to ten in Eukarya). In addition, a variant of protein-only RNase P (PRORP) lacking an RNA subunit has been identified in human mitochondria and subsequently in terrestrial plants and some protists. As the prokaryotic and eukaryotic RNase P holoenzymes differ significantly in their structure, the bacterial RNase P is a promising target for the development of novel antibiotics. The aim of this work was to evaluate, whether the bacterial protein subunit can be addressed with small molecules. Two published RNase P inhibitors with a thiosemicarbazide core and an isoflavone skeleton, respectively, served as a starting point. During initial experiments testing the thiosemicarbazide RNPA2000, solubility problems were observed, suggesting that the compound may induce protein aggregation. Small molecule-induced protein aggregation is one major source of false-positive results in drug discovery campaigns. To address this issue, the following control experiments were conducted: (i) addition of detergent to the enzyme kinetic reactions, (ii) variation of RNase P holoenzyme / protein subunit concentration, (iii) determination of the solubility of the potential inhibitor under assay conditions, (iv) analysis of cosedimentation of the protein subunit and the small molecule, and (v) testing for growth inhibition of a Bacillus subtilis strain whose RNase P activity is provided by the eukaryal PRORP. These experiments demonstrated that RNPA2000 is not a specific RNase P inhibitor, but rather induces nonspecific protein aggregation leading to the observed inhibitory effects. Additionally, it could be shown, that the isoflavone iriginolhexaacetate and purpurin, another published RNase P inhibitor, also act as protein aggregators. Therefore, this work revealed that, to date, there are no specific small molecule inhibitors known in literature that bind to the protein subunit. To address the 5'-leader cavity of the protein subunit, completely novel structures were investigated. For this purpose, a structure-based and docking-assisted approach was pursued. Initially, one compound harboring a sulfonate group showed a slight RNase P inhibition. However, during structure-activity relationship studies, solubility problems and protein aggregation reappeared, excluding this class of compounds for further investigations. In summary, the protein subunit of bacterial RNase P is highly susceptible to protein aggregation and addressing this protein with small molecules appears to be very difficult. Consequently, this protein is less suitable as an antibacterial target of small molecules. In addition to the PRORP enzyme found in Eukarya, another protein-based RNase P has been identified in Aquifex aeolicus (Aquifex aeolicus RNase P, AaRP) and related hyperthermophilic Bacteria of the family Aquificaceae. Like PRORP, AaRP also belongs to the PIN (PilT N-Terminus) domain-like superfamily, although it belongs to a different subgroup. The monomeric protein is rather small (~23 kDa), but oligomerizes to a ~280 kDa dodecamer. Within bioinformatic studies homologs of AaRP, termed HARPs (Homologs of Aquifex RNase P), were identified in some predominantly hyperthermophilic Bacteria and many Archaea. A focus of this work was to investigate the HARP of Thermodesulfatator indicus, particularly with regard to substrate recognition. Enzyme kinetic testing of substrate variants of pre-tRNAGly revealed that in the substrate, consisting of an intact acceptor stem and T-arm with 5'-leader und 3'-CCA-end, the important determinants for substrate recognition are present. Furthermore, neither 5'-leader nor 3'-end of the pre-tRNA were shown to be involved in essential enzyme interactions with the Thermodesulfatator indicus HARP. This suggests some similarity to the PRORP enzyme of Arabidopsis thaliana, which, unlike the bacterial, RNA-based RNase P, also does not interact with the 5'-leader and 3'-end of pre-tRNA substrates. However, the length-measuring mechanism of the acceptor stem and T-arm, already described for the PRORP enzyme, appears to be markedly relaxed in the Thermodesulfatator indicus HARP, such that the cleavage site is shifted to the end of the acceptor stem helix upon elongation of the T-stem.