Inhibition of enzymes in the isoprenoid biosynthetic pathway (IBP) plays an important role in the treatment of bone diseases and lowering cholesterol. The IBP begins with the enzyme HMG-CoA reductase catalyzing the conversion of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) to mevalonic acid. Mevalonic acid is then converted to isopentenyl pyrophosphate (IPP) via the intermediate mevalonate-5-diphosphate. Three molecules of IPP are joined by the enzyme farnesyl diphosphate synthase (FDPS), which yields the intermediate farnesyl pyrophosphate (FPP). FPP is an important substrate and represents the branch point in the pathway. Compounds which disrupt this pathway at FDPS include risedronate, lovastatin, and zoledronate. It is believed these compounds express their pharmacological effects on a further downstream enzyme. Further downstream from FDPS the IBP includes the key enzymes geranylgeranyl diphosphate synthase (GGDPS), which is responsible for the production of geranylgeranyl pyrophosphate (GGPP) and necessary for protein prenylation of the proteins Ras, Rho, and Rab. The second key enzyme is geranylgeranyl transferase II (GGTase II), which is responsible for the transfer of GGPP to Rab proteins. Rab proteins, which play an essential role for both protein secretion and trafficking, are of great interest as a therapeutic target for the bone disease multiple myeloma. Multiple myeloma is a disorder of malignant plasma cells, characterized by the overproduction of monoclonal protein, antibodies, or light chains, which can ultimately thicken the blood. Targeting the prevention of geranylgeranylation of the Rab proteins by the enzyme GGTase II can happen through two different strategies. The first would be an indirect route, which would aim to inhibit the enzymes upstream from GGTase II. This could include the enzymes HMG-CoA reductase, FDPS, and GGDPS. Drugs that inhibit the earlier steps in the IBP indirectly prevent protein geranylgeranylation but they also limit formation of other key processes further downstream. The second approach would be a direct inhibition of the enzyme responsible for protein prenylation, GGTase II. There are very few know inhibitors of GGTase II. One such inhibitor is a carboxyphosphonate 3-PEHPC, a mimic of the bisphosphonate risedronate. Unfortunately, 3-PEHPC does not display an attractive potency. Efforts to develop a more potent inhibitor of the enzyme GGTase II, have focused on the preparation of a family of carboxyphosphonates containing a triazole core, and these compounds were prepared via click chemistry. Their activity has been studied, but the salts that were successfully made were ultimately inactive in comparison to 3-PEHPC. Previous attempts at forming isoprene-containing triazole carboxyphosphonates, as isoprene chains were shown to help increase activity towards GGTase II, proved difficult and unsuccessful. Focus was then switched to alteration of isoprene bisphosphonate triazoles as GGDPS inhibitors in attempts to increase activity towards this enzyme. The biological activity of these bisphosphonates was found to be selective and potent inhibitors of GGDPS, with little to no activity towards the GGTase II enzyme. In an effort to develop more potent inhibitors, isoprene triazole carboxyphosphonates were produced in an attempt to enhance the biological activity towards GGTase II. Along with forming a family of isoprene bisphosphonate compounds to increase the biological activity towards GGDPS in comparison to other previously made bisphosphonates. These carboxyphosphonate and bisphosphonate compounds were prepared through click chemistry and tested for their activity toward GGTase II and GGDPS.

Farnesyl pyrophosphate synthase (FPPS) is a key branch point enzyme in the mevalonate pathway and the main molecular target of nitrogen-containing bisphosphonates (N-BPs), potent inhibitors of osteoclastic activity and the leading drug of choice for conditions characterized by excessive bone resorption. The main aim of this thesis is to investigate the interaction of N-BPs with FPPS in order to gain further insights into the mechanism of drug inhibition. Kinetic and crystallographic studies following site-directed mutagenesis of FPPS reveal key residues involved in stabilization of carbocation intermediate, substrate binding and formation of a tight enzyme-inhibitor complex. The aromatic ring of Tyr204 is involved in N-BP binding but not in the catalytic mechanism, where the hydroxyl moiety plays an important role. Lys200 is implicated in regulation of substrate binding, product specificity and enzyme isomerization which leads to a tight binding inhibition. Phe239 is considered important for the FPPS C-terminal switch which stabilizes substrate binding and promotes the inhibitor induced isomerized state. The highly conserved Arg112, Asp103 and Asp107 are pivotal for catalysis. Successful purification of the full length of Rab geranylgeranyl transferase (RGGT) complex downstream of the FPPS in the mevalonate pathway was achieved and may lead to co-crystallization with BP analogues and identification of the putative site of drug binding. Investigation of the in vitro effect of N-BPs on osteoclastogenesis suggest a correlation with FPPS inhibition kinetics for the most potent N-BPs but indicate an alternative mechanism of the disruption of bone resorption by alendronate. Together these results highlight the importance of the multiple interactions of N-BPs with side-chain residues of FPPS which dictate their strength of binding and advance the understanding of their pharmacophore effect.

Introducing effective irrigation management in arid and semi-arid regions, like most areas of South Africa, is an indispensable way of maximising crop yield and enhancing productivity of scarce freshwater resources. Holistic improvements in agricultural water management could be realised through integrating the knowledge of crop-specific water requirements. In order to develop effective irrigation schedules for rose-scented geranium (Pelargonium capitatum x P. radens), greenhouse and field experiments were conducted at the Hatfield Experimental Farm of the University of Pretoria, Pretoria, South Africa, from 28 October 2004 to 2006. Results from 20, 40, 60 and 80% maximum allowable depletion (MAD) levels of the plant available soil water (ASW) indicated that plant roots extracted most of the soil water from the top 40 cm soil layer, independent of the treatment. Both essential oil yield and fresh herbage mass responded positively to high soil water content. Increasing the MAD level to 60% and higher resulted in a significant reduction in herbage mass and essential oil yields. An increase in the degree of water stress apparently increased the essential oil concentration (percentage oil on fresh herbage mass basis), but its contribution to total essential oil yield (kg/ha oil) was limited. There was no significant relationship between MAD level and essential oil composition. For water saving without a significant reduction in essential oil yield of rose-scented geranium, a MAD of 40% of ASW is proposed. Response of rose-scented geranium to a one-month irrigation withholding period in the second or third month of regrowth cycles showed that herbage mass and oil yield were positively related. Herbage yield was significantly reduced when the water stress period was imposed during the third or fourth month of regrowth. A remarkable essential oil yield loss was observed only when the plants were stressed during the fourth month of regrowth. Essential oil content (% oil on fresh herbage mass basis) was higher in stressed plants, especially when stressed late, but oil yield dropped due to lower herbage mass. The relationship between essential oil composition and irrigation treatments was not consistent. Water-use efficiency was not significantly affected by withholding irrigation in the second or in the third month of regrowth. With a marginal oil yield loss, about 330 to 460 m3 of water per hectare per regrowth cycle could be saved by withholding irrigation during the third month of regrowth. The overall results highlighted that in water-scarce regions withholding irrigation during either the second or the third month of regrowth in rose-scented geranium could save water that could be used by other sectors of society. In greenhouse pot experiments, rose-scented geranium was grown under different irrigation frequencies, in two growth media. Irrigation was withheld on 50% of the plants (in each plot) for the week prior to harvesting. Herbage and essential oil yields were better in the sandy clay soil than in silica sand. Essential oil content (% oil on fresh herbage mass basis) apparently increased with a decrease in irrigation frequency. Both herbage and total essential oil yields positively responded to frequent irrigation. A one-week stress period prior to harvesting significantly increased essential oil content and total essential oil yield. Hence, the highest essential oil yield was obtained from a combination of high irrigation frequency and a one-week irrigation-withholding period. In the irrigation frequency treatments, citronellol and citronellyl formate contents tended to increase with an increase in the stress level, but the reverse was true for geraniol and geranyl formate. Leaf physiological data were recorded during the terminal one-week water stress in the glasshouse pot trial. Upon rewatering, stomatal conductance (Gs) and transpiration rate (Rt) were significantly lower in the less often irrigated than in the more often irrigated treatments, while leaf water potential (yw) and relative water content (RWC) were the same for all plants, indicating that water stress had an after-effect on Gs and Rt. At the end of the stress period, Gs, Rt, yw and RWC were lower in the plants from the more often irrigated than from the less often irrigated treatments. Irrespective of irrigation treatment, one type of non-glandular and two types (different in shape and size) of glandular trichomes were observed. In water stressed-conditions, stomata and trichome densities increased, while the total number of stomata and trichomes per leaf appeared to remain more or less the same. Water stress conditions resulted in stomatal closure. / Thesis (PhD)--University of Pretoria, 2009. / Plant Production and Soil Science / unrestricted