Hutchinson-Gilford progeria syndrome (HGPS), a progeroid syndrome in children, is caused by mutations in LMNA (the gene for prelamin A and lamin C) that result in the deletion of 50 aa within prelamin A. In normal cells, prelamin A is a ''CAAX protein'' that is farnesylated and then processed further to generate mature lamin A, which is a structural protein of the nuclear lamina. The mutant prelamin A in HGPS, which is commonly called progerin, retains the CAAX motif that triggers farnesylation, but the 50-aa deletion prevents the subsequent processing to mature lamin A. The presence of progerin adversely affects the integrity of the nuclear lamina, resulting in misshapen nuclei and nuclear blebs. We hypothesized that interfering with protein farnesylation would block the targeting of progerin to the nuclear envelope, and we further hypothesized that the mislocalization of progerin away from the nuclear envelope would improve the nuclear blebbing phenotype. To approach this hypothesis, we created a gene-targeted mouse model of HGPS, generated genetically identical primary mouse embryonic fibroblasts, and we then examined the effect of a farnesyltransferase inhibitor on nuclear blebbing. The farnesyltransferase inhibitor mislocalized progerin away from the nuclear envelope to the nucleoplasm, as determined by immunofluoresence microscopy, and resulted in a striking improvement in nuclear blebbing (P < 0.0001 by 2 statistic). These studies suggest a possible treatment strategy for HGPS.aging ͉ lamin A͞C ͉ laminopathy H utchinson-Gilford progeria syndrome (HGPS) is a progeroid syndrome characterized by a host of aging-like phenotypes, including a wizened appearance of the skin, osteoporosis, alopecia, and premature atherosclerosis (1). Children with HGPS die at the mean age of 13, generally from myocardial infarctions or strokes (1). This disease is caused by the accumulation of a mutant form of prelamin A that cannot be processed to mature lamin A (1). In normal cells, wild-type prelamin A is virtually undetectable because it is fully converted to mature lamin A, a structural protein of the nuclear lamina (2, 3). The nuclear lamina is an intermediate filament meshwork adjacent to the inner nuclear membrane that provides structural support for the nucleus (2, 3).Prelamin A contains a nuclear localization signal and terminates with a CAAX motif (2), in which C is a cysteine, A residues are usually aliphatic amino acids, and X can be one of many different residues. CAAX motifs are also found on lamin B1, lamin B2, the Ras family of proteins, and many other cellular proteins. The CAAX motif triggers three sequential enzymatic posttranslational modifications, beginning with protein prenylation. In the case of prelamin A, the first processing step is carried out by protein farnesyltransferase (FTase) and involves the addition of a 15-carbon farnesyl lipid to the thiol group of the cysteine within the CAAX motif. Second, the last 3 aa of the protein (i.e., ϪAAX) are removed by a prenylprotein-specific endoprotease. For p...
Hutchinson-Gilford progeria syndrome (HGPS) is a devastating premature aging disease resulting from a mutation in the LMNA gene, which encodes nuclear lamins A and C. Lamin A is synthesized as a precursor (prelamin A) with a C-terminal CaaX motif that undergoes farnesylation, endoproteolytic cleavage, and carboxylmethylation. Prelamin A is subsequently internally cleaved by the zinc metalloprotease Ste24 (Zmpste24) protease, which removes the 15 C-terminal amino acids, including the CaaX modifications, to yield mature lamin A. HGPS results from a dominant mutant form of prelamin A (progerin) that has an internal deletion of 50 aa near the C terminus that includes the Zmpste24 cleavage site and blocks removal of the CaaX-modified C terminus. Fibroblasts from HGPS patients have aberrant nuclei with irregular shapes, which we hypothesize result from the abnormal persistence of the farnesyl and͞or carboxylmethyl CaaX modifications on progerin. If this hypothesis is correct, inhibition of CaaX modification by mutation or pharmacological treatment should alleviate the nuclear morphology defect. Consistent with our hypothesis, we find that expression in HeLa cells of GFP-progerin or an uncleavable form of prelamin A with a Zmpste24 cleavage site mutation induces the formation of abnormal nuclei similar to those in HGPS fibroblasts. Strikingly, inhibition of farnesylation pharmacologically with the farnesyl transferase inhibitor rac-R115777 or mutationally by alteration of the CaaX motif dramatically reverses the abnormal nuclear morphology. These results suggest that farnesyl transferase inhibitors represent a possible therapeutic option for individuals with HGPS and͞or other laminopathies due to Zmpste24 processing defects.aging ͉ posttranslational processing ͉ laminopathy ͉ Ste24p ͉ Zarnestra
New therapeutics to combat malaria are desperately needed. Here we show that the enzyme protein farnesyltransferase (PFT) from the malaria parasite Plasmodium falciparum (P. falciparum) is an ideal drug target. PFT inhibitors (PFTIs) are well tolerated in man, but are highly cytotoxic to P. falciparum. Because of their anticancer properties, PFTIs comprise a highly developed class of compounds. PFTIs are ideal for the rapid development of antimalarials, allowing "piggy-backing" on previously garnered information. Low nanomolar concentrations of tetrahydroquinoline (THQ)-based PFTIs inhibit P. falciparum PFT and are cytotoxic to cultured parasites. Biochemical studies suggest inhibition of parasite PFT as the mode of THQ cytotoxicity. Studies with malaria-infected mice show that THQ PFTIs dramatically reduce parasitemia and lead to parasite eradication in the majority of animals. These studies validate P. falciparum PFT as a target for the development of antimalarials and describe a potent new class of THQ PFTIs with antimalaria activity.
Substituted tetrahydroquinolines (THQs) have been previously identified as inhibitors of mammalian protein farnesyltransferase (PFT). Previously we showed that blocking PFT in the malaria parasite led to cell death and that THQ-based inhibitors are the most potent among several structural classes of PFT inhibitors (PFTIs). We have prepared 266 THQ-based PFTIs and discovered several compounds that inhibit the malarial enzyme in the sub-to low-nanomolar range and that block the growth of the parasite (P. falciparum) in the low-nanomolar range. This body of structure-activity data can be rationalized in most cases by consideration of the X-ray structure of one of the THQs bound to mammalian PFT together with a homology structural model of the malarial enzyme. The results of this study provide the basis for selection of antimalarial PFTIs for further evaluation in preclinical drug discovery assays.
New antimalarials are urgently needed. We have shown that tetrahydroquinoline (THQ) protein farnesyltransferase (PFT) inhibitors (PFTIs) are effective against the Plasmodium falciparum PFT and are effective at killing P. falciparum in vitro. Previously described THQ PFTIs had limitations of poor oral bioavailability and rapid clearance from the circulation of rodents. In this paper, we validate both the Caco-2 cell permeability model for predicting THQ intestinal absorption and the in vitro liver microsome model for predicting THQ clearance in vivo. Incremental improvements in efficacy, oral absorption, and clearance rate were monitored by in vitro tests; and these tests were followed up with in vivo absorption, distribution, metabolism, and excretion studies. One compound, PB-93, achieved cure when it was given orally to P. berghei-infected rats every 8 h for a total of 72 h. However, PB-93 was rapidly cleared, and dosing every 12 h failed to cure the rats. Thus, the in vivo results corroborate the in vitro pharmacodynamics and demonstrate that 72 h of continuous high-level exposure to PFTIs is necessary to kill plasmodia. The metabolism of PB-93 was demonstrated by a novel technique that relied on double labeling with a radiolabel and heavy isotopes combined with radiometric liquid chromatography and mass spectrometry. The major liver microsome metabolite of PB-93 has the PFT Znbinding N-methyl-imidazole removed; this metabolite is inactive in blocking PFT function. By solving the X-ray crystal structure of PB-93 bound to rat PFT, a model of PB-93 bound to malarial PFT was constructed. This model suggests areas of the THQ PFTIs that can be modified to retain efficacy and protect the Zn-binding N-methyl-imidazole from dealkylation.
In the current study, injectable formulations containing Doxercalciferol as the active pharmaceutical ingredient are analyzed by using gradient-elution high-performance liquid chromatography with ultraviolet detection. Various related impurities and degradants are quantified by using solid-phase extraction (SPE) for enhanced sensitivity. The assay of possible related impurities and Doxercalciferol analogues present at trace quantities is performed by using Trans-1-α-hydroxy vitamin D2 (Doxercalciferol related degradation product/Impurity B) as standard and 1-β-hydroxy vitamin D2 (Doxercalciferol related degradation product/Impurity C) as internal standards for the SPE study. The current method is shown to be stability-indicating and free from interferences from any of the formulation excipients and potential degradation products and impurities. The validated method is shown to be reproducible, accurate, sensitive and selective.
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