Cryptococcus neoformans can be an opportunistic human fungal pathogen that can cause life-threatening respiratory and neurological infections. B-based drugs) echinocandins antimetabolites (flucytosine) and azoles. Of these only the polyenes azoles and flucytosine have activity against C. neoformans infections (4). Drug toxicity and antifungal resistance often limit the usage of these medicines (5). There’s an urgent have to develop fresh antifungal therapeutics therefore. One successful plan for developing low priced therapeutics for neglected illnesses would be to repurpose or iteratively improve existing medications originally created for various other therapies (6-9). For greater than a 10 years SN 38 manufacture inhibitors of protein farnesyltransferase (FTIs)2 have already been under analysis as tumor chemotherapeutics using a concomitant advancement of a substantial number of business lead compounds and id from the structural properties that information FTI style (10). Right here we record that FTase as a result can also be an attractive brand-new target for the introduction of low priced antifungal therapeutics targeted against C. neoformans. FTase is really a ubiquitous eukaryotic enzyme that catalyzes post-translational lipidation from the C terminus greater than 60 essential signaling proteins (10 11 Protein substrates of FTase keep a C-terminal series CAAX theme: cysteine (C) two generally aliphatic residues (AA) along with a adjustable (X) residue. The lipid substrate of FTase may be the 15-carbon isoprenoid farnesyl pyrophosphate (FPP). FTase is really a zinc-dependent metalloenzyme. Response intermediates have already been described structurally SN 38 manufacture in hFTase by way of a group of crystallographic snapshots used along the response organize and corroborated by kinetic research (12-15). Through the entire response pathway the enzyme framework continues to be rigid whereas the isoprenoid substrate undergoes conformational rearrangement (12 13 16 The gradual part of the response is item release that is accelerated with the addition FCF1 of another lipid substrate molecule (15). A structural intermediate from the individual enzyme reveals that before its discharge the lipidated peptide item is displaced right into a shallow hydrophobic “leave groove” positioned next to the active site (12). In mammals FTase is usually dispensable for adult homeostasis although it is required during stages of embryonic development (17). In Saccharomyces cerevisiae deletion of FTase results in alterations of cell fitness but the enzyme itself is not essential (18). In these organisms loss of FTase activity can be compensated for by the related enzyme protein geranylgeranyltransferase-I (19 20 By contrast the C. neoformans FTase (CnFTase) is essential for viability (21). Here we show that several previously developed anticancer FTIs inhibit CnFTase and exhibit C. neoformans fungicidal activity. Phase III clinical candidate tipifarnib (22 23 (R115777 or Zarnestra) and a recently described ethylenediamine scaffold inhibitor (24-26) demonstrate significant growth inhibition effects on C. neoformans. Manumycin A a natural product FTI (27) is the most potent FTI tested and exhibits fungicidal activity. Treatment with manumycin A interferes with localization of Ras1 at the cell membrane of C. neoformans. We report the x-ray structures of CnFTase that define its reaction cycle and inhibitor binding modes and identify regions that are structurally divergent relative to hFTase. A substrate-induced conformational change further mechanistically distinguishes CnFTase from hFTase along the reaction pathway. Taken together these data provide compelling evidence that the essential FTase of C. neoformans is an attractive drug target. Additionally these studies offer a paradigm by which known FTI scaffolds may be re-purposed to accelerate and lower the cost of novel antifungal inhibitor development. EXPERIMENTAL PROCEDURES Antifungal Compound Testing Six FTIs were tested for antifungal activity: L-778 123 (28) L-744 832 (29) manumycin A (27) tipifarnib (23) and two ethylenediamine-scaffold inhibitors (24-26). Inhibitor stocks were 20 mm in DMSO. Amphotericin B was used as a positive control (30); its stock concentration was 5.4 mm in DMSO. Determination of minimal inhibitor concentration (MIC) was performed according to the Clinical and Laboratory Standards Institute/National Committee for Clinical Laboratory Standards standard assay with modification of the growth medium made based on published protocols because of poor development.