Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.
Ontology highlight
ABSTRACT:
SUBMITTER: Bukhtiyarova M
PROVIDER: S-EPMC7812668 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The identification of activating mutations in NOTCH1 in 50% of T cell acute lymphoblastic leukemia has generated interest in elucidating how these mutations contribute to oncogenic transformation and in targeting the pathway. A phenotypic screen identified compounds that interfere with trafficking of Notch and induce apoptosis via an endoplasmic reticulum (ER) stress mechanism. Target identification approaches revealed a role for SLC39A7 (ZIP7), a zinc transport family member, in governing Notch trafficking and signaling. Generation and sequencing of a compound-resistant cell line identified a V430E mutation in ZIP7 that confers transferable resistance to the compound NVS-ZP7-4. NVS-ZP7-4 altered zinc in the ER, and an analog of the compound photoaffinity labeled ZIP7 in cells, suggesting a direct interaction between the compound and ZIP7. NVS-ZP7-4 is the first reported chemical tool to probe the impact of modulating ER zinc levels and investigate ZIP7 as a novel druggable node in the Notch pathway.
| S-EPMC7251565 | biostudies-literature
Project description:Prenylation is a posttranslational modification whereby C-terminal lipidation leads to protein localization to membranes. A C-terminal "Ca(1)a(2)X" sequence has been proposed as the recognition motif for two prenylation enzymes, protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I. To define the parameters involved in recognition of the a(2) residue, we performed structure-activity analysis which indicates that FTase discriminates between peptide substrates based on both the hydrophobicity and steric volume of the side chain at the a(2) position. For nonpolar side chains, the dependence of the reactivity on side chain volume at this position forms a pyramidal pattern with a maximal activity near the steric volume of valine. This discrimination occurs at a step in the kinetic mechanism that is at or before the farnesylation step. Furthermore, a(2) selectivity is also affected by the identity of the adjacent X residue, leading to context-dependent substrate recognition. Context-dependent a(2) selectivity suggests that FTase recognizes the sequence downstream of the conserved cysteine as a set of two or three cooperative, interconnected recognition elements as opposed to three independent amino acids. These findings expand the pool of proposed FTase substrates in cells. A better understanding of the molecular recognition of substrates performed by FTase will aid in both designing new FTase inhibitors as therapeutic agents and characterizing proteins involved in prenylation-dependent cellular pathways.
| S-EPMC2765569 | biostudies-literature
Project description:With 1.6 million casualties annually and 2 billion people being infected, tuberculosis is still one of the most pressing healthcare challenges. Here we report on the new computational docking algorithm FRIGATE which unites continuous local optimization techniques (conjugate gradient method) with an inherently discrete computational approach in forcefield computation, resulting in equal or better scoring accuracies than several benchmark docking programs. By utilizing FRIGATE for a virtual screen of the ZINC library against the Mycobacterium tuberculosis (Mtb) enzyme antigen 85C, we identified novel small molecule inhibitors of multiple drug-resistant Mtb, which bind in vitro to the catalytic site of antigen 85C.
| S-EPMC3229595 | biostudies-literature
Project description:Endotoxemia plays an important role in the pathogenesis of sepsis and is accompanied by dysregulated apoptosis of immune and non-immune cells. Treatment with statins reduces mortality in rodent models of sepsis and endotoxemia. Inhibition of protein isoprenylation, including farnesylation, has been proposed as a mechanism to mediate the lipid-lowering-independent effects of statins. Nonetheless, the effects of the inhibition of isoprenylation have not yet been studied. To investigate the role of farnesylation, we evaluated the effects of farnesyltransferase inhibitor and statin on survival following lipopolysaccharide (LPS) challenge in mice. Both simvastatin (2mg/kg BW) and FTI-277 (20mg/kg BW) treatment improved survival by twofold after LPS injection, as compared with vehicle alone (p<0.01). LPS-induced cleavage (activation) of caspase-3, an indicator of apoptotic change, and increased protein expression of proapoptotic molecules, Bax and Bim, and activation of c-Jun NH(2)-terminal kinase (JNK/SAPK) in the liver and spleen were attenuated by both simvastatin and FTI-277. These results demonstrate that farnesyltransferase inhibitor as well as statin significantly reduced LPS-induced mortality in mice. Our findings also suggest that inhibition of protein farnesylation may contribute to the lipid-lowering-independent protective effects of statins in endotoxemia, and that protein farnesylation may play a role in LPS-induced stress response, including JNK/SAPK activation, and apoptotic change. Our data argue that farnesyltransferase may be a potential molecular target for treating patients with endotoxemia.
| S-EPMC2813732 | biostudies-literature
Project description:Internalization and intracellular trafficking of G protein-coupled receptors (GPCRs) play pivotal roles in cell responsiveness. Dysregulation in receptor trafficking can lead to aberrant signaling and cell behavior. Here, using an endosomal BRET-based assay in a high-throughput screen with the prototypical GPCR angiotensin II type 1 receptor (AT1R), we sought to identify receptor trafficking inhibitors from a library of ~115,000 small molecules. We identified a novel dual Ras and ARF6 inhibitor, which we named Rasarfin, that blocks agonist-mediated internalization of AT1R and other GPCRs. Rasarfin also potently inhibits agonist-induced ERK1/2 signaling by GPCRs, and MAPK and Akt signaling by EGFR, as well as prevents cancer cell proliferation. In silico modeling and in vitro studies reveal a unique binding modality of Rasarfin within the SOS-binding domain of Ras. Our findings unveil a class of dual small G protein inhibitors for receptor trafficking and signaling, useful for the inhibition of oncogenic cellular responses.
| S-EPMC8333425 | biostudies-literature
Project description:The parasite Plasmodium falciparum causes severe malaria and is the most dangerous to humans. However, it exhibits resistance to their drugs. Farnesyltransferase has been identified in pathogenic protozoa of the genera Plasmodium and the target of farnesyltransferase includes Ras family. Therefore, the inhibition of farnesyltransferase has been suggested as a new strategy for the treatment of malaria. However, the exact functional mechanism of this agent is still unknown. In addition, the effect of farnesyltransferase inhibitor (FTIs) on mitochondrial level of malaria parasites is not fully understood. In this study, therefore, the effect of a FTI R115777 on the function of mitochondria of P. falciparum was investigated experimentally. As a result, FTI R115777 was found to suppress the infection rate of malaria parasites under in vitro condition. It also reduces the copy number of mtDNA-encoded cytochrome c oxidase III. In addition, the mitochondrial membrane potential (ΔΨm) and the green fluorescence intensity of MitoTracker were decreased by FTI R115777. Chloroquine and atovaquone were measured by the mtDNA copy number as mitochondrial non-specific or specific inhibitor, respectively. Chloroquine did not affect the copy number of mtDNA-encoded cytochrome c oxidase III, while atovaquone induced to change the mtDNA copy number. These results suggest that FTI R115777 has strong influence on the mitochondrial function of P. falciparum. It may have therapeutic potential for malaria by targeting the mitochondria of parasites.
| S-EPMC4566515 | biostudies-literature
Project description:Protein prenylation is a post-translational modification required for proper cellular localization and activity of many important eukaryotic proteins. Farnesyltransferase inhibitors (FTIs) have been explored extensively for their antitumor activity. To assist in identifying potentially new and more useful markers for therapeutic applications, we developed a strategy that uses a combination of metabolic labeling and 2D DIGE (differential gel electrophoresis) to discover new prenylated proteins whose cellular levels are influenced by FTIs. In this approach, metabolic labeling of prenylated proteins was first carried out with an alkyne-modified isoprenoid analog, C15Alk, in the presence or absence of the FTI L-744,832. The resulting alkyne-tagged proteins were then labeled with Cy3-N3 and Cy5-N3 and subjected to 2D-DIGE. Multiple spots having altered levels of labeling in presence of the FTI were observed. Mass spectrometric analysis of some of the differentially labeled spots identified several known prenylated proteins, along with HisRS, PACN-3, GNAI-1 and GNAI-2, which are not known to be prenylated. In vitro farnesylation of a C-terminal peptide sequence derived from GNAI-1 and GNAI-2 produced a farnesylated product, suggesting GNAI-1 and GNAI-2 are potential novel farnesylated proteins. These results suggest that this new strategy could be useful for the identification of prenylated proteins whose level of post-translational modification has been modulated by the presence of an FTI. Additionally, this approach, which decreases sample complexity and thereby facilitates analysis, should be applicable to studies of other post-translational modifications as well.
| S-EPMC4058996 | biostudies-literature
Project description:HIV-1 Nef is a viral accessory protein critical for AIDS progression. Nef lacks intrinsic catalytic activity and binds multiple host cell signaling proteins, including Hck and other Src-family tyrosine kinases. Nef binding induces constitutive Hck activation that may contribute to HIV pathogenesis by promoting viral infectivity, replication and downregulation of cell-surface MHC-I molecules. In this study, we developed a yeast-based phenotypic screen to identify small molecules that inhibit the Nef-Hck complex.Nef-Hck interaction was faithfully reconstituted in yeast cells, resulting in kinase activation and growth arrest. Yeast cells expressing the Nef-Hck complex were used to screen a library of small heterocyclic compounds for their ability to rescue growth inhibition. The screen identified a dihydrobenzo-1,4-dioxin-substituted analog of 2-quinoxalinyl-3-aminobenzene-sulfonamide (DQBS) as a potent inhibitor of Nef-dependent HIV-1 replication and MHC-I downregulation in T-cells. Docking studies predicted direct binding of DQBS to Nef which was confirmed in differential scanning fluorimetry assays with recombinant purified Nef protein. DQBS also potently inhibited the replication of HIV-1 NL4-3 chimeras expressing Nef alleles representative of all M-group HIV-1 clades.Our findings demonstrate the utility of a yeast-based growth reversion assay for the identification of small molecule Nef antagonists. Inhibitors of Nef function discovered with this assay, such as DQBS, may complement the activity of current antiretroviral therapies by enabling immune recognition of HIV-infected cells through the rescue of cell surface MHC-I.
| S-EPMC3874621 | biostudies-literature
Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes severe health problems. Despite intensive investigation, many aspects of microbial virulence remain poorly understood. We used a high-throughput, high-content, whole-organism, phenotypic screen to identify small molecules that inhibit P. aeruginosa virulence in Caenorhabditis elegans. Approximately half of the hits were known antimicrobials. A large number of hits were nonantimicrobial bioactive compounds, including the cancer chemotherapeutic 5-fluorouracil. We determined that 5-fluorouracil both transiently inhibits bacterial growth and reduces pyoverdine biosynthesis. Pyoverdine is a siderophore that regulates the expression of several virulence determinants and is critical for pathogenesis in mammals. We show that 5-fluorouridine, a downstream metabolite of 5-fluorouracil, is responsible for inhibiting pyoverdine biosynthesis. We also show that 5-fluorouridine, in contrast to 5-fluorouracil, is a genuine antivirulence compound, with no bacteriostatic or bactericidal activity. To our knowledge, this is the first report utilizing a whole-organism screen to identify novel compounds with antivirulent properties effective against P. aeruginosa. IMPORTANCE Despite intense research effort from scientists and the advent of the molecular age of biomedical research, many of the mechanisms that underlie pathogenesis are still understood poorly, if at all. The opportunistic human pathogen Pseudomonas aeruginosa causes a variety of soft tissue infections and is responsible for over 50,000 hospital-acquired infections per year. In addition, P. aeruginosa exhibits a striking degree of innate and acquired antimicrobial resistance, complicating treatment. It is increasingly important to understand P. aeruginosa virulence. In an effort to gain this information in an unbiased fashion, we used a high-throughput phenotypic screen to identify small molecules that disrupted bacterial pathogenesis and increased host survival using the model nematode Caenorhabditis elegans. This method led to the unexpected discovery that addition of a modified nucleotide, 5-fluorouridine, disrupted bacterial RNA metabolism and inhibited synthesis of pyoverdine, a critical toxin. Our results demonstrate that this compound specifically functions as an antivirulent.
| S-EPMC4999921 | biostudies-literature
Project description:In this study, we used high-throughput computational screening to discover drug-like inhibitors of the host MyD88 protein-protein signaling interaction implicated in the potentially lethal immune response associated with Staphylococcal enterotoxins. We built a protein-protein dimeric docking model of the Toll-interleukin receptor (TIR)-domain of MyD88 and identified a binding site for docking small molecules. Computational screening of 5 million drug-like compounds led to testing of 30 small molecules; one of these molecules inhibits the TIR-TIR domain interaction and attenuates pro-inflammatory cytokine production in human primary cell cultures. Compounds chemically similar to this hit from the PubChem database were observed to be more potent with improved drug-like properties. Most of these 2(nd) generation compounds inhibit Staphylococcal enterotoxin B (SEB)-induced TNF-?, IFN-?, IL-6, and IL-1? production at 2-10 ?M in human primary cells. Biochemical analysis and a cell-based reporter assay revealed that the most promising compound, T6167923, disrupts MyD88 homodimeric formation, which is critical for its signaling function. Furthermore, we observed that administration of a single dose of T6167923 completely protects mice from lethal SEB-induced toxic shock. In summary, our in silico approach has identified anti-inflammatory inhibitors against in vitro and in vivo toxin exposure with promise to treat other MyD88-related pro-inflammatory diseases.
| S-EPMC4585646 | biostudies-literature