ABSTRACT
Polycystic kidney disease (PKD) is a common hereditary kidney disease. Although PKD occurrence is associated with certain gene mutations, its onset regulatory mechanisms are still not well understood. Here, we first report that the key enzyme geranylgeranyl diphosphate synthase (GGPPS) is specifically expressed in renal tubular epithelial cells of mouse kidneys. We aimed to explore the role of GGPPS in PKD. In this study, we established a Ggppsfl/fl:Cdh16cre mouse model and compared its phenotype with that of wild-type mice. A Ggpps-downregulation HK2 cell model was also used to further determine the role of GGPPS. We found that GGPPS was specifically expressed in renal tubular epithelial cells of mouse kidneys. Its expression also increased with age. Low GGPPS expression was observed in human ADPKD tissues. In the Ggppsfl/fl:Cdh16cre mouse model, Ggpps deletion in renal tubular epithelial cells induced the occurrence and development of renal tubule cystic dilation and caused the death of mice after birth due to abnormal renal function. Enhanced proliferation of cyst-lining epithelial cells was also observed after the knockout of Ggpps. These processes were related to the increased rate of Rheb on membrane/cytoplasm and hyperactivation of mTORC1 signaling. In conclusion, the deficiency of GGPPS in kidney tubules induced the formation of renal cysts. It may play a critical role in PKD pathophysiology. A novel therapeutic strategy could be designed according to this work.
Subject(s)
Kidney Tubules , Animals , Mice , Kidney Tubules/metabolism , Kidney Tubules/pathology , Humans , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/genetics , Epithelial Cells/metabolism , Epithelial Cells/pathology , Polycystic Kidney Diseases/genetics , Polycystic Kidney Diseases/pathology , Polycystic Kidney Diseases/metabolism , Male , Disease Models, Animal , Mice, Inbred C57BL , Kidney Diseases, Cystic/genetics , Kidney Diseases, Cystic/metabolism , Kidney Diseases, Cystic/pathology , Mice, Knockout , Cell Line , Multienzyme ComplexesABSTRACT
In the clinical development of farnesyltransferase inhibitors (FTIs) for HRAS-mutant tumors, responses varied by cancer type. Co-occurring mutations may affect responses. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and to study their effect on sensitivity to FTIs. Using targeted sequencing data from the MSK-IMPACT and Dana-Farber Cancer Institute Genomic Evidence Neoplasia Information Exchange databases, we identified comutations that were observed predominantly in HRAS-mutant versus KRAS-mutant or NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of coaltered mutations (48.8%) in the MAPK, PI3K, or RTK pathway genes, compared with KRAS-mutant (41.4%) and NRAS-mutant (38.4%) cancers (p < 0.05). Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effects of comutations on sensitivity to FTIs, HrasG13R was transfected into "RASless" (Kraslox/lox/Hras-/-/Nras-/-/RERTert/ert) mouse embryonic fibroblasts (MEFs), which sensitized nontransfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion and Pik3caH1047R transduction led to resistance to tipifarnib in HrasG13R-transfected MEFs in the presence or absence of KrasWT, whereas BrafG466E transduction led to resistance to tipifarnib only in the presence of KrasWT. Combined treatment with tipifarnib and MEK inhibition sensitized cells to tipifarnib in all settings, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage-dependent MAPK or PI3K pathway alterations, which confer resistance to tipifarnib. The combined use of FTIs and MEK inhibition is a promising strategy for HRAS-mutant tumors.
Subject(s)
Drug Resistance, Neoplasm , Farnesyltranstransferase , Mutation , Proto-Oncogene Proteins p21(ras) , Humans , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/genetics , Drug Resistance, Neoplasm/genetics , Proto-Oncogene Proteins p21(ras)/genetics , Animals , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Neoplasms/genetics , Neoplasms/drug therapy , Neoplasms/pathology , Cell Line, Tumor , Mice , Quinolones/pharmacology , Signal Transduction/drug effects , Signal Transduction/genetics , Genomics/methodsABSTRACT
Geranylgeranyl diphosphate synthase (GGPPS) is the crucial bottleneck in carotenoid biosynthesis. However, low activity limits the broad application of GGPPS. In this study, OsGGPPS1 in rice was engineered based on ancestral sequence reconstruction (ASR) and semirational design to improve the catalytic performances of existing GGPPS. The better mutant of A22R/A26P with improved enzyme activity was generated based on ASR. Additionally, the improved enzyme activity of mutants as V162A/M218S/F227Y was designed using a semirational design. The combinatorial assembly of the d-OsGGPPS1 mutant (A22R/A26P/V162A/M218S/F227Y) exhibited higher conversion of IPP and each cosubstrate of DMAPP for 9.8-fold in GPP production, GPP for 6.4-fold in FPP production, and FPP for 1.4-fold in GGPP production relative to wild-type OsGGPPS1 at 25 °C, which showed higher conversion than wild-type OsGGPPS1 at temperatures as high as 50 °C. The successful design of OsGGPPS1 was representative of protein engineering, which will shed new light on GGPPS engineering and active plant pigment resource utilization.
Subject(s)
Farnesyltranstransferase , Oryza , Plant Proteins , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Proteins/chemistry , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/chemistry , Oryza/genetics , Oryza/enzymology , Kinetics , Protein Engineering , BiocatalysisABSTRACT
ABSTRACT: A phase 2, international, open-label, nonrandomized, single-arm trial was conducted to evaluate the efficacy and safety of tipifarnib, a farnesyltransferase inhibitor, as monotherapy for relapsed/refractory peripheral T-cell lymphoma (PTCL) and to evaluate tumor mutation profile as a biomarker of response. Adults with relapsed/refractory PTCL received tipifarnib 300 mg orally twice daily for 21 days in a 28-day cycle. The primary end point was objective response rate (ORR); secondary end points included ORR, progression-free survival (PFS), duration of response (DOR), and adverse events (AEs) in specific subtypes. Sixty-five patients with PTCL were enrolled: n = 38 angioimmunoblastic T-cell lymphoma (AITL), n = 25 PTCL not otherwise specified, and n = 2 other T-cell lymphomas. The ORR was 39.7% (95% confidence interval [CI], 28.1-52.5) in all patients and 56.3% (95% CI, 39.3-71.8) for AITL. Median PFS was 3.5 months overall (954% CI, 2.1-4.4), and 3.6 months (95% CI, 1.9-8.3) for AITL. Median DOR was 3.7 months (95% CI, 2.0-15.3), and greatest in patients with AITL (7.8 months; 95% CI, 2.0-16.3). The median overall survival was 32.8 months (95% CI, 14.4 to not applicable). Tipifarnib-related hematologic AEs were manageable and included neutropenia (43.1%), thrombocytopenia (36.9%), and anemia (30.8%); other tipifarnib-related AEs included nausea (29.2%) and diarrhea (27.7%). One treatment-related death occurred. Mutations in RhoA, DNMT3A, and IDH2 were seen in 60%, 33%, and 27%, respectively, in the AITL tipifarnib responder group vs 36%, 9%, and 9% in the nonresponder group. Tipifarnib monotherapy demonstrated encouraging clinical activity in heavily pretreated relapsed/refractory PTCL, especially in AITL, with a manageable safety profile. This trial was registered at www.ClinicalTrials.gov as #NCT02464228.
Subject(s)
Farnesyltranstransferase , Lymphoma, T-Cell, Peripheral , Quinolones , Humans , Lymphoma, T-Cell, Peripheral/drug therapy , Lymphoma, T-Cell, Peripheral/mortality , Quinolones/therapeutic use , Quinolones/adverse effects , Female , Male , Middle Aged , Aged , Farnesyltranstransferase/antagonists & inhibitors , Adult , Aged, 80 and over , Treatment Outcome , Enzyme Inhibitors/therapeutic use , Enzyme Inhibitors/adverse effects , Neoplasm Recurrence, Local/drug therapy , Antineoplastic Agents/therapeutic use , Antineoplastic Agents/adverse effectsABSTRACT
Drug-tolerance has emerged as one of the major non-genetic adaptive processes driving resistance to targeted therapy (TT) in non-small cell lung cancer (NSCLC). However, the kinetics and sequence of molecular events governing this adaptive response remain poorly understood. Here, we combine real-time monitoring of the cell-cycle dynamics and single-cell RNA sequencing in a broad panel of oncogenic addiction such as EGFR-, ALK-, BRAF- and KRAS-mutant NSCLC, treated with their corresponding TT. We identify a common path of drug adaptation, which invariably involves alveolar type 1 (AT1) differentiation and Rho-associated protein kinase (ROCK)-mediated cytoskeletal remodeling. We also isolate and characterize a rare population of early escapers, which represent the earliest resistance-initiating cells that emerge in the first hours of treatment from the AT1-like population. A phenotypic drug screen identify farnesyltransferase inhibitors (FTI) such as tipifarnib as the most effective drugs in preventing relapse to TT in vitro and in vivo in several models of oncogenic addiction, which is confirmed by genetic depletion of the farnesyltransferase. These findings pave the way for the development of treatments combining TT and FTI to effectively prevent tumor relapse in oncogene-addicted NSCLC patients.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Drug Resistance, Neoplasm , Farnesyltranstransferase , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Humans , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/genetics , Lung Neoplasms/genetics , Lung Neoplasms/drug therapy , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Drug Resistance, Neoplasm/genetics , Cell Line, Tumor , Animals , Mice , Oncogene Addiction/genetics , Molecular Targeted Therapy , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Female , Xenograft Model Antitumor Assays , Oncogenes/genetics , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , QuinolonesABSTRACT
Respiratory syncytial virus (RSV) is the major cause of bronchiolitis and pneumonia in young children and the elderly. There are currently no approved RSV-specific therapeutic small molecules available. Using high-throughput antiviral screening, we identified an oral drug, the prenylation inhibitor lonafarnib, which showed potent inhibition of the RSV fusion process. Lonafarnib exhibited antiviral activity against both the RSV A and B genotypes and showed low cytotoxicity in HEp-2 and human primary bronchial epithelial cells (HBEC). Time-of-addition and pseudovirus assays demonstrated that lonafarnib inhibits RSV entry, but has farnesyltransferase-independent antiviral efficacy. Cryo-electron microscopy revealed that lonafarnib binds to a triple-symmetric pocket within the central cavity of the RSV F metastable pre-fusion conformation. Mutants at the RSV F sites interacting with lonafarnib showed resistance to lonafarnib but remained fully sensitive to the neutralizing monoclonal antibody palivizumab. Furthermore, lonafarnib dose-dependently reduced the replication of RSV in BALB/c mice. Collectively, lonafarnib could be a potential fusion inhibitor for RSV infection.
Subject(s)
Pyridines , Respiratory Syncytial Virus Infections , Respiratory Syncytial Virus, Human , Viral Fusion Proteins , Humans , Respiratory Syncytial Virus Infections/drug therapy , Respiratory Syncytial Virus Infections/genetics , Pyridines/pharmacology , Mice , Animals , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/genetics , Viral Fusion Proteins/genetics , Viral Fusion Proteins/antagonists & inhibitors , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/genetics , Antiviral Agents/pharmacology , Antiviral Agents/chemistry , Piperidines/pharmacology , Piperidines/chemistry , Mice, Inbred BALB C , Protein Conformation , DibenzocycloheptenesABSTRACT
As is the case for most solid tumours, chemotherapy remains the backbone in the management of metastatic disease. However, the occurrence of chemotherapy resistance is a cause to worry, especially in bladder cancer. Extensive evidence indicates molecular changes in bladder cancer cells to be the underlying cause of chemotherapy resistance, including the reduced expression of farnesyl-diphosphate farnesyltransferase 1 (FDFT1) - a gene involved in cholesterol biosynthesis. This can likely be a hallmark in examining the resistance and sensitivity of chemotherapy drugs. This work performs spectroscopic analysis and metabolite characterization on resistant, sensitive, stable-disease and healthy bladder tissues. Raman spectroscopy has detected peaks at around 1003 cm-1 (squalene), 1178 cm-1 (cholesterol), 1258 cm-1 (cholesteryl ester), 1343 cm-1 (collagen), 1525 cm-1 (carotenoid), 1575 cm-1 (DNA bases) and 1608 cm-1 (cytosine). The peak parameters were examined, and statistical analysis was performed on the peak features, attaining significant differences between the sample groups. Small-angle x-ray scattering (SAXS) measurements observed the triglyceride peak together with 6th, 7th and 8th - order collagen peaks; peak parameters were also determined. Neutron activation analysis (NAA) detected seven trace elements. Carbon (Ca), magnesium (Mg), chlorine (Cl) and sodium (Na) have been found to have the greatest concentration in the sample groups, suggestive of a role as a biomarker for cisplatin resistance studies. Results from the present research are suggested to provide an important insight into understanding the development of drug resistance in bladder cancer, opening up the possibility of novel avenues for treatment through personalised interventions.
Subject(s)
Cisplatin , Drug Resistance, Neoplasm , Spectrum Analysis, Raman , Urinary Bladder Neoplasms , Humans , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Cisplatin/pharmacology , Cisplatin/therapeutic use , Farnesyltranstransferase/metabolism , Spectrum Analysis, Raman/methods , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology , X-Ray Diffraction , Farnesyl-Diphosphate Farnesyltransferase/metabolismABSTRACT
Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.
Subject(s)
Computational Biology , Peptide Library , Humans , Computational Biology/methods , Substrate Specificity , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/chemistry , Oligopeptides/chemistry , Oligopeptides/metabolism , Amino Acid Sequence , Crystallography, X-Ray , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Protein BindingABSTRACT
Bioorthogonal chemistry has gained widespread use in the study of many biological systems of interest, including protein prenylation. Prenylation is a post-translational modification, in which one or two 15- or 20-carbon isoprenoid chains are transferred onto cysteine residues near the C-terminus of a target protein. The three main enzymesâprotein farnesyltransferase (FTase), geranylgeranyl transferase I (GGTase I), and geranylgeranyl transferase II (GGTase II)âthat catalyze this process have been shown to tolerate numerous structural modifications in the isoprenoid substrate. This feature has previously been exploited to transfer an array of farnesyl diphosphate analogues with a range of functionalities, including an alkyne-containing analogue for copper-catalyzed bioconjugation reactions. Reported here is the synthesis of an analogue of the isoprenoid substrate embedded with norbornene functionality (C10NorOPP) that can be used for an array of applications, ranging from metabolic labeling to selective protein modification. The probe was synthesized in seven steps with an overall yield of 7% and underwent an inverse electron demand Diels-Alder (IEDDA) reaction with tetrazine-containing tags, allowing for copper-free labeling of proteins. The use of C10NorOPP for the study of prenylation was explored in the metabolic labeling of prenylated proteins in HeLa, COS-7, and astrocyte cells. Furthermore, in HeLa cells, these modified prenylated proteins were identified and quantified using label-free quantification (LFQ) proteomics with 25 enriched prenylated proteins. Additionally, the unique chemistry of C10NorOPP was utilized for the construction of a multiprotein-polymer conjugate for the targeted labeling of cancer cells. That construct was prepared using a combination of norbornene-tetrazine conjugation and azide-alkyne cycloaddition, highlighting the utility of the additional degree of orthogonality for the facile assembly of new protein conjugates with novel structures and functions.
Subject(s)
Click Chemistry , Farnesyltranstransferase , Norbornanes , Protein Prenylation , Norbornanes/chemistry , Farnesyltranstransferase/metabolism , Humans , AnimalsABSTRACT
African swine fever (ASF) is an acute, hemorrhagic, and severe infectious disease caused by the ASF virus (ASFV). ASFV has evolved multiple strategies to escape host antiviral immune responses. Here, we reported that ASFV pB318L, a trans-geranylgeranyl-diphosphate synthase, reduced the expression of type I interferon (IFN-I) and IFN-stimulated genes (ISGs). Mechanically, pB318L not only interacted with STING to reduce the translocation of STING from the endoplasmic reticulum to the Golgi apparatus but also interacted with IFN receptors to reduce the interaction of IFNAR1/TYK2 and IFNAR2/JAK1. Of note, ASFV with interruption of B318L gene (ASFV-intB318L) infected PAMs produces more IFN-I and ISGs than that in PAMs infected with its parental ASFV HLJ/18 at the late stage of infection. Consistently, the pathogenicity of ASFV-intB318L is attenuated in piglets compared with its parental virus. Taken together, our data reveal that B318L gene may partially affect ASFV pathogenicity by reducing the production of IFN-I and ISGs. This study provides a clue to design antiviral agents or live attenuated vaccines to prevent and control ASF.
Subject(s)
African Swine Fever Virus , African Swine Fever , Interferon Type I , Animals , Swine , Farnesyltranstransferase/metabolism , Viral Proteins/metabolism , Nucleotidyltransferases/genetics , Nucleotidyltransferases/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Signal TransductionABSTRACT
Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.
Subject(s)
Anaplastic Lymphoma Kinase , Dibenzocycloheptenes , Farnesyltranstransferase , GTP Phosphohydrolases , MicroRNAs , Neuroblastoma , Piperidines , Protein Kinase Inhibitors , Pyridines , Animals , Female , Humans , Mice , Anaplastic Lymphoma Kinase/genetics , Anaplastic Lymphoma Kinase/metabolism , Anaplastic Lymphoma Kinase/antagonists & inhibitors , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Drug Resistance, Neoplasm/genetics , Drug Resistance, Neoplasm/drug effects , Drug Synergism , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/metabolism , Gene Expression Regulation, Neoplastic/drug effects , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , Membrane Proteins/metabolism , Membrane Proteins/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Mutation , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Neuroblastoma/pathology , Neuroblastoma/metabolism , Piperidines/pharmacology , Piperidines/therapeutic use , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Pyridines/pharmacology , Pyridines/therapeutic use , Xenograft Model Antitumor AssaysABSTRACT
Hutchinson-Gilford Progeria syndrome (HGPS) is a severe premature ageing disorder caused by a 50 amino acid truncated (Δ50AA) and permanently farnesylated lamin A (LA) mutant called progerin. On a cellular level, progerin expression leads to heterochromatin loss, impaired nucleocytoplasmic transport, telomeric DNA damage and a permanent growth arrest called cellular senescence. Although the genetic basis for HGPS has been elucidated 20 years ago, the question whether the Δ50AA or the permanent farnesylation causes cellular defects has not been addressed. Moreover, we currently lack mechanistic insight into how the only FDA-approved progeria drug Lonafarnib, a farnesyltransferase inhibitor (FTI), ameliorates HGPS phenotypes. By expressing a variety of LA mutants using a doxycycline-inducible system, and in conjunction with FTI, we demonstrate that the permanent farnesylation, and not the Δ50AA, is solely responsible for progerin-induced cellular defects, as well as its rapid accumulation and slow clearance. Importantly, FTI does not affect clearance of progerin post-farnesylation and we demonstrate that early, but not late FTI treatment prevents HGPS phenotypes. Collectively, our study unravels the precise contributions of progerin's permanent farnesylation to its turnover and HGPS cellular phenotypes, and how FTI treatment ameliorates these. These findings are applicable to other diseases associated with permanently farnesylated proteins, such as adult-onset autosomal dominant leukodystrophy.
Subject(s)
Lamin Type A , Progeria , Humans , Dibenzocycloheptenes , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/antagonists & inhibitors , Farnesyltranstransferase/genetics , Lamin Type A/metabolism , Lamin Type A/genetics , Piperidines , Progeria/metabolism , Progeria/genetics , Progeria/pathology , Progeria/drug therapy , Protein Prenylation , PyridinesABSTRACT
Depletion of cellular levels of geranylgeranyl diphosphate by inhibition of the enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential strategy for disruption of protein transport by limiting the geranylgeranylation of the Rab proteins that regulate intracellular trafficking. As such, there is interest in the development of GGDPS inhibitors for the treatment of malignancies characterized by abnormal protein production, including multiple myeloma. Our previous work has explored the structure-function relationship of a series of isoprenoid triazole bisphosphonate-based GGDPS inhibitors, with modifications having impact on enzymatic, cellular and in vivo activities. We have synthesized a new series of α-amino bisphosphonates to understand the impact of modifying the alpha position with a moiety that is potentially linkable to other agents. Bioassays evaluating the enzymatic and cellular activities of these compounds demonstrate that incorporation of the α-amino group affords compounds with GGDPS inhibitory activity which is modulated by isoprenoid tail chain length and olefin stereochemistry. These studies provide further insight into the complexity of the structure-function relationship and will enable future efforts focused on tumor-specific drug delivery.
Subject(s)
Diphosphonates , Enzyme Inhibitors , Diphosphonates/pharmacology , Diphosphonates/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Farnesyltranstransferase , Triazoles/chemistry , Terpenes/chemistryABSTRACT
Geranylgeranyl diphosphate synthase (GGPPS) as the short-chain prenyltransferases for catalyzing the formation of the acyclic precursor (E)-GGPP has been extensively investigated in mammals, plants, and microbes, but its functional plasticity is poorly understood in insect species. Here, a single GGPPS in leaf beetle Monolepta hieroglyphica, MhieGGPPS, was functionally investigated. Phylogenetic analysis showed that MhieGGPPS was clustered in one clade with homologs and had six conserved motifs. Molecular docking results indicated that binding sites of dimethylallyl diphosphate (DMAPP), (E)-geranyl pyrophosphate (GPP), and (E)-farnesyl pyrophosphate (FPP) were in the chain-length determination region of MhieGGPPS, respectively. In vitro, recombiant MhieGGPPS could catalyze the formation of (E)-geranylgeraniol against different combinations of substrates including isopentenyl pyrophosphate (IPP)/DMAPP, IPP/(E)-GPP, and IPP/(E)-FPP, suggesting that MhieGGPPS could not only use (E)-FPP but also (E)-GPP and DMAPP as the allylic cosubstrates. In kinetic analysis, the (E)-FPP was most tightly bound to MhieGGPPS than that of others. It was proposed that MhieGGPPS as a multifunctional enzyme is differentiated from the other GGPPSs in the animals and plants, which only accepted (E)-FPP as the allylic cosubstrate. These findings provide valuable insights into understanding the functional plasticity of GGPPS in M. hieroglyphica and the novel biosynthesis mechanism in the isoprenoid pathway.
Subject(s)
Coleoptera , Hemiterpenes , Organophosphorus Compounds , Polyisoprenyl Phosphates , Sesquiterpenes , Animals , Farnesyltranstransferase , Kinetics , Molecular Docking Simulation , Phylogeny , MammalsABSTRACT
Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Carotenoids , Gene Expression Regulation, Plant , Carotenoids/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Nudix Hydrolases , Chloroplasts/metabolism , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/metabolism , Geranylgeranyl-Diphosphate Geranylgeranyltransferase/genetics , Farnesyltranstransferase/metabolism , Farnesyltranstransferase/genetics , Pyrophosphatases/metabolism , Pyrophosphatases/genetics , Protein Processing, Post-Translational , Plants, Genetically Modified , Plant Leaves/metabolism , Plant Leaves/geneticsABSTRACT
Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.
Subject(s)
Enzyme Inhibitors , Neoplasms , Humans , Farnesyltranstransferase/chemistry , Farnesyltranstransferase/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Enzyme Inhibitors/chemistry , Terpenes/chemistry , Terpenes/pharmacology , Protein Prenylation , Neoplasms/drug therapyABSTRACT
Geranylgeranyl pyrophosphate (diphosphate) synthase (GGPPS) plays an important role in various physiological processes in insects, such as isoprenoid biosynthesis and protein prenylation. Here, we functionally characterised the GGPPS from the major agricultural lepidopteran pests Spodoptera frugiperda and Helicoverpa armigera. Partial disruption of GGPPS by CRISPR in S. frugiperda decreased embryo hatching rate and larval survival, suggesting that this gene is essential. Functional expression in vitro of Helicoverpa armigera GGPPS in Escherichia coli revealed a catalytically active enzyme. Next, we developed and optimised an enzyme assay to screen for potential inhibitors, such as the zoledronate and the minodronate, which showed a dose-dependent inhibition. Phylogenetic analysis of GGPPS across insects showed that GGPPS is highly conserved but also revealed several residues likely to be involved in substrate binding, which were substantially different in bee pollinator and human GGPPS. Considering the essentiality of GGPPS and its putative binding residue variability qualifies a GGPPS as a novel pesticide target. The developed assay may contribute to the identification of novel insecticide leads.
Subject(s)
Pesticides , Humans , Animals , Bees/genetics , Farnesyltranstransferase/genetics , Farnesyltranstransferase/metabolism , Phylogeny , Zoledronic AcidABSTRACT
Congenital muscular dystrophies are a group of progressive disorders with wide range of symptoms associated with diverse cellular mechanisms. Recently, biallelic variants in GGPS1 were linked to a distinct autosomal recessive form of muscular dystrophy associated with hearing loss and ovarian insufficiency. In this report, we present a case of a young patient with a homozygous variant in GGPS1. The patient presented with only proximal muscle weakness, and elevated liver transaminases with spared hearing function. The hepatic involvement in this patient caused by a novel deleterious variant in the gene extends the phenotypic and genotypic spectrum of GGPS1 related muscular dystrophy.
Subject(s)
Deafness , Dimethylallyltranstransferase , Hearing Loss , Muscular Dystrophies , Primary Ovarian Insufficiency , Female , Humans , Muscular Dystrophies/diagnosis , Muscular Dystrophies/genetics , Homozygote , Dimethylallyltranstransferase/genetics , Geranyltranstransferase/genetics , Farnesyltranstransferase/geneticsABSTRACT
Acute myeloid leukemia (AML) is a heterogeneous disease and the most common form of acute leukemia with a poor prognosis. Due to its complexity, the disease requires the identification of biomarkers for reliable prognosis. To identify potential disease genes that regulate patient prognosis, we used differential co-expression network analysis and transcriptomics data from relapsed, refractory, and previously untreated AML patients based on their response to treatment in the present study. In addition, we combined functional genomics and transcriptomics data to identify novel and therapeutically potential systems biomarkers for patients who do or do not respond to treatment. As a result, we constructed co-expression networks for response and non-response cases and identified a highly interconnected group of genes consisting of SECISBP2L, MAN1A2, PRPF31, VASP, and SNAPC1 in the response network and a group consisting of PHTF2, SLC11A2, PDLIM5, OTUB1, and KLRD1 in the non-response network, both of which showed high prognostic performance with hazard ratios of 4.12 and 3.66, respectively. Remarkably, ETS1, GATA2, AR, YBX1, and FOXP3 were found to be important transcription factors in both networks. The prognostic indicators reported here could be considered as a resource for identifying tumorigenesis and chemoresistance to farnesyltransferase inhibitor. They could help identify important research directions for the development of new prognostic and therapeutic techniques for AML.
Subject(s)
Leukemia, Myeloid, Acute , Humans , Farnesyltranstransferase/genetics , Farnesyltranstransferase/therapeutic use , Leukemia, Myeloid, Acute/drug therapy , Leukemia, Myeloid, Acute/genetics , Prognosis , Gene Expression Profiling/methods , Enzyme Inhibitors/therapeutic use , Transcription Factors/genetics , Biomarkers, Tumor/geneticsABSTRACT
Head and neck squamous cell carcinomas (HNSCCs) are often associated with poor outcomes, due at least in part to the limited number of treatment options available for those patients who develop recurrent and/or metastatic disease (R/M HNSCC). Even with the recent validation and approval of immunotherapies in the first-line setting for these patients, the need for the development of new and alternative precision medicine strategies with survival benefit is clear. Oncogenic alterations in the HRAS (Harvey rat sarcoma virus) proto-oncogene are seen in approximately 4-8% of R/M HNSCC tumors. Recently, several preclinical and clinical advancements have been made in the implementation of small-molecule inhibitors that block post-translational farnesylation of HRas, thereby abrogating its downstream oncogenic activity. In this review, we focus on the biology of wild-type and mutant HRas signaling in HNSCC, and rationale for use and outcomes of farnesyltransferase inhibitors in patients with HRAS-mutant tumors.