FTase Inhibitors Research Articles

Development of erythrina-based PARP-1/FTase dual-target inhibitors against lung cancer epithelial-mesenchymal transition (EMT) in vivo and in vitro

A novel series of erythrina derivatives as PARP-1/FTase inhibitors were synthesized, and evaluated for their biological activities. Compound T9 had excellent inhibitory effects on cell viability (A549: IC50 = 1.74 μM; A549/5-Fu: IC50 = 1.03 μM) and in vitro enzyme activities (PARP-1: IC50 = 0.40 μM; FTase: IC50 = 0.067 μM). Molecular docking and point mutation assays demonstrated the interaction of compound T9 with key amino acid residues. The compound T9 exhibited potent anti-proliferation and anti-migration capabilities against A549 and A549/5-Fu cells. PCR array and western blot results showed that compound T9 could effectively inhibit EMT-related proteins in A549 and A549/5-Fu cells, thereby inhibiting the development of lung cancer. Importantly, compound T9 could significantly inhibit tumor growth in the A549 xenograft tumor model (TGI = 65.3 %). In conclusion, this study was the first presentation of the concept of dual-target inhibitors of the PARP-1/FTase enzymes. It also provides the basis for further research and development of novel PARP-1/FTase inhibitors.

FTase inhibitors and cancer: prospects for use in targeted therapies

FTase inhibitors and cancer: prospects for use in targeted therapies

Discovery of farnesyltransferase inhibitors (FTIs) with high brain exposure for treatment of Alzheimer’s disease and other tauopathies (T‐PEP Related Presentation)

AbstractBackgroundFarnesyltransferase (FTase) is a prenyltransferase enzyme that adds farnesyldiphosphate to the cysteine of CaaX proteins. Compelling evidence suggests that FTase inhibitors (FTIs) cause lysosome activation and a dramatic increase in tau degradation (Hernandez, et al. Science Translational Medicine 11.485 (2019): eaat3005)MethodNovel small molecule inhibitors of FTase were synthesized and tested in a luminescence‐based enzymatic assay of FTase inhibition. Active compounds were tested in ADME assays including MDCK‐MDR1 cells to evaluate permeability and transporter‐mediated efflux. Optimized compounds were evaluated in pharmacokinetics and in vivo pharmacodynamics (PD) experiments measuring enzymatic farnesylation of farnesyl alcohol azide in the mouse brain.ResultWe report the first highly brain‐penetrant FTase inhibitors. These compounds show nanomolar potency and achieve high exposure in both plasma and brain. PD experiments show that the compounds inhibit farnesylation in the CNS of our mouse modelConclusionBrain penetrant small molecule FTase inhibitors show promising preclinical pharmacology and are currently in the lead optimization stage of drug discovery.

Prenylated PALM2 Promotes the Migration of Esophageal Squamous Cell Carcinoma Cells Through Activating Ezrin

Proteins containing a CAAX motif at the C-terminus undergo prenylation for localization and activity and include a series of key regulatory proteins, such as RAS superfamily members, heterotrimeric G proteins, nuclear lamina protein, and several protein kinases and phosphatases. However, studies of prenylated proteins in esophageal cancer are limited. Here, through research on large-scale proteomic data of esophageal cancer in our laboratory, we found that paralemmin-2 (PALM2), a potential prenylated protein, was upregulated and associated with poor prognosis in patients. Low-throughput verification showed that the expression of PALM2 in esophageal cancer tissues was higher than that in their paired normal esophageal epithelial tissues, and it was generally expressed in the membrane and cytoplasm of esophageal cancer cells. PALM2 interacted with the two subunits of farnesyl transferase (FTase), FNTA and FNTB. Either the addition of an FTase inhibitor or mutation in the CAAX motif of PALM2 (PALM2C408S) impaired its membranous localization and reduced the membrane location of PALM2, indicating PALM2 was prenylated by FTase. Overexpression of PALM2 enhanced the migration of esophageal squamous cell carcinoma cells, whereas PALM2C408S lost this ability. Mechanistically, PALM2 interacted with the N-terminal FERM domain of ezrin of the ezrin/radixin/moesin (ERM) family. Mutagenesis indicated that lysine residues K253/K254/K262/K263 in ezrin’s FERM domain and C408 in PALM2’s CAAX motif were important for PALM2/ezrin interaction and ezrin activation. Knockout of ezrin prevented enhanced cancer cell migration by PALM2 overexpression. PALM2, depending on its prenylation, increased both ezrin membrane localization and phosphorylation of ezrin at Y146. In summary, prenylated PALM2 enhances the migration of cancer cells by activating ezrin.

Abstract 6149: STAT3 activation promotes invasiveness and cancer stemness in lung cancer

Abstract Lung cancer is the leading cause of cancer-related death worldwide, featuring a high incidence and low 5-year survival rate. Nearly 40% of patients are diagnosed with metastatic disease when they are first presented to the clinic. However, drug intervention targeting established metastatic tumors remains challenging, a better understanding of therapeutically exploitable mechanisms is urgently needed. This present study aims to explore novel therapeutic intervention against metastatic lung cancer. To understand the tumor-cell-intrinsic pathway that drives metastatic progression, we generated a highly invasive subline of lung cancer cell - A549 (ATCC, USA) by serial selections for Matrigel-coated transwells. The invasive subline A549-i3 shows about 5 times higher invasion potential than the parental cell line A549-Par (p<0.05, Student t-test). Colony-formation Assay and suspension growth in ultra-low attachment plates were used to measure the 2D proliferation ability and anoikis resistance, albeit with no difference. The differential efficacies between A549-Par and A549-i3 against multiple pathway inhibitors were screened using MTT assay in vitro. Notably, A549-i3 is more sensitive towards napabucasin (STAT3 inhibitor) and tipifarnib (FTase inhibitor) when compared with A549-Par, suggesting their potency in targeting cancer stemness and addicted oncogenic driver. Furthermore, napabucasin reduced the migration and invasion abilities of A549-i3 to similar extent as their parent counterpart. Overall, our preliminary data identified STAT3 as a potential therapeutic vulnerability in highly invasive cancer cells. Future work will be focused on identifying pathways responsible to sensitize highly invasive NSCLC cells toward STAT3 inhibition using multiomics approach. This project was supported by the Research Impact Fund (Ref. No.: R4015-19) from the Research Grants Council in Hong Kong. Citation Format: Sai Fung Yeung, Sze Man Chan, Chun Ho Law, Hoi Yin Chan, Kwok Wing Tsui. STAT3 activation promotes invasiveness and cancer stemness in lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6149.

Impaired Autophagic-Lysosomal Fusion in Parkinson's Patient Midbrain Neurons Occurs through Loss of ykt6 and Is Rescued by Farnesyltransferase Inhibition.

Macroautophagy is a catabolic process that coordinates with lysosomes to degrade aggregation-prone proteins and damaged organelles. Loss of macroautophagy preferentially affects neuron viability and is associated with age-related neurodegeneration. We previously found that α-synuclein (α-syn) inhibits lysosomal function by blocking ykt6, a farnesyl-regulated soluble NSF attachment protein receptor (SNARE) protein that is essential for hydrolase trafficking in midbrain neurons. Using Parkinson's disease (PD) patient iPSC-derived midbrain cultures, we find that chronic, endogenous accumulation of α-syn directly inhibits autophagosome-lysosome fusion by impairing ykt6-SNAP-29 complexes. In wild-type (WT) cultures, ykt6 depletion caused a near-complete block of autophagic flux, highlighting its critical role for autophagy in human iPSC-derived neurons. In PD, macroautophagy impairment was associated with increased farnesyltransferase (FTase) activity, and FTase inhibitors restored macroautophagic flux through promoting active forms of ykt6 in human cultures, and male and female mice. Our findings indicate that ykt6 mediates cellular clearance by coordinating autophagic-lysosomal fusion and hydrolase trafficking, and that macroautophagy impairment in PD can be rescued by FTase inhibitors.SIGNIFICANCE STATEMENT The pathogenic mechanisms that lead to the death of neurons in Parkinson's disease (PD) and Dementia with Lewy bodies (LBD) are currently unknown. Furthermore, disease modifying treatments for these diseases do not exist. Our study indicates that a cellular clearance pathway termed autophagy is impaired in patient-derived culture models of PD and in vivo We identified a novel druggable target, a soluble NSF attachment protein receptor (SNARE) protein called ykt6, that rescues autophagy in vitro and in vivo upon blocking its farnesylation. Our work suggests that farnesyltransferase (FTase) inhibitors may be useful therapies for PD and DLB through enhancing autophagic-lysosomal clearance of aggregated proteins.

The identities of insulin signaling pathway are affected by overexpression of Tau and its phosphorylation form.

Hyperphosphorylated Tau formed neurofibrillary tangles was one of the major neuropathological hallmarks of Alzheimer's disease (AD). Dysfunctional insulin signaling in brain is involved in AD. However, the effect of Tau pathology on brain insulin resistance remains unclear. This study explored the effects of overexpressing wild-type Tau (WTau) or Tau with pseudo-phosphorylation at AT8 residues (PTau) on the insulin signaling pathway (ISP). 293T cells or SY5Y cells overexpressing WTau or PTau were treated with or without insulin. The elements in ISP or the regulators of IPS were analyzed by immunoblotting, immunofluorescent staining and co-immunoprecipitation. Akt inhibitor MK2206 was used for evaluating the insulin signaling to downstream of mTOR in Tau overexpressing cells. The effects of anti-aging drug lonafarnib on ISP in WTau or PTau cells were also analyzed with immunoblotting. Considering lonafarnib is an inhibitor of FTase, the states of Rhes, one of FTase substrate in WTau or PTau cells were analyzed by drug affinity responsive target stability (DARTS) assay and the cellular thermal shift assay (CETSA). WTau or PTau overexpression in cells upregulated basal activity of elements in ISP in general. However, overexpression of WTau or PTau suppressed the ISP signaling transmission responses induced by insulin simulation, appearing relative higher response of IRS-1 phosphorylation at tyrosine 612 (IRS-1 p612) in upstream IPS, but a lower phosphorylation response of downstream IPS including mTOR, and its targets 4EPB1 and S6. This dysregulation of insulin evoked signaling transmission was more obvious in PTau cells. Suppressing Akt with MK2206 could compromise the levels of p-S6 and p-mTOR in WTau or PTau cells. Moreover, the changes of phosphatases detected in WTau and PTau cells may be related to ISP dysfunction. In addition, the effects of lonafarnib on the ISP in SY5Y cells with WTau and PTau overexpression were tested, which showed that lonafarnib treatment resulted in reducing the active levels of ISP elements in PTau cells but not in WTau cells. The differential effects are probably due to Tau phosphorylation modulating lonafarnib-induced alterations in Rhes, as revealed by DARTS assay. Overexpression of Tau or Tau with pseudo-phosphorylation at AT8 residues could cause an upregulation of the basal/tonic ISP, but a suppression of insulin induced the phasic activation of ISP. This dysfunction of ISP was more obvious in cells overexpressing pseudo-phosphorylated Tau. These results implied that the dysfunction of ISP caused by Tau overexpression might impair the physiological fluctuation of neuronal functions in AD. The different effects of lonafarnib on ISP between WTau and PTau cells, indicating that Tau phosphorylation mediates an additional effect on ISP. This study provided a potential linkage of abnormal expression and phosphorylation of Tau to the ISP dysfunction in AD.

Analyzing the postulated inhibitory effect of Manumycin A on farnesyltransferase.

Manumycin A is postulated to be a specific inhibitor against the farnesyltransferase (FTase) since this effect has been shown in 1993 for yeast FTase. Since then, plenty of studies investigated Manumycin A in human cells as well as in model organisms like Caenorhabditis elegans. Some studies pointed to additional targets and pathways involved in Manumycin A effects like apoptosis. Therefore, these studies created doubt whether the main mechanism of action of Manumycin A is FTase inhibition. For some of these alternative targets half maximal inhibitory concentrations (IC50) of Manumycin A are available, but not for human and C. elegans FTase. So, we aimed to 1) characterize missing C. elegans FTase kinetics, 2) elucidate the IC50 and Ki values of Manumycin A on purified human and C. elegans FTase 3) investigate Manumycin A dependent expression of FTase and apoptosis genes in C. elegans. C. elegans FTase has its temperature optimum at 40°C with KM of 1.3µM (farnesylpyrophosphate) and 1.7µM (protein derivate). Whilst other targets are inhibitable by Manumycin A at the nanomolar level, we found that Manumycin A inhibits cell-free FTase in micromolar concentrations (Ki human 4.15μM; Ki C. elegans 3.16μM). Furthermore, our gene expression results correlate with other studies indicating that thioredoxin reductase 1 is the main target of Manumycin A. According to our results, the ability of Manumycin A to inhibit the FTase at the micromolar level is rather neglectable for its cellular effects, so we postulate that the classification as a specific FTase inhibitor is no longer valid.

Impact of a conserved N-terminal proline-rich region of the α-subunit of CAAX-prenyltransferases on their enzyme properties

BackgroundThe CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique β-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far.MethodsTo determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein–protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays.ResultsThe PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological β-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The KM values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species.ConclusionsOur data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I.83PbrUwLcXbuUPceRJ4UzrVideo

Targeting farnesylation as a novel therapeutic approach in HRAS-mutant rhabdomyosarcoma

Activating RAS mutations are found in a subset of fusion-negative rhabdomyosarcoma (RMS), and therapeutic strategies to directly target RAS in these tumors have been investigated, without clinical success to date. A potential strategy to inhibit oncogenic RAS activity is the disruption of RAS prenylation, an obligate step for RAS membrane localization and effector pathway signaling, through inhibition of farnesyltransferase (FTase). Of the major RAS family members, HRAS is uniquely dependent on FTase for prenylation, whereas NRAS and KRAS can utilize geranylgeranyl transferase as a bypass prenylation mechanism. Tumors driven by oncogenic HRAS may therefore be uniquely sensitive to FTase inhibition. To investigate the mutation-specific effects of FTase inhibition in RMS we utilized tipifarnib, a potent and selective FTase inhibitor, in in vitro and in vivo models of RMS genomically characterized for RAS mutation status. Tipifarnib reduced HRAS processing, and plasma membrane localization leading to decreased GTP-bound HRAS and decreased signaling through RAS effector pathways. In HRAS-mutant cell lines, tipifarnib reduced two-dimensional and three-dimensional cell growth, and in vivo treatment with tipifarnib resulted in tumor growth inhibition exclusively in HRAS-mutant RMS xenografts. Our data suggest that small molecule inhibition of FTase is active in HRAS-driven RMS and may represent an effective therapeutic strategy for a genomically-defined subset of patients with RMS.

Lonafarnib Inhibits Farnesyltransferase via Suppressing ERK Signaling Pathway to Prevent Osteoclastogenesis in Titanium Particle-Induced Osteolysis.

Wear debris after total joint arthroplasty can attract the recruitment of macrophages, which release pro-inflammatory substances, triggering the activation of osteoclasts, thereby leading to periprosthetic osteolysis (PPOL) and aseptic loosening. However, the development of pharmacological strategies targeting osteoclasts to prevent periprosthetic osteolysis has not been fruitful. In this study, we worked toward researching the effects and mechanisms of a farnesyltransferase (FTase) inhibitor Lonafarnib (Lon) on receptor activator of nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis and bone resorption, as well as the impacts of Lon on titanium particle-induced osteolysis. To investigate the impacts of Lon on bone resorption and osteoclastogenesis in vitro, bone marrow macrophages were incubated and stimulated with RANKL and macrophage colony-stimulating factor (M-CSF). The influence of Lon on osteolysis prevention in vivo was examined utilizing a titanium particle-induced mouse calvarial osteolysis model. The osteoclast-relevant genes expression was explored by real-time quantitative PCR. Immunofluorescence was used to detect intracellular localization of nuclear factor of activated T cells 1 (NFATc1). SiRNA silence assay was applied to examine the influence of FTase on osteoclasts activation. Related signaling pathways, including NFATc1 signaling, NF-κB, mitogen-activated protein kinases pathways were identified by western blot assay. Lon was illustrated to suppress bone resorptive function and osteoclastogenesis in vitro, and it also reduced the production of pro-inflammatory substances and prevented titanium particle-induced osteolysis in vivo. Lon decreased the expression of osteoclast-relevant genes and suppressed NFATc1 nuclear translocation and auto-amplification. Mechanistically, Lon dampened FTase, and inhibition of FTase reduced osteoclast formation by suppressing ERK signaling. Lon is a promising treatment option for osteoclast-related osteolysis diseases including periprosthetic osteolysis by targeted inhibition of FTase through suppressing ERK signaling.

Inhibiting the "Undruggable" RAS/Farnesyltransferase (FTase) Cancer Target by Manumycin-related Natural Products.

Cancer is an uncontrolled cell growth that can generate diverse types of cancer, in which these will also present a different behavior in the face of pharmacological treatment. These cancers' types are found in one of the three categories, leukemias (also named lymphomas), carcinomas, and sarcomas. In general, cancer's pathogenesis is associated with three genetic mutations, where could emerge from oncogenes, tumor suppressor genes, and/or genes responsible for regulating DNA replication. The term "undruggable" is frequently related to the difficulty to design drugs to specific targets, such as MYC, MYB, NF-κB, and RAS family of proteins. This last comprises more than 140 proteins, and these are responsible for 30% of mutations in human cancers. Also, there are three ras genes transcribed in human cells, called H-, K-, and N-ras oncogenes. Still, the RAS proteins (farnesyltransferase (FTase) and geranylgeranyltransferase (GGTase) enzymes) perform essential steps in post-translational modification of eukaryotes cells, such as (1) the farnesylation of the cysteine residue at the C-terminal tetrapeptide CAAX; (2) proteolytic cleavage of the three C-terminal AAX oligopeptide; and (3) carboxymethylation of the new C-terminal prenylated cysteine. Thus, the inhibition of this undruggable RAS family of proteins has been considered a promising alternative to design new anticancer agents since they are responsible for many types of human cancers. Then, the manumycin A (obtained from the Streptomyces parvulus Tü64) and its analogs (epoxyquinol core with or without their southern and eastern side chains; and dihydroxycyclohexenones core) have been described as promising FTase inhibitors, which have demonstrated their benefits against several types of cancer. In this review, a complete introduction about cancer and its relation with RAS proteins is provided, as well as, the prenylation mechanism of the cysteine residue is discussed in detail. Posteriorly, studies involving manumycin-related compounds are described, showing some synthetic routes for obtaining them and utilizing these natural products in monotherapies or combined therapies with other anticancer drugs.

Peptide scaffold‐derived peptidomimetic farnesyltransferase inhibitors

AbstractHuman protein farnesyltransferase (FTase) is involved in many essential signal transduction proteins and has been explored as druggable targets of diverse diseases such as cancer and inflammation. Here, we propose a new strategy termed as Peptide Scaffold‐derived Peptidomimetic FTase Inhibitors (PSPFI) to discover peptidomimetic lead molecular entities that can inhibit FTase. The strategy first defines a PSPFI‐oriented peptidomimetic library that contains >30,000 sulfhydryl/thioether‐carrying peptidomimetics, and then employs molecular docking calculation, multiple similarity analysis, structural minimization, and affinity scoring to screen against the library, in order to discover those peptidomimetics that share high similarity with the core binding module (CBM) of FTase Rap2a peptide substrate in 2D‐chemistry, 3D‐conformation, pharmacophore, and nonbonded pattern, as well as exhibit high theoretical affinity to FTase. Totally 56 hit peptidomimetics were identified from the library, from which we manually select 6 structurally diverse, purchasable peptidomimetics to perform FTase assays. Consequently, four are determined as potent FTase inhibitors (IC50 < 1 μM); their inhibitory activity is moderately lower than the sophisticated FTase inhibitor Tipifarnib and considerably higher than the native CBM peptide. These peptidomimetic ligands share similar extended conformation, binding mode, and Zn2+ coordination with Rap2a CBM peptide in FTase active site, but would have a different inhibition mechanism to Tipifarnib.

Computational investigation of novel farnesyltransferase inhibitors using 3D-QSAR pharmacophore modeling, virtual screening, molecular docking and molecular dynamics simulation studies: A new insight into cancer treatment

Farnesyltransferase (FTase) is considered as an effective target in treating a variety of cancers. In this investigation, a 3D-QSAR pharmacophore search was performed to identify potential inhibitors against FTase. Correspondingly, 3D-QSAR pharmacophore models for FTase inhibitors were generated utilizing the training set compounds and validated through the test set, cost analysis and Fischer's randomization test. The created pharmacophore models were evaluated based on Debnath's analysis. Then, the pharmacophore model Hypo1 was selected to perform pharmacophore-based screening. Subsequently, the identified compounds were filtered considering Lipinski's Rule of Five (RoF) and Absorption, Distribution, Metabolism, Excretion and Toxicity (ADMET) properties. In the next step, the inter-molecular interactions of the retrieved compounds and the most active compound of the training set with FTase were analyzed utilizing the flexible docking technique. Eventually, the best two compounds, ZINC08952683 and ZINC08918035, along with the reference compound, ZINC13559176, were selected for Molecular Dynamics (MD) studies in order to conduct an in-depth study on their complexes with FTase. MD results confirmed docking studies and showed that these two compounds could make stable complexes with the FTase active site and may have great inhibitory effects on FTase.

Scaffold-based analysis of nonpeptide oncogenic FTase inhibitors using multiple similarity matching, binding affinity scoring and enzyme inhibition assay

Oncogenic protein farnesyltransferase (FTase) is a key enzyme responsible for the lipid modification of a large and important number of proteins including Ras, which has been recognized as a druggable target of diverse cancers. Here, we report a systematic scaffold-based analysis to investigate the affinity, selectivity and cross-reactivity of nonpeptide inhibitors across ontology-enriched, disease-associated FTase mutants, by integrating multiple similarity matching, binding affinity scoring and enzyme inhibition assay. It is revealed that nonpeptide inhibitors are generally insensitive to FTase mutations; many of them cannot definitely select for wild-type target over mutant enzymes. Therefore, off-target is observed as a common phenomenon for the untargeted consequence of targeted therapies with FTase inhibition. This is not unexpected if considering that the enzyme active site is highly conserved in composition, configuration and function. The off-target, on the one hand, causes nonpeptide inhibitors with adverse drug reactions and, on the other hand, makes the inhibitors as promising candidates for the new use of old drugs. To practice the latter, a number of unexpected mutant–inhibitor interactions involved in cancer signaling pathways are uncovered in the created profile, from which several nonpeptide inhibitors are identified as insensitive to a drug-resistant mutation. Structural analysis suggests that the inhibitor ligands can bind to the mutant active site in a similar manner with wild-type target, although their nonbonded interactions appear to be impaired moderately upon the mutation.

Discovery of an Anion-Dependent Farnesyltransferase Inhibitor from a Phenotypic Screen.

By employing a phenotypic screen, a set of compounds, exemplified by 1, were identified which potentiate the ability of histone deacetylase inhibitor vorinostat to reverse HIV latency. Proteome enrichment followed by quantitative mass spectrometric analysis employing a modified analogue of 1 as affinity bait identified farnesyl transferase (FTase) as the primary interacting protein in cell lysates. This ligand-FTase binding interaction was confirmed via X-ray crystallography and temperature dependent fluorescence studies, despite 1 lacking structural and binding similarity to known FTase inhibitors. Although multiple lines of evidence established the binding interaction, these ligands exhibited minimal inhibitory activity in a cell-free biochemical FTase inhibition assay. Subsequent modification of the biochemical assay by increasing anion concentration demonstrated FTase inhibitory activity in this novel class. We propose 1 binds together with the anion in the active site to inhibit farnesyl transferase. Implications for phenotypic screening deconvolution and HIV reactivation are discussed.

Tipifarnib as a Precision Therapy for HRAS-Mutant Head and Neck Squamous Cell Carcinomas.

Tipifarnib is a potent and highly selective inhibitor of farnesyltransferase (FTase). FTase catalyzes the posttranslational attachment of farnesyl groups to signaling proteins that are required for localization to cell membranes. Although all RAS isoforms are FTase substrates, only HRAS is exclusively dependent upon farnesylation, raising the possibility that HRAS-mutant tumors might be susceptible to tipifarnib-mediated inhibition of FTase. Here, we report the characterization of tipifarnib activity in a wide panel of HRAS-mutant and wild-type head and neck squamous cell carcinoma (HNSCC) xenograft models. Tipifarnib treatment displaced both mutant and wild-type HRAS from membranes but only inhibited proliferation, survival, and spheroid formation of HRAS-mutant cells. In vivo, tipifarnib treatment induced tumor stasis or regression in all six HRAS-mutant xenografts tested but displayed no activity in six HRAS wild-type patient-derived xenograft (PDX) models. Mechanistically, drug treatment resulted in the reduction of MAPK pathway signaling, inhibition of proliferation, induction of apoptosis, and robust abrogation of neovascularization, apparently via effects on both tumor cells and endothelial cells. Bioinformatics and quantitative image analysis further revealed that FTase inhibition induces progressive squamous cell differentiation in tipifarnib-treated HNSCC PDXs. These preclinical findings support that HRAS represents a druggable oncogene in HNSCC through FTase inhibition by tipifarnib, thereby identifying a precision therapeutic option for HNSCCs harboring HRAS mutations.

Overexpression of FNTB and the activation of Ras induce hypertrophy and promote apoptosis and autophagic cell death in cardiomyocytes.

Farnesyltransferase (FTase) is an important enzyme that catalyses the modification of protein isoprene downstream of the mevalonate pathway. Previous studies have shown that the tissue of the heart in the suprarenal abdominal aortic coarctation (AAC) group showed overexpression of FTaseβ (FNTB) and the activation of the downstream protein Ras was enhanced. FTase inhibitor (FTI) can alleviate myocardial fibrosis and partly improve cardiac remodelling in spontaneously hypertensive rats. However, the exact role and mechanism of FTase in myocardial hypertrophy and remodelling are not fully understood. Here, we used recombinant adenovirus to transfect neonatal rat ventricular cardiomyocytes to study the effect of FNTB overexpression on myocardial remodelling and explore potential mechanisms. The results showed that overexpression of FNTB induces neonatal rat ventricular myocyte hypertrophy and reduces the survival rate of cardiomyocytes. FNTB overexpression induced a decrease in mitochondrial membrane potential and increased apoptosis in cardiomyocytes. FNTB overexpression also promotes autophagosome formation and the accumulation of autophagy substrate protein, LC3II. Transmission electron microscopy (TEM) and mCherry‐GFP tandem fluorescent‐tagged LC3 (tfLC3) showed that FNTB overexpression can activate autophagy flux by enhancing autophagosome conversion to autophagolysosome. Overactivated autophagy flux can be blocked by bafilomycin A1. In addition, salirasib (a Ras farnesylcysteine mimetic) can alleviate the hypertrophic phenotype of cardiomyocytes and inhibit the up‐regulation of apoptosis and autophagy flux induced by FNTB overexpression. These results suggest that FTase may have a potential role in future treatment strategies to limit the adverse consequences of cardiac hypertrophy, cardiac dysfunction and heart failure.

Design, Synthesis and Biological Evaluation of Novel Piperazinone Derivatives as Cytotoxic Agents.

Purpose: In this study, a series of piperazin-2-one derivatives were prepared through bioisosteric substitution of the imidazole ring of L-778,123 (imidazole-containing FTase inhibitor) and rearrangement of groups based on the tipifarnib structure. Final compounds were evaluated for their cytotoxic activities on cancer and normal cell lines by MTT assay. Methods: Methyl α-bromophenylacetic acid and 1-(3-chlorophenyl) piperazin-2-one were synthesized using previously described methods. Methyl 2-(4-chlorophenyl)-2-(4-(3- chlorophenyl)-3-oxopiperazin-1-yl) acetate was prepared by reaction between these two compounds in presence of potassium carbonate. Finally, methoxy group of ester was substituted by various amines such as guanidine, thiourea, urea and hydrazide. The synthesized compounds were tested for their cytotoxicity against colon cancer (HT-29) and lung cancer (A549) cell lines as well as MRC-5 (normal fetal lung fibroblasts) cells as a healthy cell line using MTT colorimetric assay method. Results: Replacement of imidazole moiety with guanidine, thiourea, and hydrazide could increase cytotoxicity toward all three cell lines. Some substituents, such as amine, urea, and hydroxylamine exhibited significant cytotoxicity (<500 µM) but lower than L-778,123 as standard compound. Hydroxyl and methoxy substituents did not show significant cytotoxicity. Imidazole substituent group revealed cytotoxicity similar to L-778,123 All compounds showed lower cytotoxic activity against normal cell lines compared with cancer cell lines. Conclusion: It seems the electron density of substituted groups and rearrangement of groups may significantly increase cytotoxic activity

Farnesyltransferase Inhibition Exacerbates Eosinophilic Inflammation and Airway Hyperreactivity in Mice with Experimental Asthma: The Complex Roles of Ras GTPase and Farnesylpyrophosphate in Type 2 Allergic Inflammation.

Ras, a small GTPase protein, is thought to mediate Th2-dependent eosinophilic inflammation in asthma. Ras requires cell membrane association for its biological activity, and this requires the posttranslational modification of Ras with an isoprenyl group by farnesyltransferase (FTase) or geranylgeranyltransferase (GGTase). We hypothesized that inhibition of FTase using FTase inhibitor (FTI)-277 would attenuate allergic asthma by depleting membrane-associated Ras. We used the OVA mouse model of allergic inflammation and human airway epithelial (HBE1) cells to determine the role of FTase in inflammatory cell recruitment. BALB/c mice were first sensitized then exposed to 1% OVA aerosol or filtered air, and half were injected daily with FTI-277 (20 mg/kg per day). Treatment of mice with FTI-277 had no significant effect on lung membrane-anchored Ras, Ras protein levels, or Ras GTPase activity. In OVA-exposed mice, FTI-277 treatment increased eosinophilic inflammation, goblet cell hyperplasia, and airway hyperreactivity. Human bronchial epithelial (HBE1) cells were pretreated with 5, 10, or 20 μM FTI-277 prior to and during 12 h IL-13 (20 ng/ml) stimulation. In HBE1 cells, FTase inhibition with FTI-277 had no significant effect on IL-13-induced STAT6 phosphorylation, eotaxin-3 peptide secretion, or Ras translocation. However, addition of exogenous FPP unexpectedly augmented IL-13-induced STAT6 phosphorylation and eotaxin-3 secretion from HBE1 cells without affecting Ras translocation. Pharmacological inhibition of FTase exacerbates allergic asthma, suggesting a protective role for FTase or possibly Ras farnesylation. FPP synergistically augments epithelial eotaxin-3 secretion, indicating a novel Ras-independent farnesylation mechanism or direct FPP effect that promotes epithelial eotaxin-3 production in allergic asthma.