Non-Heme Iron Enzymes Catalyze Heterobicyclic and Spirocyclic Isoquinolone Core Formation in Piperazine Alkaloid Biosynthesis.

We report the discovery and biosynthesis of new piperazine alkaloids-arizonamides, and their derived compounds-arizolidines, featuring heterobicyclic and spirocyclic isoquinolone skeletons, respectively. Their biosynthetic pathway involves two crucial non-heme iron enzymes, ParF and ParG, for core skeleton construction. ParF has a dual function facilitating 2,3-alkene formation of helvamide, as a substrate for ParG, and oxidative cleavage of piperazine. Notably, ParG exhibits catalytic versatility in multiple oxidative reactions, including cyclization and ring reconstruction. A key amino acid residue Phe67 was characterized to control the formation of the constrained arizonamide B backbone by ParG.

BPR3P0128, a non-nucleoside RNA-dependent RNA polymerase inhibitor, inhibits SARS-CoV-2 variants of concern and exerts synergistic antiviral activity in combination with remdesivir.

Viral RNA-dependent RNA polymerase (RdRp), a highly conserved molecule in RNA viruses, has recently emerged as a promising drug target for broad-acting inhibitors. Through a Vero E6-based anti-cytopathic effect assay, we found that BPR3P0128, which incorporates a quinoline core similar to hydroxychloroquine, outperformed the adenosine analog remdesivir in inhibiting RdRp activity (EC50 = 0.66 µM and 3 µM, respectively). BPR3P0128 demonstrated broad-spectrum activity against various severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern. When introduced after viral adsorption, BPR3P0128 significantly decreased SARS-CoV-2 replication; however, it did not affect the early entry stage, as evidenced by a time-of-drug-addition assay. This suggests that BPR3P0128's primary action takes place during viral replication. We also found that BPR3P0128 effectively reduced the expression of proinflammatory cytokines in human lung epithelial Calu-3 cells infected with SARS-CoV-2. Molecular docking analysis showed that BPR3P0128 targets the RdRp channel, inhibiting substrate entry, which implies it operates differently-but complementary-with remdesivir. Utilizing an optimized cell-based minigenome RdRp reporter assay, we confirmed that BPR3P0128 exhibited potent inhibitory activity. However, an enzyme-based RdRp assay employing purified recombinant nsp12/nsp7/nsp8 failed to corroborate this inhibitory activity. This suggests that BPR3P0128 may inhibit activity by targeting host-related RdRp-associated factors. Moreover, we discovered that a combination of BPR3P0128 and remdesivir had a synergistic effect-a result likely due to both drugs interacting with separate domains of the RdRp. This novel synergy between the two drugs reinforces the potential clinical value of the BPR3P0128-remdesivir combination in combating various SARS-CoV-2 variants of concern.

Complexation and evolution of cis-prenyltransferase homologues in Cinnamomum kanehirae deduced from kinetic and functional characterizations.

Eukaryotic dehydrodolichyl diphosphate synthases (DHDDSs), cis-prenyltransferases (cis-PTs) synthesizing precursors of dolichols to mediate glycoprotein biosynthesis require partners, for eample Nus1 in yeast and NgBR in animals, which are cis-PTs homologues without activity but to boost the DHDDSs activity. Unlike animals, plants have multiple cis-PT homologues to pair or stand alone to produce various chain-length products with less known physiological roles. We chose Cinnamomum kanehirae, a tree that contains two DHDDS-like and three NgBR-like proteins from genome analysis, and found that one DHDDS-like protein acted as a homodimeric cis-PT to make a medium-chain C55 product, while the other formed heterodimeric complexes with either one of two NgBR homologues to produce longer-chain products. Both complexes were functional to complement the growth defect of the yeast rer2 deficient strain at a higher temperature. From the roles for the polyprenol and dolichol biosynthesis and sequence motifs, their homologues in various species were compared to reveal their possible evolutionary paths.

Synthesis, evaluation, and mechanism of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones as potent reversible SARS-CoV-2 entry inhibitors.

A class of 1-(4-(arylethylenylcarbonyl)phenyl)-4-carboxy-2-pyrrolidinones were designed and synthesized via Michael addition, cyclization, aldol condensation, and deprotonation to inhibit the human transmembrane protease serine 2 (TMPRSS2) and Furin, which are involved in priming the SARS-CoV-2 Spike for virus entry. The most potent inhibitor 2f (81) was found to efficiently inhibit the replication of various SARS-CoV-2 delta and omicron variants in VeroE6 and Calu-3 cells, with EC50 range of 0.001-0.026 µM by pre-incubation with the virus to avoid the virus entry. The more potent antiviral activities than the proteases inhibitory activities led to discovery that the synthesized compounds also inhibited Spike's receptor binding domain (RBD):angiotensin converting enzyme 2 (ACE2) interaction as a main target, and their antiviral activities were enhanced by inhibiting TMPRSS2 and/or Furin. To further confirm the blocking effect of 2f (81) on virus entry, SARS-CoV-2 Spike pseudovirus was used in the entry assay and the results showed that the compound inhibited the pseudovirus entry in a ACE2-dependent pathway, via mainly inhibiting RBD:ACE2 interaction and TMPRSS2 activity in Calu-3 cells. Finally, in the in vivo animal model of SARS-CoV-2 infection, the oral administration of 25 mg/kg 2f (81) in hamsters resulted in reduced bodyweight loss and 5-fold lower viral RNA levels in nasal turbinate three days post-infection. Our findings demonstrated the potential of the lead compound for further preclinical investigation as a potential treatment for SARS-CoV-2.

SARS-CoV-2 3CLpro displays faster self-maturation in vitro than SARS-CoV 3CLpro due to faster C-terminal cleavage.

The coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a worldwide pandemic. The 3C-like protease (3CLpro ), which cleaves 11 sites including its own N- and C-termini on the viral polyproteins, is essential for SARS-CoV-2 replication. In this study, we constructed the full-length inactive 3CLpro with N- and C-terminal extensions as substrates for monitoring self-cleavage by wild-type 3CLpro . We found that the rate-limiting C-terminal self-cleavage rate of SARS-CoV-2 3CLpro was 35-fold faster than that of SARS-CoV 3CLpro using the Trx/GST-tagged C145A 3CLpro substrates. Since self-cleavage of 3CLpro is the initial step for maturation of other viral proteins, our study suggests more facile SARS-CoV-2 replication than that of SARS-CoV.

Drug Repurposing for the Identification of Compounds with Anti-SARS-CoV-2 Capability via Multiple Targets.

Since 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been rapidly spreading worldwide, causing hundreds of millions of infections. Despite the development of vaccines, insufficient protection remains a concern. Therefore, the screening of drugs for the treatment of coronavirus disease 2019 (COVID-19) is reasonable and necessary. This study utilized bioinformatics for the selection of compounds approved by the U.S. Food and Drug Administration with therapeutic potential in this setting. In addition, the inhibitory effect of these compounds on the enzyme activity of transmembrane protease serine 2 (TMPRSS2), papain-like protease (PLpro), and 3C-like protease (3CLpro) was evaluated. Furthermore, the capability of compounds to attach to the spike-receptor-binding domain (RBD) was considered an important factor in the present assessment. Finally, the antiviral potency of compounds was validated using a plaque reduction assay. Our funnel strategy revealed that tamoxifen possesses an anti-SARS-CoV-2 property owing to its inhibitory performance in multiple assays. The proposed time-saving and feasible strategy may accelerate drug screening for COVID-19 and other diseases.

Simeprevir Potently Suppresses SARS-CoV-2 Replication and Synergizes with Remdesivir.

The outbreak of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global threat to human health. Using a multidisciplinary approach, we identified and validated the hepatitis C virus (HCV) protease inhibitor simeprevir as an especially promising repurposable drug for treating COVID-19. Simeprevir potently reduces SARS-CoV-2 viral load by multiple orders of magnitude and synergizes with remdesivir in vitro. Mechanistically, we showed that simeprevir not only inhibits the main protease (Mpro) and unexpectedly the RNA-dependent RNA polymerase (RdRp) but also modulates host immune responses. Our results thus reveal the possible anti-SARS-CoV-2 mechanism of simeprevir and highlight the translational potential of optimizing simeprevir as a therapeutic agent for managing COVID-19 and future outbreaks of CoV.

Identification of non-covalent 3C-like protease inhibitors against severe acute respiratory syndrome coronavirus-2 via virtual screening of a Korean compound library.

The outbreak of coronavirus (CoV) disease 2019 (COVID-19) caused by the severe acute respiratory syndrome CoV-2 (SARS-CoV-2) has turned into a pandemic. The enzyme 3C-like protease (3CLpro) is essential for the maturation of viral polyproteins in SARS-CoV-2 and is therefore regarded as a key drug target for treating the disease. To identify 3CLpro inhibitors that can suppress SARS-CoV-2 replication, we performed a virtual screening of 500,282 compounds in a Korean compound bank. We then subjected the top computational hits to inhibitory assays against 3CLpro in vitro, leading to the identification of a class of non-covalent inhibitors. Among these inhibitors, compound 7 showed an EC50 of 39.89 µM against SARS-CoV-2 and CC50 of 453.5 µM. This study provides candidates for the optimization of potent 3CLpro inhibitors showing antiviral effects against SARS-CoV-2.

Kinetic Characterization and Inhibitor Screening for the Proteases Leading to Identification of Drugs against SARS-CoV-2.

Coronavirus (CoV) disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has claimed many lives worldwide and is still spreading since December 2019. The 3C-like protease (3CLpro) and papain-like protease (PLpro) are essential for maturation of viral polyproteins in SARS-CoV-2 life cycle and thus regarded as key drug targets for the disease. In this study, 3CLpro and PLpro assay platforms were established, and their substrate specificities were characterized. The assays were used to screen collections of 1,068 and 2,701 FDA-approved drugs. After excluding the externally used drugs which are too toxic, we totally identified 12 drugs as 3CLpro inhibitors and 36 drugs as PLpro inhibitors active at 10 µM. Among these inhibitors, six drugs were found to suppress SARS-CoV-2 with the half-maximal effective concentration (EC50) below or close to 10 µM. This study enhances our understanding on the proteases and provides FDA-approved drugs for prevention and/or treatment of COVID-19.

Uncovering Flexible Active Site Conformations of SARS-CoV-2 3CL Proteases through Protease Pharmacophore Clusters and COVID-19 Drug Repurposing.

The infectious SARS-CoV-2 causes COVID-19, which is now a global pandemic. Aiming for effective treatments, we focused on the key drug target, the viral 3C-like (3CL) protease. We modeled a big dataset with 42 SARS-CoV-2 3CL protease-ligand complex structures from ∼98.7% similar SARS-CoV 3CL protease with abundant complex structures. The diverse flexible active site conformations identified in the dataset were clustered into six protease pharmacophore clusters (PPCs). For the PPCs with distinct flexible protease active sites and diverse interaction environments, we identified pharmacophore anchor hotspots. A total of 11 "PPC consensus anchors" (a distinct set observed in each PPC) were observed, of which three "PPC core anchors" EHV2, HV1, and V3 are strongly conserved across PPCs. The six PPC cavities were then applied in virtual screening of 2122 FDA drugs for repurposing, using core anchor-derived "PPC scoring S" to yield seven drug candidates. Experimental testing by SARS-CoV-2 3CL protease inhibition assay and antiviral cytopathic effect assays discovered active hits, Boceprevir and Telaprevir (HCV drugs) and Nelfinavir (HIV drug). Specifically, Boceprevir showed strong protease inhibition with micromolar IC50 of 1.42 µM and an antiviral activity with EC50 of 49.89 µM, whereas Telaprevir showed moderate protease inhibition only with an IC50 of 11.47 µM. Nelfinavir solely showed antiviral activity with a micromolar EC50 value of 3.28 µM. Analysis of binding mechanisms of protease inhibitors revealed the role of PPC core anchors. Our PPCs revealed the flexible protease active site conformations, which successfully enabled drug repurposing.

Chaperonin-Containing TCP-1 Promotes Cancer Chemoresistance and Metastasis through the AKT-GSK3ß-ß-Catenin and XIAP-Survivin Pathways.

Chaperonin-containing TCP-1 (CCT) is a chaperonin composed of eight subunits that participates in intracellular protein folding. Here, we showed that increased levels of subunits of CCT, particularly CCT-ß, were significantly correlated with lower survival rates for cancer patients. Endogenously high expression of CCT-ß was found in cancer cell lines, such as the triple-negative breast cancer cell line MDA-MB-231 and the highly metastatic non-small-cell lung cancer cell line CL1-5. Knocking down CCT-ß in these cancer cells led to decreased levels of anti-apoptotic proteins, such as XIAP, as well as inhibited phosphorylation of Ser473-AKT and GSK3, resulting in decrease of the nucleus-entering form of ß-catenin; these changes reduced the chemoresistance and migration/invasion of the cells. Conversely, overexpression of CCT-ß recovered the chemoresistance and cell migration/invasion by promoting the AKT-GSK3ß-ß-catenin and XIAP-Survivin pathways. Coimmunoprecipitation data revealed that the CCT complex might directly bind and stabilize XIAP and ß-catenin. This study not only elucidates the roles of CCT in chemoresistance and metastasis, which are two major obstacles for current cancer therapy, but also provides a possible therapeutic strategy against cancers with overexpressed CCT-ß.

Targeting ß-tubulin/CCT-ß complex induces apoptosis and suppresses migration and invasion of highly metastatic lung adenocarcinoma.

Metastasis, the movement of cancer cells from one site to another, is responsible for the highest number of cancer deaths, especially in lung cancer patients. In this study, we first identified a prognostic marker of lung adenocarcinoma, TCP-1 ß subunit (chaperonin-containing TCP-1ß; CCT-ß). We showed a compound that disrupted the interaction of CCT-ß with ß-tubulin killed a highly metastatic non-small cell lung cancer cell line CL1-5 through inducing Endoplasmic reticulum stress and caspases activation. Moreover, at the dosage of EC20, the compound inhibited migration and invasion of the lung cancer cells by suppressing matrix metalloproteinase (MMP)-2/9 and epithelial-mesenchymal transition (EMT)-related proteins through downregulating mitogen-activated protein kinases (MAPKs), Akt/ß-catenin and integrin-focal adhesion kinase signaling pathways. Unlike the anticancer drugs, such as Taxol, that target the adenosine triphosphate site of ß-tubulin, this study reveals a therapeutic target, ß-tubulin/CCT-ß complex, for metastatic human lung adenocarcinoma. The study demonstrated CCT-ß as a prognostic marker. Targeting ß-tubulin/CCT-ß complex caused apoptosis and inhibited invasion/migration of CCT-ß overexpressed, highly metastatic lung adenocarcinoma.

Characterization of a Cis-Prenyltransferase from Lilium longiflorum Anther.

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths via consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). cis-Prenyltransferases, which catalyze cis-double bond formation during IPP condensation, usually synthesize long-chain products as lipid carriers to mediate peptidoglycan biosynthesis in prokaryotes and protein glycosylation in eukaryotes. Unlike only one or two cis-prenyltransferases in bacteria, yeast, and animals, plants have several cis-prenyltransferases and their functions are less understood. As reported here, a cis-prenyltransferase from Lilium longiflorum anther, named LLA66, was expressed in Saccharomyces cerevisiae and characterized to produce C40/C45 products without the capability to restore the growth defect from Rer2-deletion, although it was phylogenetically categorized as a long-chain enzyme. Our studies suggest that evolutional mutations may occur in the plant cis-prenyltransferase to convert it into a shorter-chain enzyme.

The Penetration Depth for Hanatoxin Partitioning into the Membrane Hydrocarbon Core Measured with Neutron Reflectivity.

Hanatoxin (HaTx) from spider venom works as an inhibitor of Kv2.1 channels, most likely by interacting with the voltage sensor (VS). However, the way in which this water-soluble peptide modifies the gating remains poorly understood as the VS is deeply embedded within the bilayer, although it would change its position depending on the membrane potential. To determine whether HaTx can indeed bind to the VS, the depth at which HaTx penetrates into the POPC membranes was measured with neutron reflectivity. Our results successfully demonstrate that HaTx penetrates into the membrane hydrocarbon core (∼9 Šfrom the membrane surface), not lying on the membrane-water interface as reported for another voltage sensor toxin (VSTx). This difference in penetration depth suggests that the two toxins fix the voltage sensors at different positions with respect to the membrane normal, thereby explaining their different inhibitory effects on the channels. In particular, results from MD simulations constrained by our penetration data clearly demonstrate an appropriate orientation for HaTx to interact with the membranes, which is in line with the biochemical information derived from stopped-flow analysis through delineation of the toxin-VS binding interface.

Caspase-3, a key apoptotic protein, as a prognostic marker in gastric cancer after curative surgery.

BACKGROUND: Caspase-3 acts as a major executioner protein in proteolytic degradation during apoptosis. The role of Caspase-3 in gastric cancer remains controversial. METHODS: A total of 366 gastric cancer patients who received curative surgery were enrolled. Caspase-3 expression in gastric tumors was examined by immunohistochemical staining. Correlations between Caspase-3 expression and the survival rates and between Caspase-3 expression and the clinicopathological parameters of the gastric cancer patients were analyzed. RESULTS: The 5-year overall survival rates of gastric cancer patients with and without Caspase-3 expression were 51.2% and 37.3%, respectively (P = 0.030). The 5-year disease-free survival rates of gastric cancer patients with and without Caspase-3 expression were 49.2% and 34.6%, respectively (P = 0.029). Analyses of the clinicopathological features showed that larger tumor size (P = 0.030), more advanced Borrmann type (P = 0.012), more aggressive stromal reaction (P = 0.001), higher classification using Ming's infiltrating histology type (P = 0.018), more lymph node involvement (P = 0.019), and more lymphovascular involvement (P = 0.045) were significantly correlated with a lack of Caspase-3 expression. The multivariate analysis showed that age (P = 0.001), Borrmann classification (P = 0.032), stromal reaction type (P = 0.018), TNM pathological T category (P = 0.002), TNM pathological N category (P < 0.001), and Caspase-3 expression (P = 0.041) were significantly correlated with the overall survival of gastric cancer patients. CONCLUSION: Caspase-3 expression in gastric cancer patients is related to favorable clinicopathological features and a positive prognosis after curative surgery. Caspase-3 may act as a tumor suppressor in human gastric cancer.

A flexible loop for mannan recognition and activity enhancement in a bifunctional glycoside hydrolase family 5.

BACKGROUND: An array of glycoside hydrolases with multiple substrate specificities are required to digest plant cell wall polysaccharides. Cel5E from Clostridium thermocellum and Cel5A from Thermotoga maritima are two glycoside hydrolase family 5 (GH5) enzymes with high sequence and structural similarity, but notably possess different substrate specificities; the former is a bifunctional cellulase/xylanase and the latter is a cellulase/mannanase. A specific loop in TmCel5A, Tmloop, is one of the most structurally divergent regions compared to CtCel5E and interacts with substrates, suggesting the importance for mannan recognition. METHOD: A Tmloop inserted CtCel5E and its related mutants were produced to investigate the role of Tmloop in catalysis. Crystal structure of CtCel5E-TmloopF267A followed by site-direct mutagenesis reveals the mechanism. RtCelB, a homolog with Tmloop was identified to have mannanase activity. RESULT: Tmloop incorporation enables CtCel5E to gain mannanase activity. Tyr270, His277, and Trp282 in the Tmloop are indispensable for CtCel5E-Tmloop catalysis, and weakening hydrophobic environment near the Tmloop enhances enzyme kcat. Using our newly identified loop motif to search for structurally conserved homologs in other subfamilies of GH5, we identified RtCelB. This homolog, originally annotated as a cellulase also possesses mannanase and xylanase activities. CONCLUSION: Our studies show that Tmloop enhances GH5 enzyme promiscuity and plays a role in catalysis. GENERAL SIGNIFICANCE: The study identified a loop of GH5 for mannan recognition and catalysis. Weakening the hydrophobic environment near the loop can also enhance the enzyme catalytic rate. Our findings provide a new insight on mannan recognition and activity enhancement of GH5.

Highly sensitive fluorogenic sensing of L-Cysteine in live cells using gelatin-stabilized gold nanoparticles decorated graphene nanosheets.

A highly sensitive and selective fluorogenic sensing of L-Cysteine (L-Cys) was implemented based on gelatin stabilized gold nanoparticles decorated reduced graphene oxide (rGO/Au) nanohybrid. The rGO/Au nanohybrid was prepared by the one-pot hydrothermal method and well characterized by different physiochemical techniques. The nanohybrid exhibits a weak fluorescence of rGO due to the energy transfer from the rGO to Au NPs. The rGO/Au nanohybrid shows enhanced fluorescence activity due to the restoration of quenched fluorescence of rGO/Au nanohybrid in presence of L-Cys. The rGO/Au nanohybrid exhibits much lower detection limit of 0.51 nM for L-Cys with higher selectivity. The fluorescence sensing mechanism arose from the fluorescence recovery due to the stronger interaction between Au NPs and L-Cys, and consequently, the energy transfer was prevented between rGO and Au NPs. The practicability of rGO/Au sensor was implemented by invitro bioimaging measurements in Colo-205 (colorectal adenocarcinoma) and MKN-45 (gastric carcinoma) cancer live cells with excellent biocompatibility.

Disrupting CCT-ß : ß-tubulin selectively kills CCT-ß overexpressed cancer cells through MAPKs activation.

We have previously demonstrated the ability of I-Trp to disrupt the protein-protein interaction of ß-tubulin with chaperonin-containing TCP-1ß (CCT-ß). This caused more severe apoptosis in multidrug-resistant MES-SA/Dx5, compared to MES-SA, due to its higher CCT-ß overexpression. In this study, we screened a panel of cancer cell lines, finding CCT-ß overexpression in the triple-negative breast cancer cell line MDA-MB-231, colorectal cancer cell lines Colo205 and HCT116, and a gastric cancer cell line MKN-45. Thus, I-Trp killed these cancers with sub- to low-µM EC50, whereas it was non-toxic to MCF-10A. We then synthesized analogs of I-Trp and evaluated their cytotoxicity. Furthermore, apoptotic mechanism investigations revealed the activation of both protein ubiquitination/degradation and ER-associated protein degradation pathways. These pathways proceeded through activation of MAPKs at the onset of CCT-ß : ß-tubulin complex disruption. We thus establish an effective strategy to treat CCT-ß overexpressed cancers by disrupting the CCT-ß : ß-tubulin complex.

Identification and evaluation of potent Middle East respiratory syndrome coronavirus (MERS-CoV) 3CLPro inhibitors.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649 deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 µM and from 0.2 to 0.7 µM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50 values ranging from 0.6 to 1.4 µM, by downregulating the viral protein production in cells as well as reducing secretion of infectious viral particles into culture supernatants. They also suppressed other α- and ß-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70 against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against emerging CoVs and picornaviruses.

Fluorescent Farnesyl Diphosphate Analogue: A Probe To Validate trans-Prenyltransferase Inhibitors.

Some trans-prenyltransferases, such as long-chain C40 octaprenyl diphosphate synthase (OPPS), short-chain C15 farnesyl diphosphate synthase (FPPS), and C20 geranylgeranyl diphosphate synthase (GGPPS), are important drug targets. These enzymes catalyze chain elongation of FPP or geranyl diphosphate (GPP) through condensation reactions with isopentenyl diphosphate (IPP), forming designated numbers of trans-double bonds in the final products. To facilitate drug discovery, we report here a sensitive and reliable fluorescence-based assay for monitoring their activities in real time. MANT-O-GPP, a fluorescent analogue of FPP, was used as an alternative substrate and converted by the wild-type OPPS and the engineered FPPS and GGPPS into sufficiently long products with enhanced fluorescence intensities. This fluorescence probe was used to reveal the inhibitory mechanism of zoledronate, a bisphosphonate drug that targets human FPPS and possibly GGPPS.