Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2023 Oct 21.
Published in final edited form as: ACS Chem Biol. 2022 Oct 4;17(10):2945–2953. doi: 10.1021/acschembio.2c00645

Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation

Johannes Morstein a,b,#, Taysir Bader c,#, Ariana L Cardillo d,#, Julian Schackmann b, Sudhat Ashok e, James L Hougland e,f,g, Christine A Hrycyna d,*, Dirk H Trauner b,h,*, Mark D Distefano c,*
PMCID: PMC9799063  NIHMSID: NIHMS1860028  PMID: 36194691

Abstract

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility this approach for the optical control of protein prenylation.

Graphical Abstract

graphic file with name nihms-1860028-f0001.jpg

Introduction

Approximately 10 to 20% of all mammalian proteins are thought to undergo protein lipidation.1 Among the most common types of posttranslational modifications are fatty acylation and isoprenylation.2 The latter consists of the attachment of an isoprenoid lipid with 3 isoprene repeats (farnesylation, 15 carbons) or 4 isoprene repeats (geranylgeranylation, 20 carbons) by either protein farnesyltransferase (FTase), or types 1, 2 or 3 geranylgeranyltransferase (GGTase I, II or III) to specific protein substrates. These groups are linked via a thioether bond to one or two cysteine residues positioned near the C-terminus of a target protein.3 This step is followed by removal of a C-terminal tripeptide sequence by either Ste24 or Ras Converting CAAX Endopeptidase 1 (RCE1) enzymes,4,5 and finally methylation of the newly exposed carboxyl cysteine by Protein-S-isoprenylcysteine O-methyltransferase (ICMT).6 Combined, these modifications generate the functional/active states of the lipidated protein through modulation of their cellular localization and/or protein-protein interactions.7

Several chemical probes have been developed to study and inhibit protein isoprenylation as a means to disrupt the processing of CaaX proteins implicated in disease pathways.8 Farnesyltransferase inhibitors (FTIs) have been explored in several trials for cancer therapy9 and have recently been approved for the treatment of hepatitis D virus infections, progeria, and progeroid laminopathies.10 To allow for improved spatiotemporal control of protein farnesylation, we have previously synthesized FTIs with photocleavable protecting groups that enable the UVA light-triggered activation of these caged molecules.11 However, this light-induced inhibition of farnesyltransferase (FTase) is an indirect method to control the function of isoprenylated proteins and it does not allow for reversibility or activation of the process. We envisioned that reversible control of the structure of the isoprenoid lipid and, in turn its function, could be achieved through incorporation of a reversibly photoswitchable moiety, such as a hydrophobic azobenzene, into an isoprenoid substrate.

In recent years, this approach has been extensively explored for photoswitchable sphingolipids and glycerolipids.12 These photolipids have been used to control biological targets of signaling lipids, including GPCRs,13–15 ion channels,16–18 enzymes,19–22 nuclear hormone receptors,23,24 and immunoreceptors,25 and as a means to control membrane biophysics in model membranes26–28 and cells.29 However, to date, this approach has not been extended to other important classes of lipids, such as steroids or isoprenoids. The development of photoswitchable isoprenoid lipids was further motivated by previously reported arene-rich analogs that proved to be efficient substrates for FTase (Figure 1A).30 These included a benzyl phenyl ether, which is a structural isostere (‘azostere’31,32) of azobenzenes. Photoswitchable analogs could, in principle, allow for the optical control of substrate prenylation, processing, and bioactivity (Figure 1B). Herein, we systematically explore the use of photoswitchable FPP analogs, termed AzoFPPs, for the optical control of protein isoprenylation, the subsequent processing of isoprenylated peptides, and the bioactivity of a prenylated fully processed, bioactive peptide (Figure 1C). Each enzymatic step in the CaaX pathway was explored with a peptide substrate in either the trans- or cis-forms to probe the relative sensitivity of lipid structure on the protein isoprenylation processing steps. Lastly, the bioactivity of the mature, fully processed, peptide a-factor containing the photoswitchable lipid moiety was assessed using a yeast growth arrest assay. Our results suggest, that AzoFPPs enable optical control of the isoprenylation step catalyzed by FTases without significantly affecting subsequent processing steps.

Figure 1 |. Design of photoswitchable FPP analogs and optical probing of prenylation processing.

Figure 1 |

Open in a new tab

(A) ‘Azologization’ of FPP and arene-rich analog. (B) Schematic illustration of protein farnesylation and subsequent processing. (C) Schematic representation of optical probing of peptide prenylation and processing with photoswitchable FPP analogs.

Results and Discussion

Design, Synthesis, and Photophysical Characterization of Photoswitchable FPP Analogs.

Initially, two photoswitchable analogs of FPP (Figure 2A) were designed and synthesized (see Figure S1 for the synthetic routes). The first analog, AzoFPP-1, was based on direct incorporation of an azobenzene into an isoprenoid-like allylic scaffold. The second analog, AzoFPP-2, was inspired by a previously reported Aryl-FPP derivative developed by Spielmann et al. that shows better steady-state kinetic parameters for isoprenylating an H-Ras sequence compared with FPP, the physiological substrate.30 This Aryl-FPP analog allowed for straight-forward azologization31,32 to obtain a photoswitchable analog. Briefly, 3-hydroxy azobenzene (2) was coupled to a prenol-derived alcohol (1) via a Mitsunobu reaction, followed by deprotection, which was then transformed into a chloride under Appel conditions.33 This allowed for the introduction of the diphosphate functionality into 4. Ion exchange chromatography and further purification gave AzoFPP-1 (5). The azobenzene precursor 6 for AzoFPP-2 (7) was generated under Baeyer-Mills conditions34,35 followed by a similar reaction sequence to yield the diphosphate (Figure 2A). UV-Vis spectroscopy showed that AzoFPP-1 behaved similarly to an unsubstituted azobenzene (Figure 2B). Isomerization from the thermodynamically favored trans-configuration to the cis-form was triggered with UV irradiation (λ = 365 nm), as evidenced by monitoring via reversed-phase HPLC (Figure S2). This process was reversible using blue light (λ = 460 nm) over multiple cycles (Figure 2C). Both compounds underwent slow thermal relaxation to the trans-isomer with t 1/2 = 25 h for Azo-FPP-1 and t1/2 = 29 h for Azo-FPP-2, measured in PBS buffer at 37 °C.

Figure 2 |. Synthesis, photophysical characterization, and molecular docking of photoswitchable farnesyl diphosphate analogs.

Figure 2 |

Open in a new tab

(A) Chemical synthesis of AzoFPP-1 and AzoFPP-2. (B) The UV-Vis spectra of AzoFPP-1 in varying wavelength-adapted photostationary states obtained using 50 μM AzoFPP-1 in PBS. (C) Reversible cycling between photoisomers with alternating illumination at the two distinct wavelengths, 365 nm and 460 nm, demonstrated the rapid rate of isomerization. The reactions were performed using 50 μM AzoFPP-1 in PBS. (D) Crystal structure of FTase (grey) bound to farnesyl diphosphate (green) and peptide substrate (grey sticks). Spheres shown for the leucine residue in the CVLS substrate sequence. PDB 1JCR (E) Molecular docking of AzoFPP-1 in trans (cyan) and cis (purple) into FTase. Spheres shown for the leucine residue in the CVLS substrate sequence.

Optical control of peptide farnesylation

Molecular docking studies of the photoswitchable FPP analogs into the structure of Rattus norvegicus FTase (rFTase, PDB 1JCR) suggested that the trans isomer of the analogs would be accepted by the enzyme better than the cis isomer. Notably, we found that trans-AzoFPP-1 (Figure 2E) exhibited a similar binding pose relative to the peptide substrate compared to endogenous FPP (Figure 2D), while cis-AzoFPP-1 exhibited some visible steric clash with leucine of the substrate peptide CVLS, suggesting that this photoisomer may be a less effective substrate for transfer by interfering with the binding of the peptide substrate, the second step in the kinetic mechanism of the enzyme after farnesyl diphosphate (FPP) binding.36–38

Based on these promising docking results, the in vitro farnesylation of a model peptide (8a) with AzoFPP-1 by yeast farnesyltransferase (yFTase) was explored (Figure 3A & B). The model peptide contained a dansyl fluorophore (λex = 335 nm; λem = 518 nm) for visualization, an RAG sequence to increase solubility and ionization in mass spectrometry, and a CVIA sequence derived from the precursor to the prenylated yeast mating pheromone a-factor. The ratio of 8a to the corresponding farnesylated peptide 8b (with FPP) or 8c/d (with AzoFPP-1) was monitored by LC-MS. While the substrate 8a exhibited a single peak with a retention time of 27.9 min in the absence of enzyme, incubation with yFTase and FPP resulted in formation of a new peak with a longer retention time of 58.8 min and a mass consistent with the formation of the farnesylated product 8b (Figure 3C). Similarly, in the presence of yFTase and AzoFPP-1, a new product eluting at 55.1 min and with a mass consistent with the formation of 8c was observed (Figure 3D). At saturating substrate concentrations (22 μM FPP, 2.4 uM peptide), 63% of 8a was converted to 8b, and this conversion was not significantly affected by irradiation with UV-A light (Figure 3E). For comparison, 51% of 8a was converted to 8c under the same conditions with AzoFPP-1. Thus, AzoFPP-1 appears to be an efficient substrate for yFTase, reacting at approximately 80% the rate observed with FPP. Most importantly, upon irradiation with UV-A light prior to enzyme addition, the conversion to product was markedly reduced from 51% to 10% (5-fold), demonstrating that trans-AzoFPP-1 undergoes significantly more effective transfer to the peptide substrate allowing for optical control of substrate farnesylation. It is important to note that the reaction mixtures containing AzoFPP-1 were allowed to relax for 12 hours after quenching and prior to analysis, thus only 8c was observed and not 8d. This step simplified the analysis because while 8d has a distinct retention time that can be detected, it slowly converts to 8c; allowing complete relaxation eliminated the need to analyze the enzymatic reactions immediately upon completion. To examine whether this marked reduction in rate manifested by cis-AzoFPP-1 was attributable to an effect on KM or kcat, similar experiments were performed at lower isoprenoid concentrations near KM. Under those conditions, trans-AzoFPP-1 again yielded 5-fold greater conversion than cis-AzoFPP-1 (Figure S3). Since the rates measured at high substrate concentration should reflect differences in kcat while the rates observed at low substrate concentrations can reflect effects on both kcat and KM, these results suggest that the major impact of isomerization is on kcat. Parallel experiments performed with a mammalian farnesyltransferase (R. norvegicus, rFTase) exhibited a similar preference for the trans-AzoFPP-1 isomer (Figures S4 and S5). Substrate AzoFPP-2 did not undergo detectable yFTase- or rFTase-catalyzed transfer to a peptide substrate and was therefore not further pursued in this study.

Figure 3 |. Optical control of peptide farnesylation.

Figure 3 |

Open in a new tab

(A) Schematic representation of model peptide substrate farnesylation with AzoFPP-1 in the trans and cis form. (B) Chemical structure of peptide substrate for FTase (8) and a-factor variants (9–11) with various functionalizations (a–d). (C & D) HPLC chromatograms showing conversion of 8a to 8b (C) or 8c (D) upon incubation of 8a with FPP and yFTase in the dark (top) or after UV-A irradiation (bottom). Substrate concentrations were at saturating levels. Absorbance was monitored at 220 nm. (E) Quantification of (C) and (D). Error bars represent SEM.

Optical probing of prenylation processing

Given the substantial (5-fold) optical control of the prenyltransferase-catalyzed reaction obtained with AzoFPP-1, we then decided to investigate the subsequent steps in the isoprenylation processing pathway including proteolysis and carboxyl methylation, and the bioactivity of peptides containing the photoswitchable isoprenoid group. For this purpose, the yeast mating pheromone a-factor was employed because it is a well-established substrate for these enzymes and it has a simple bioactive cellular assay. a-Factor has been extensively studied for its three posttranslational modifications (isoprenylation, proteolysis and carboxyl methylation) that are required for proper mating between two haploid yeast (S. cerevisae) cells.39–43 a-Factor precursors 9a and 10a containing VIA and Cys-COOH C-termini were synthesized by standard solid phase peptide synthetic methods. a-Factor precursor 11a with a C-terminal methyl ester was prepared using a side chain anchoring methodology where Fmoc-Cys-OMe linked to trityl resin via its thiol group (Figure S7) was employed for subsequent solid phase peptide synthesis.44,45 Subsequent peptides were then prenylated chemically with trans,trans-farnesyl bromide or the corresponding chloride precursor used to prepare AzoFPP-1 at pH 5.0 in the presence of Zn(OAc)2 and NaI. These conditions were optimized (Figure S8) based on previously reported procedures.44–48 Peptides containing a VIA (9b and 9c), Cys-COOH (10b and 10c), or Cys-COMe (11b and 11c) termini were obtained in this manner. Using these model peptides, each processing enzyme was assayed for activity with its respective a-factor substrate in either the trans-form (dark) or cis-form (after UV-A irradiation) for light-dependent conversion. Compounds 9b and 9c were used in experiments with the proteases Rce1 and Ste24 and 10b and 10c were used with the isoprenylcysteine carboxyl methyltransferase, Ste14, the Icmt from S. cerevisiae (Figure 4A). To accomplish this, samples were irradiated using using our Cell DISCO system49,50 (5 ms irradiation every 15 s at 370 nm). Generally, azobenzene-containing peptides were converted to products at rates similar to their farnesylated counterpart in each of the enzymatic steps studied, except in the case of Rce1 where a 2-fold decrease was observed with the photolipid-containing peptide (Figure 4B). Each enzyme exhibited only minimal light-dependent activity differences when treated with saturating concentrations of substrate (Figure 4B). These enzymes were further tested at substrate concentrations nearer to the KM to test for possible KM effects, such as changes in binding affinity between the two isoprenoid conformations (Figure S8). No significant differences were observed suggesting that cis/trans isomerization of the diazo-arene had little effect on these enzymatic transformations.

Figure 4 |. Optical probing of prenylation processing pathway.

Figure 4 |

Open in a new tab

(A) Schematic representation of prenylation processing with photoswitchable a-factor analogs in the trans and cis form. (B) Quantification with and without UV-A irradiation of Rce1 and Ste24 activity with compounds 9b and 9c/d (15 μM), and Ste14 activity with compounds 10b and 10c/d (25 μM). (C) Yeast growth arrest halo assay with and without UV-A irradiation of compounds 11b and 11c/d. The amount of substrate spotted is listed in table above with solution controls, ddH2O, YPD and the YPD/BSA mixture used to dilute and make the compound solution, are in the first row. Quantified growth end-point values listed in table below. Error bars represent SEM.

Finally, the bioactivity of the “fully processed” peptides 11b (a-factor), 11c, and 11d was assessed in a yeast growth arrest halo assay employing the DISCO system adapted to a 24 well format (Figure 4C).42,51 All three peptides were found to be active in this receptor-mediated growth arrest assay and exhibited similar potencies. These data suggest that the bioactivity of a-factor is not sensitive to the structural prenyl-group variations explored. Overall, the optical probing of the prenylation processing pathway reported here suggests that the photoswitchable analog permits selective control of peptide lipidation by farnesyltransferase but exhibits little effect on the subsequent processing steps.

Concluding Remarks

Here, we show that isoprenoid lipids can be modified to contain a molecular photoswitch to function as photoswitchable substrates for peptide lipidation by farnesyltransferase. This work enlarges the classes of lipids that can be modified with photoswitches to isoprenoids, which have not been previously investigated using this approach.12,52 The development of the photoswitchable FPP analog AzoFPP-1 and its integration into a series of photoswitchable a-factor analogs enabled us to systematically test the utility of these compounds for optical control of various steps in the CaaX processing pathway, including protein isoprenylation, proteolytic processing, carboxyl methylation, and a-factor bioactivity. This study demonstrated that peptide lipidation with AzoFPP-1 could be effectively modulated through switching between its trans and cis form, while proteolysis, carboxymethylation, and bioactivity were not sensitive to photoisomerization. These findings suggest that the initial lipidation step is more tightly controlled by lipid structure than the subsequent processing steps and that the tool developed here enables selective optical control of this initial step. Given the importance of isoprenylated proteins in signal transduction pathways, these photoswitchable isoprenoids could be particularly useful for decelerating protein prenylation in a temporally controllable manner or for probing cellular signaling processes that sense or are tightly controlled by isoprenoid structure.3,53,54

To date, 2213 isoprenoid lipids have been described (LIPID MAPS55,56). Many of these exhibit linear isoprenoid chains that could be functionalized with an azobenzene in an analogous fashion to yield optical control of their function. Linear isoprenoid lipids with interesting bioactivity include the tocotrienols (Vitamin E),57 cannabinoids (cannabigerol or cannabigerolic acid),58 the moenomycin antibiotics,59 or other natural products such as auraptene and umbelliprenin.60 Isoprenoid lipids have further been used in the design of synthetic pharmacophores, such as the Ras inhibitor Salirasib.61,62 Future efforts will address the development of photoswitchable isoprenoids based on these and other bioactive metabolites to assess how modular the described approach is for the class of isoprenoid lipids.

Materials and Methods

Photophysical Characterization of AzoFPP-1 and AzoFPP-2

UV-Vis spectra were recorded using a Varian Cary 50 Bio UV-Visible Spectrophotometer. Photoswitching was achieved using 365 nm or 460 nm LED light sources. The LEDs were pointed directly onto the top of the sample cuvette with photoswitch (50 μM in DMSO). An initial spectrum was recorded (dark-adapted state, black) and then again following illumination at 365 nm for 30 s (cis-adapted state, gray). A third spectrum was recorded after irradiation at 470 nm for 30 s (trans-adapted state, blue). Absorption at 340 nm was recorded over several switching cycles whilst alternating illumination at 365 nm and 460 nm with. The light source was directly pointed onto the top of the sample cuvette.

Molecular Docking

For modeling of AzoFPP1 in cis and trans confirmations into the active site of PFTase (pdb file 1JCR), docking was performed using MacroModel v #9.9 and its program Glide. The FTase crystal structure was prepared and minimized using the default settings in the protein preparation wizard as part of the Maestro (Schrodinger Release 2021 – 03, Maestro Version 12.9.137package. Prime function was used to fill in missing loops and side chains. Afterwards, a receptor grid large enough to encompass the entire binding site for AzoFPP1 was generated from the prepared PFTase enzyme. An extra precision docking parameter was set and 10 000 ligand poses per docking were run per AzoFPP1 confirmation. The conformations with the overall highest binding score were chosen for display here.

yFTase Mediated Prenylation of Peptide Dns-CVIA peptide with AzoFPP-1 and AzoFPP-2

yFTase was expressed and purified as previously described.63,64 To test if yFTase would process AzoFPP-1, a solution of 2.4 μM 8a was prepared in yFTase prenylation buffer (50 mM Tris Ph 7.5, 15 mM DTT, 10 mM MgCl2, 50 μM ZnCl2, 20 mM KCl) along with either AzoFPP-1 or AzoFPP-2 at 22 μM and placed in a low adhesion microcentrifuge tube. Afterwards the enzymatic reactions were initiated by adding yFTase to a final enzyme concentration of 0.100 mM and a final volume of 250 μL, then incubating at RT for 20 h. The reactions were quenched by the addition of 50 μL of glacial CH3COOH before subjecting to LCMS analysis. LCMS analysis was performed on an Agilent 1200 series system (Windows 10, ChemStation Software, G1322A Degasser, G1312A binary pump, G1329A autosampler, G1315B diode array detector, 6130 quadrupole) equipped with a C18 column (Agilent ZORBAX 300-SB-C18, 5 μM, 4.6 × 250 mm). Samples were not filtered as filtration caused the observation of no peptide products.

Kinetic analysis to determine reactivity of trans- and cis-Azo-FPP1 with yFTase

A solution of 2.4 μM 8a was prepared in yFTase prenylation buffer along with either farnesyl diphosphate (FPP) or AzoFPP-1 at 22 μM (high concentration) or 1 μM (low concentration). UV irradiation of select samples was done by placing the solution in round quartz tubes (10 × 50 mm) with 1 mm wall thickness and irradiating in a Rayonet reactor using 3 × 350 nm bulbs (14 W, RPR-3500 Å) for 2 min. To confirm that Azo-FPP-1 was completely isomerized after 2 min and had not relaxed within the timeframe required to carry out the enzymatic reaction, a solution containing only AzoFPP-1 at 22 μM in prenylation buffer was analyzed by LCMS before and after irradiation with incubation at RT for one h. Complete shift in retention time was observed (Figure S2). Afterwards the enzymatic reactions were carried out in low adhesion microcentrifuge tubes, and initiated by adding yFTase to a final enzyme concentration of 0.175 μM and a final volume of 450 μL, then incubating at RT for 15 min. The reactions were quenched by the addition of 50 μL of glacial CH3COOH before subjecting to LCMS analysis. LCMS analysis was performed on an Agilent 1200 series system (Windows 10, ChemStation Software, G1322A Degasser, G1312A binary pump, G1329A autosampler, G1315B diode array detector, 6130 quadrupole) equipped with a C18 column (Agilent ZORBAX 300-SB-C18, 5 μM, 4.6 X 250 mm). Samples were not filtered as filtration caused the observation of no peptide products. All reactions were run in triplicates. Extent of enzymatic conversion was determined by integration of the starting material and product peaks in 220 nm absorbance chromatograms. This assumes that 8a and 8c have a similar e220 since all the amide bonds as well as the Dansyl group exhibit absorbance at that wavelength. To confirm the validity of this assumption, a master mix containing all the reaction component except the enzyme was prepared (2.4 μM 8a, 22 μM, 50 mM Tris Ph 7.5, 15 mM DTT, 10 mM MgCl2, 50 μM ZnCl2, 20 mM KCl). This solution was split into two equal aliquots each in two low adhesion microcentrifuge tubes. One aliquot received yFTase enzyme in Tris buffer to a final concentration of 0.175 μM and a final volume of 450 μL, while the other received equal volume of only Tris buffer. After incubating at RT until ~50% conversion was observed each solution received 50 μL of glacial CH3COOH and both were subjected to LC-MS analysis. The integrated 220 nm absorbance of the 8a peak in the case of the no enzyme solution was 1765.3 units, while the sum of the integrated areas of 8a and 8c peak was 1735.2 units in the case of the sample with yFTase enzyme, which are within 2% of each other.

rFTase Mediated Prenylation of Peptide Dns-CVIS peptide with AzoFPP-1 and AzoFPP-2

To ascertain if mammalian FTase would process Azo-FPP-1 and AzoFPP-2, Rattus norvegicus FTase (rFTase) was expressed and purified as previously described.65,66 dns-CVLS peptide, representing the native sequence of the enzyme with the addition of a Dansyl fluorophore for detection and quantification, was incubated at 3 μM in an rFTase prenylation buffer (50 mM HEPPSO-NaOH, pH 7.8, 5 mM TCEP, and 5 mM MgCl2) (50 μL total) for 20 minutes in 0.65 mL low-adhesion Eppendorf tubes. 50 μL of an enzyme solution containing 100 nM rFTase and either 10 μM AzoFPP-1 or AzoFPP-2 was then incubated at RT for 16 hours before adding an equal volume of 20% CH3COOH in (CH3)2CHOH and subjecting to HPLC analysis. HPLC analysis was performed at ambient temperature on an Agilent 1260 HPLC system with auto-sampler, UV-Vis, and fluorescence detection using a C18 reversed-phase analytical column (Zorbax XDB-C18). a linear gradient from 30% acetonitrile in 25 mM ammonium acetate to 100% acetonitrile flowing at 1 mL/min over 30 minutes was used. Peptides and products were detected by fluorescence (λex 340 nm, λem 496 nm). In the case of AzoFPP-1, complete conversion was observed. In the case of AzoFPP-2, no conversion was observed (data not shown).

Kinetic analysis to determine reactivity of trans- and cis-Azo-FPP1 with rFTase

To test if there would be a difference in the rate of processing of tras-AziFPP-1 vs cis-AzoFPP-1 by rFTase, solutions of 3 μM of Dns-GCVLS peptide in rFTase prenylation buffer were incubated for 20 minutes in 0.65 mL low-adhesion Eppendorf tubes. These solutions were then either non-illuminated (trans isomer) or illuminated with 365 nm LED light (cis isomer) for 3 minutes in the dark. To initiate the reaction, 50 μL solutions containing 100 nM rFTase and 10 μM AzoFPP-1 in rFTase prenylation buffer were added to each tube. Reactions were incubated at RT for different time points; 30 minutes, 60 minutes, 120 minutes, 240 minutes, or 360 minutes before quenching and running HPLC analysis as described above. Reaction progress, expressed as % conversion, was calculated by dividing the product integral by the sum of the product and substrate integrals followed my multiplication by 100.

Growth Arrest Assay

Growth arrest assays were performed as previously described with modifications.42,51 Briefly, supersensitive, ss2 MATα cells (strain SM2375) were grown overnight at 30°C in yeast peptone dextrose (YPD) media. Cells were pelleted at 2,000 × g and washed twice with ddH2O prior to resuspension in ddH2O at 1×106 cells/mL and combined with Bacto agar (1.1% in YPD) for a final concentration of 250,000 cells/mL. Cells were spread onto solid YPD medium in each well of a 24-well plate to form a lawn of MATα cells at 20,000 cells/well. Dilutions of FPP (11b) and AzoFPP-1 (11c/d) a-factor analogs were prepared in 0.5% bovine serum albumin (BSA)/YPD. UV-treated samples were irradiated for 2 min at 365 nm. For all samples, 2.5 μL of diluted a-factor analog were spotted onto the lawn in 3000, 300, 30, 15, 7.5 and 3.8 pg amounts. Plates were incubated for 24 hrs at 30 °C in the dark or under UV-A irradiation using the Cell DISCO system (5 ms irradiation every 15s at 370 nm).49,50 The assay end point was determined for each a-factor analog and UV treatment condition to be the lowest concentration at which agar clearance was detectable. Each experiment was performed in triplicate.

Protease and Methyltransferase Assays

Proteolytic and methylation assays were performed using crude membrane preparations as previously described.6,67,68 Briefly, proteolysis by Rce1 and Ste24 were measured using a coupled proteolysis/methylation assay in which crude membrane preparations from S. cerevisiae overexpressing Rce1 or Ste24 (5 μg) were combined with excess amounts of Ste14 overexpressing crude membranes (10 ug per condition). FPP (9b) or AzoFPP-1 (9c/d) a-factor analogs were assayed at saturating (maximal velocity, Vmax) conditions of 15 μM (Figure 4B). These compounds were also tested below established KM values for the enzymes (Figure S7).42 Samples were pre-irradiated with UV-A (370 nm) light for 2 min. Subsequently, 20 μM of S-adensoyl [14C-methyl]-L-Methionine (52.6 mCi/mmol) (PerkinElmer, USA) in 100 mM Tris-HCl, pH 7.5 was added to the reaction. Reactions were incubated at 30°C for 30 min under dark or UV-A conditions using the Cell DISCO, as described above. Reactions were terminated with the addition of 50 μL of 1M NaOH/ 1% SDS. Reaction mixtures were spotted onto filter paper, which was placed in the neck of a closed vial above 10 mL of scintillation fluid. [14C]-methanol vapors were allowed to diffuse into the scintillation fluid for 3 h at RT and subsequently quantified by liquid scintillation counting. Sample counts were corrected using background in the absence of enzyme. For the evaluation of methylation by Ste14, similar conditions were used, with FPP (10b) or AzoFPP-1 (10c/d) a-factor analogs at saturating (maximal velocity, Vmax) conditions of 25 μM (Figure 4B).42 5 μM substrate was used for conditions below KM of Ste14 (Figure S7). Each reaction was performed in duplicate and counted three times. Assays were repeated in triplicate. Enzyme specific activity is reported as pmol methyl groups transferred per min per mg of enzyme.

Supplementary Material

Supporting Information

Acknowledgments

J. M. thanks the NCI for a K00 award (K00CA253758). J. L. H. thank the NIH for funding (R01GM132606). D.T. thanks the National Institutes of Health for financial support (R01NS108151). M. D. D. and C. A. H. thank the National Science Foundation for funding (NSF/CHE 1905204). The authors thank Ian M. Ahearn and Mark R. Philips for their helpful comments and experimental support in the early stages of this study.

Footnotes

Supporting Information

Supplementary Figures, experimental procedures and compound characterization including 1H NMR, 13C NMR, and 31P NMR spectra and HPLC chromatograms.

The authors declare no competing financial interests.

References

  • (1).Khoury GA; Baliban RC; Floudas CA Proteome-Wide Post-Translational Modification Statistics: Frequency Analysis and Curation of the Swiss-Prot Database. Sci. Rep 2011, 1 (1), 90. 10.1038/srep00090. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (2).Jiang H; Zhang X; Chen X; Aramsangtienchai P; Tong Z; Lin H Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies. Chem. Rev 2018, 118 (3), 919–988. 10.1021/acs.chemrev.6b00750. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (3).Wang M; Casey PJ Protein Prenylation: Unique Fats Make Their Mark on Biology. Nat. Rev. Mol. Cell Biol 2016, 17 (2), 110–122. 10.1038/nrm.2015.11. [DOI] [PubMed] [Google Scholar]
  • (4).Ma YT; Chaudhuri A; Rando RR Substrate Specificity of the Isoprenylated Protein Endoprotease. Biochemistry 1992, 31 (47), 11772–11777. 10.1021/bi00162a014. [DOI] [PubMed] [Google Scholar]
  • (5).Ashby MN; King DS; Rine J Endoproteolytic Processing of a Farnesylated Peptide in Vitro. Proc. Natl. Acad. Sci. U. S. A 1992, 89 (10), 4613–4617. 10.1073/pnas.89.10.4613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (6).Hrycyna CA; Clarke S Farnesyl Cysteine C-Terminal Methyltransferase Activity Is Dependent upon the STE14 Gene Product in Saccharomyces Cerevisiae. Mol. Cell. Biol 1990, 10 (10), 5071–5076. 10.1128/mcb.10.10.5071-5076.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (7).Gelb MH; Brunsveld L; Hrycyna CA; Michaelis S; Tamanoi F; Van Voorhis WC; Waldmann H Therapeutic Intervention Based on Protein Prenylation and Associated Modifications. Nat. Chem. Biol 2006, 2 (10), 518–528. 10.1038/nchembio818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (8).Palsuledesai CC; Distefano MD Protein Prenylation: Enzymes, Therapeutics, and Biotechnology Applications. ACS Chem. Biol 2015, 10 (1), 51–62. 10.1021/cb500791f. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (9).William Pass DVM FTase Inhibition Holds Promise for RAS Targeting and Beyond 2018.
  • (10).Dhillon S Lonafarnib: First Approval. Drugs 2021, 81 (2), 283–289. 10.1007/s40265-020-01464-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (11).Abate-Pella D; Zeliadt NA; Ochocki JD; Warmka JK; Dore TM; Blank DA; Wattenberg EV; Distefano MD Photochemical Modulation of Ras-Mediated Signal Transduction Using Caged Farnesyltransferase Inhibitors: Activation by One- and Two-Photon Excitation. ChemBioChem 2012, 13 (7), 1009–1016. 10.1002/cbic.201200063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (12).Trauner D; Morstein J Optical Control of Glycerolipids and Sphingolipids. Chimia 2021, 75 (12), 1022–1025. 10.2533/chimia.2021.1022. [DOI] [PubMed] [Google Scholar]
  • (13).Frank JA; Yushchenko DA; Fine NHF; Duca M; Citir M; Broichhagen J; Hodson DJ; Schultz C; Trauner D Optical Control of GPR40 Signalling in Pancreatic β-Cells. Chem. Sci 2017, 8 (11), 7604–7610. 10.1039/C7SC01475A. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (14).Morstein J; Hill RZ; Novak AJE; Feng S; Norman DD; Donthamsetti PC; Frank JA; Harayama T; Williams BM; Parrill AL; Tigyi GJ; Riezman H; Isacoff EY; Bautista DM; Trauner D Optical Control of Sphingosine-1-Phosphate Formation and Function. Nat. Chem. Biol 2019, 15 (6), 623. 10.1038/s41589-019-0269-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (15).Morstein J; Dacheux MA; Norman DD; Shemet A; Donthamsetti PC; Citir M; Frank JA; Schultz C; Isacoff EY; Parrill AL; Tigyi GJ; Trauner D Optical Control of Lysophosphatidic Acid Signaling. J. Am. Chem. Soc 2020, 142 (24), 10612–10616. 10.1021/jacs.0c02154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (16).Frank JA; Moroni M; Moshourab R; Sumser M; Lewin GR Photoswitchable Fatty Acids Enable Optical Control of TRPV1 2015, 6, 7118. 10.1038/ncomms8118. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (17).Lichtenegger M; Tiapko O; Svobodova B; Stockner T; Glasnov TN; Schreibmayer W; Platzer D; de la Cruz GG; Krenn S; Schober R; Shrestha N; Schindl R; Romanin C; Groschner K An Optically Controlled Probe Identifies Lipid-Gating Fenestrations within the TRPC3 Channel. Nat. Chem. Biol 2018, 14 (4), 396–404. 10.1038/s41589-018-0015-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (18).Leinders-Zufall T; Storch U; Bleymehl K; Schnitzler M. M. y; Frank JA; Konrad DB; Trauner D; Gudermann T; Zufall F PhoDAGs Enable Optical Control of Diacylglycerol-Sensitive Transient Receptor Potential Channels. Cell Chem. Biol 2018, 25 (2), 215–223.e3. 10.1016/j.chembiol.2017.11.008. [DOI] [PubMed] [Google Scholar]
  • (19).Frank JA; Yushchenko DA; Hodson DJ; Lipstein N; Nagpal J; Rutter GA; Rhee J-S; Gottschalk A; Brose N; Schultz C; Trauner D Photoswitchable Diacylglycerols Enable Optical Control of Protein Kinase C. Nat Chem Biol 2016, 12 (9), 755–762. 10.1038/nchembio.2141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (20).Kol M; Williams B; Toombs-Ruane H; Franquelim HG; Korneev S; Schroeer C; Schwille P; Trauner D; Holthuis JC; Frank JA Optical Manipulation of Sphingolipid Biosynthesis Using Photoswitchable Ceramides. eLife 2019, 8, e43230. 10.7554/eLife.43230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (21).Morstein J; Kol M; Novak AJE; Feng S; Khayyo S; Hinnah K; Li-Purcell N; Pan G; Williams BM; Riezman H; Atilla-Gokcumen GE; Holthuis JCM; Trauner D Short Photoswitchable Ceramides Enable Optical Control of Apoptosis. ACS Chem. Biol 2021, 16 (3), 452–456. 10.1021/acschembio.0c00823. [DOI] [PubMed] [Google Scholar]
  • (22).Tei R; Morstein J; Shemet A; Trauner D; Baskin JM Optical Control of Phosphatidic Acid Signaling. ACS Cent. Sci 2021, 7 (7), 1205–1215. 10.1021/acscentsci.1c00444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (23).Morstein J; Trads JB; Hinnah K; Willems S; Barber DM; Trauner M; Merk D; Trauner D Optical Control of the Nuclear Bile Acid Receptor FXR with a Photohormone. Chem. Sci 2020, 11 (2), 429–434. 10.1039/C9SC02911G. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (24).Hinnah K; Willems S; Morstein J; Heering J; Hartrampf FWW; Broichhagen J; Leippe P; Merk D; Trauner D Photohormones Enable Optical Control of the Peroxisome Proliferator-Activated Receptor γ (PPARγ). J. Med. Chem 2020, 63 (19), 10908–10920. 10.1021/acs.jmedchem.0c00654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (25).Hartrampf N; Seki T; Baumann A; Watson P; Vepřek NA; Hetzler BE; Hoffmann-Röder A; Tsuji M; Trauner D Optical Control of Cytokine Production Using Photoswitchable Galactosylceramides. Chem. – Eur. J 2020, 26 (20), 4476–4479. 10.1002/chem.201905279. [DOI] [PubMed] [Google Scholar]
  • (26).Pernpeintner C; Frank JA; Urban P; Roeske CR; Pritzl SD; Trauner D; Lohmüller T Light-Controlled Membrane Mechanics and Shape Transitions of Photoswitchable Lipid Vesicles. Langmuir 2017, 33 (16), 4083–4089. 10.1021/acs.langmuir.7b01020. [DOI] [PubMed] [Google Scholar]
  • (27).Doroudgar M; Morstein J; Becker-Baldus J; Trauner D; Glaubitz C How Photoswitchable Lipids Affect the Order and Dynamics of Lipid Bilayers and Embedded Proteins. J. Am. Chem. Soc 2021, 143 (25), 9515–9528. 10.1021/jacs.1c03524. [DOI] [PubMed] [Google Scholar]
  • (28).Chander N; Morstein J; Bolten JS; Shemet A; Cullis PR; Trauner D; Witzigmann D Optimized Photoactivatable Lipid Nanoparticles Enable Red Light Triggered Drug Release. Small 2021, 17 (21), 2008198. 10.1002/smll.202008198. [DOI] [PubMed] [Google Scholar]
  • (29).Jiménez-Rojo N; Feng S; Morstein J; Pritzl SD; Harayama T; Asaro A; Vepřek NA; Arp CJ; Reynders M; Novak AJE; Kanshin E; Ueberheide B; Lohmüller T; Riezman H; Trauner D Optical Control of Membrane Fluidity Modulates Protein Secretion. bioRxiv February 14, 2022, p 2022.02.14.480333. 10.1101/2022.02.14.480333. [DOI] [Google Scholar]
  • (30).Subramanian T; Pais JE; Liu S; Troutman JM; Suzuki Y; Leela Subramanian K; Fierke CA; Andres DA; Spielmann HP Farnesyl Diphosphate Analogues with Aryl Moieties Are Efficient Alternate Substrates for Protein Farnesyltransferase. Biochemistry 2012, 51 (41), 8307–8319. 10.1021/bi3011362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (31).Broichhagen J; Frank JA; Trauner D A Roadmap to Success in Photopharmacology. Acc. Chem. Res 2015, 48 (7), 1947–1960. 10.1021/acs.accounts.5b00129. [DOI] [PubMed] [Google Scholar]
  • (32).Morstein J; Awale M; Reymond J-L; Trauner D Mapping the Azolog Space Enables the Optical Control of New Biological Targets. ACS Cent. Sci 2019, 5 (4), 607–618. 10.1021/acscentsci.8b00881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (33).Appel R Tertiary Phosphane/Tetrachloromethane, a Versatile Reagent for Chlorination, Dehydration, and P N Linkage. Angew. Chem. Int. Ed. Engl 1975, 14 (12), 801–811. 10.1002/anie.197508011. [DOI] [Google Scholar]
  • (34).Baeyer A Nitrosobenzol Und Nitrosonaphtalin. Berichte Dtsch. Chem. Ges 1874, 7 (2), 1638–1640. 10.1002/cber.187400702214. [DOI] [Google Scholar]
  • (35).Mills C XCIII.—Some New Azo-Compounds. J. Chem. Soc. Trans 1895, 67 (0), 925–933. 10.1039/CT8956700925. [DOI] [Google Scholar]
  • (36).Dolence JM; Poulter CD A Mechanism for Posttranslational Modifications of Proteins by Yeast Protein Farnesyltransferase. Proc. Natl. Acad. Sci 1995, 92 (11), 5008–5011. 10.1073/pnas.92.11.5008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (37).Dolence JM; Cassidy PB; Mathis JR; Poulter CD Yeast Protein Farnesyltransferase: Steady-State Kinetic Studies of Substrate Binding. Biochemistry 1995, 34 (51), 16687–16694. 10.1021/bi00051a017. [DOI] [PubMed] [Google Scholar]
  • (38).Pompliano DL; Rands E; Schaber MD; Mosser SD; Anthony NJ; Gibbs JB Steady-State Kinetic Mechanism of Ras Farnesyl:Protein Transferase. Biochemistry 1992, 31 (15), 3800–3807. 10.1021/bi00130a010. [DOI] [PubMed] [Google Scholar]
  • (39).Anderegg RJ; Betz R; Carr SA; Crabb JW; Duntze W Structure of Saccharomyces Cerevisiae Mating Hormone A-Factor. Identification of S-Farnesyl Cysteine as a Structural Component. J. Biol. Chem 1988, 263 (34), 18236–18240. [PubMed] [Google Scholar]
  • (40).Diaz-Rodriguez V; Distefano MD A-Factor: A Chemical Biology Tool for the Study of Protein Prenylation. Curr. Top. Pept. Protein Res 2017, 18, 133–151. [PMC free article] [PubMed] [Google Scholar]
  • (41).Diaz-Rodriguez V; Mullen DG; Ganusova E; Becker JM; Distefano MD Synthesis of Peptides Containing C-Terminal Methyl Esters Using Trityl Side-Chain Anchoring: Application to the Synthesis of a-Factor and a-Factor Analogs. Org. Lett 2012, 14 (22), 5648–5651. 10.1021/ol302592v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (42).Diaz-Rodriguez V; Hsu E-T; Ganusova E; Werst ER; Becker JM; Hrycyna CA; Distefano MD A-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity. Bioconjug. Chem 2018, 29 (2), 316–323. 10.1021/acs.bioconjchem.7b00648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (43).Marcus S; Caldwell GA; Miller D; Xue CB; Naider F; Becker JM Significance of C-Terminal Cysteine Modifications to the Biological Activity of the Saccharomyces Cerevisiae a-Factor Mating Pheromone. Mol. Cell. Biol 1991, 11 (7), 3603–3612. 10.1128/MCB.11.7.3603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (44).Bader TK; Rappe TM; Veglia G; Distefano MD Chapter Eight - Synthesis and NMR Characterization of the Prenylated Peptide, a-Factor. In Methods in Enzymology; Wand AJ, Ed.; Biological NMR Part A; Academic Press, 2019; Vol. 614, pp 207–238. 10.1016/bs.mie.2018.09.025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (45).Diaz-Rodriguez V; Ganusova E; Rappe TM; Becker JM; Distefano MD Synthesis of Peptides Containing C-Terminal Esters Using Trityl Side-Chain Anchoring: Applications to the Synthesis of C-Terminal Ester Analogs of the Saccharomyces Cerevisiae Mating Pheromone a-Factor. J. Org. Chem 2015, 80 (22), 11266–11274. 10.1021/acs.joc.5b01376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (46).Yang CC; Marlowe CK; Kania R Efficient Method for Regioselective Isoprenylation of Cysteine Thiols in Unprotected Peptides. J. Am. Chem. Soc 1991, 113 (8), 3177–3178. 10.1021/ja00008a059. [DOI] [Google Scholar]
  • (47).Yang CC; Marlowe CK; Kania R Efficient Method for Regioselective Isoprenylation of Cysteine Thiols in Unprotected Peptides. J. Am. Chem. Soc 1991, 113 (8), 3177–3178. 10.1021/ja00008a059. [DOI] [Google Scholar]
  • (48).Wollack JW; Zeliadt NA; Ochocki JD; Mullen DG; Barany G; Wattenberg EV; Distefano MD Investigation of the Sequence and Length Dependence for Cell-Penetrating Prenylated Peptides. Bioorg. Med. Chem. Lett 2010, 20 (1), 161–163. 10.1016/j.bmcl.2009.11.026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (49).Borowiak M; Nahaboo W; Reynders M; Nekolla K; Jalinot P; Hasserodt J; Rehberg M; Delattre M; Zahler S; Vollmar A; Trauner D; Thorn-Seshold O Photoswitchable Inhibitors of Microtubule Dynamics Optically Control Mitosis and Cell Death. Cell 2015, 162 (2), 403–411. 10.1016/j.cell.2015.06.049. [DOI] [PubMed] [Google Scholar]
  • (50).Morstein J; Trauner D Chapter Eleven - Photopharmacological Control of Lipid Function. In Methods in Enzymology; Chenoweth DM, Ed.; Chemical Tools for Imaging, Manipulating, and Tracking Biological Systems: Diverse Methods for Prokaryotic and Eukaryotic Systems; Academic Press, 2020; Vol. 638, pp 219–232. 10.1016/bs.mie.2020.04.025. [DOI] [PubMed] [Google Scholar]
  • (51).Nijbroek GL; Michaelis S Functional Assays for Analysis of Yeast Ste6 Mutants. Methods Enzymol 1998, 292, 193–212. 10.1016/s0076-6879(98)92016-x. [DOI] [PubMed] [Google Scholar]
  • (52).Morstein J; Impastato AC; Trauner D Photoswitchable Lipids. ChemBioChem 2021, 22 (1), 73–83. 10.1002/cbic.202000449. [DOI] [PubMed] [Google Scholar]
  • (53).Hancock JF; Cadwallader K; Marshall CJ Methylation and Proteolysis Are Essential for Efficient Membrane Binding of Prenylated P21K-Ras(B). EMBO J 1991, 10 (3), 641–646. 10.1002/j.1460-2075.1991.tb07992.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (54).Wright LP; Philips MR Thematic Review Series: Lipid Posttranslational Modifications. CAAX Modification and Membrane Targeting of Ras. J. Lipid Res 2006, 47 (5), 883–891. 10.1194/jlr.R600004-JLR200. [DOI] [PubMed] [Google Scholar]
  • (55).Mullard A Finding the Way with LIPID MAPS. Nat. Rev. Mol. Cell Biol 2008, 9 (2), 92–92. 10.1038/nrm2342. [DOI] [Google Scholar]
  • (56).O’Donnell VB; Dennis EA; Wakelam MJO; Subramaniam S LIPID MAPS: Serving the next Generation of Lipid Researchers with Tools, Resources, Data, and Training. Sci. Signal 2019, 12 (563), eaaw2964. 10.1126/scisignal.aaw2964. [DOI] [PubMed] [Google Scholar]
  • (57).Pearce BC; Parker RA; Deason ME; Qureshi AA; Wright JJ Hypocholesterolemic Activity of Synthetic and Natural Tocotrienols. J. Med. Chem 1992, 35 (20), 3595–3606. 10.1021/jm00098a002. [DOI] [PubMed] [Google Scholar]
  • (58).Nachnani R; Raup-Konsavage WM; Vrana KE The Pharmacological Case for Cannabigerol. J. Pharmacol. Exp. Ther 2021, 376 (2), 204–212. 10.1124/jpet.120.000340. [DOI] [PubMed] [Google Scholar]
  • (59).Ostash B; Walker S Moenomycin Family Antibiotics: Chemical Synthesis, Biosynthesis, and Biological Activity. Nat. Prod. Rep 2010, 27 (11), 1594–1617. 10.1039/C001461N. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (60).Fiorito S; Preziuso F; Sharifi-Rad M; Marchetti L; Epifano F; Genovese S Auraptene and Umbelliprenin: A Review on Their Latest Literature Acquisitions. Phytochem. Rev 2020. 10.1007/s11101-020-09713-5. [DOI] [Google Scholar]
  • (61).Furuse J; Kurata T; Okano N; Fujisaka Y; Naruge D; Shimizu T; Kitamura H; Iwasa T; Nagashima F; Nakagawa K An Early Clinical Trial of Salirasib, an Oral RAS Inhibitor, in Japanese Patients with Relapsed/Refractory Solid Tumors. Cancer Chemother. Pharmacol 2018, 82 (3), 511–519. 10.1007/s00280-018-3618-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (62).Rotblat B; Ehrlich M; Haklai R; Kloog Y The Ras Inhibitor Farnesylthiosalicylic Acid (Salirasib) Disrupts The Spatiotemporal Localization Of Active Ras: A Potential Treatment For Cancer. In Methods in Enzymology; Small GTPases in Disease, Part B; Academic Press, 2008; Vol. 439, pp 467–489. 10.1016/S0076-6879(07)00432-6. [DOI] [PubMed] [Google Scholar]
  • (63).Mayer MP; Prestwich GD; Dolence JM; Bond PD; Wu HY; Poulter CD Protein Farnesyltransferase: Production in Escherichia Coli and Immunoaffinity Purification of the Heterodimer from Saccharomyces Cerevisiae. Gene 1993, 132 (1), 41–47. 10.1016/0378-1119(93)90512-2. [DOI] [PubMed] [Google Scholar]
  • (64).Gaon I; Turek TC; Weller VA; Edelstein RL; Singh SK; Distefano MD Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase. J. Org. Chem 1996, 61 (22), 7738–7745. 10.1021/jo9602736. [DOI] [PubMed] [Google Scholar]
  • (65).Gangopadhyay SA; Losito EL; Hougland JL Targeted Reengineering of Protein Geranylgeranyltransferase Type I Selectivity Functionally Implicates Active-Site Residues in Protein-Substrate Recognition. Biochemistry 2014, 53 (2), 434–446. 10.1021/bi4011732. [DOI] [PubMed] [Google Scholar]
  • (66).Blanden MJ; Suazo KF; Hildebrandt ER; Hardgrove DS; Patel M; Saunders WP; Distefano MD; Schmidt WK; Hougland JL Efficient Farnesylation of an Extended C-Terminal C(x)3X Sequence Motif Expands the Scope of the Prenylated Proteome. J. Biol. Chem 2018, 293 (8), 2770–2785. 10.1074/jbc.M117.805770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • (67).Anderson JL; Frase H; Michaelis S; Hrycyna CA Purification, Functional Reconstitution, and Characterization of the Saccharomyces Cerevisiae Isoprenylcysteine Carboxylmethyltransferase Ste14p. J. Biol. Chem 2005, 280 (8), 7336–7345. 10.1074/jbc.M410292200. [DOI] [PubMed] [Google Scholar]
  • (68).Coffinier C; Hudon SE; Farber EA; Chang SY; Hrycyna CA; Young SG; Fong LG HIV Protease Inhibitors Block the Zinc Metalloproteinase ZMPSTE24 and Lead to an Accumulation of Prelamin A in Cells. Proc. Natl. Acad. Sci. U. S. A 2007, 104 (33), 13432–13437. 10.1073/pnas.0704212104. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information
NIHMS1860028-supplement-Supporting_Information.pdf (2.2MB, pdf)

RESOURCES