Engineering of a Plant Isoprenyl Diphosphate Synthase for Development of Irregular Coupling Activity

We performed mutagenesis on a regular isoprenyl diphosphate synthase (IDS), neryl diphosphate synthase from Solanum lycopersicum (SlNPPS), that has a structurally related analogue performing non-head-to-tail coupling of two dimethylallyl diphosphate (DMAPP) units, lavandulyl diphosphate synthase from Lavandula x intermedia (LiLPPS). Wild-type SlNPPS catalyses regular coupling of isopentenyl diphosphate (IPP) and DMAPP in cis-orientation resulting in the formation of neryl diphosphate. However, if the enzyme is fed with DMAPP only, it is able to catalyse the coupling of two DMAPP units and synthesizes two irregular monoterpene diphosphates; their structures were elucidated by the NMR analysis of their dephosphorylation products. One of the alcohols is lavandulol. The second compound is the trans-isomer of planococcol, the first example of an irregular cyclobutane monoterpene with this stereochemical configuration. The irregular activity of SlNPPS constitutes 0.4 % of its regular activity and is revealed only if the enzyme is supplied with DMAPP in the absence of IPP. The exchange of asparagine 88 for histidine considerably enhanced the non-head-to-tail coupling. While still only observed in the absence of IPP, irregular activity of the mutant reaches 13.1 % of its regular activity. The obtained results prove that regular IDS are promising starting points for protein engineering aiming at the development of irregular activities and leading to novel monoterpene structures.     

Structural and Mechanistic Insight into Terpene Synthases that Catalyze the Irregular Non-Head-to-Tail Coupling of Prenyl Substrates

Terpenoids have diverse structures and thus represent an important class of biologically active natural products. The structural diversity of terpenoids originates from the coupling of prenyl diphosphate substrates, such as isopentenyl diphosphate and dimethylallyl diphosphate. These isoprenyl diphosphates undergo canonical and sequential “head-to-tail” coupling catalyzed by terpene synthases, followed by modifications such as cyclization, hydroxylation, and glycosylation. In recent years, several terpene synthases that catalyze irregular “non-head-to-tail” couplings to afford branched terpenoids have been identified. This minireview describes structural and mechanistic insights into these unusual coupling reactions that provide a new strategy for the structural diversification of natural products.     

Membrane Permeant Analogs for Independent Cellular Introduction of the Terpene Precursors Isopentenyl- and Dimethylallyl-Pyrophosphate

Isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) are the central five-carbon precursors to all terpenes. Despite their significance, exogenous, independent delivery of IPP and DMAPP to cells is impossible as the negatively charged pyrophosphate makes these molecules membrane impermeant. Herein, we demonstrate a facile method to circumvent this challenge through esterification of the β-phosphate with two self-immolative esters (SIEs) that neutralize the negatively charged pyrophosphate to yield membrane-permeant analogs of IPP and DMAPP. Following cellular incorporation, general esterase activity initiates cleavage of the SIEs, resulting in traceless release of IPP and DMAPP for metabolic utilization. Addition of the synthesized IPP and DMAPP precursor analogs rescued cell growth of glioblastoma (U-87MG) cancer cells concurrently treated with the HMG-CoA reductase inhibitor pitavastatin, which otherwise abrogates cell growth via blocking production of IPP and DMAPP. This work demonstrates a new application of a prodrug strategy to incorporate a metabolic intermediate and promises to enable future interrogation of the distinct biological roles of IPP and DMAPP.     

Generation of Stable Isopentenyl Monophosphate Aryloxy Triester Phosphoramidates as Activators of Vγ9Vδ2 T Cells

Aryloxy triester phosphoramidate prodrugs of the monophosphate derivatives of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) were synthesized as lipophilic derivatives that can improve cell uptake. Despite the structural similarity of IPP and DMAPP, it was noted that their phosphoramidate prodrugs exhibited distinct stability profiles in aqueous environments, which we show is due to the position of the allyl bond in the backbones of the IPP and DMAPP monophosphates. As the IPP monophosphate aryloxy triester phosphoramidates showed favorable stability, they were subsequently investigated for their ability to activate Vγ9/Vδ2 T cells and they showed promising activation of this subset of T cells. Together, these findings represent the first report of IPP and DMAPP monophosphate prodrugs and the ability of IPP aryloxy triester phosphoramidate prodrugs to activate Vγ9/Vδ2 T cells highlighting their potential as possible immunotherapeutics.     

Sesquiterpene Synthase‐Catalysed Formation of a New Medium‐Sized Cyclic Terpenoid Ether from Farnesyl Diphosphate Analogues

Terpene synthases catalyse the first step in the conversion of prenyl diphosphates to terpenoids. They act as templates for their substrates to generate a reactive conformation, from which a Mg²⁺‐dependent reaction creates a carbocation–PP_i ion pair that undergoes a series of rearrangements and (de)protonations to give the final terpene product. This tight conformational control was exploited for the (R)‐germacrene A synthase– and germacradien‐4‐ol synthase–catalysed formation of a medium‐sized cyclic terpenoid ether from substrates containing nucleophilic functional groups. Farnesyl diphosphate analogues with a 10,11‐epoxide or an allylic alcohol were efficiently converted to a 11‐membered cyclic terpenoid ether that was characterised by HRMS and NMR spectroscopic analyses. Further experiments showed that other sesquiterpene synthases, including aristolochene synthase, δ‐cadinene synthase and amorphadiene synthase, yielded this novel terpenoid from the same substrate analogues. This work illustrates the potential of terpene synthases for the efficient generation of structurally and functionally novel medium‐sized terpene ethers.     

Bis(benzoyloxybenzyl)‐DiPPro Nucleoside Diphosphates of Anti‐HIV Active Nucleoside Analogues

Nucleoside analogues are extensively used as antiviral and anticancer agents. Their efficiency is dependent on their metabolism into the ultimately active nucleoside triphosphates. Often one step or even more in the metabolism of the nucleoside to the triphosphate is inefficient. To overcome this hurdle, prodrugs of the nucleotides are needed. Bis(acyloxybenzyl)nucleoside diphosphates have been reported by us as a first example of an efficient nucleoside diphosphate prodrug (DiPPro nucleotides). Here, the synthesis and the properties of bis(benzoyloxybenzyl)nucleoside diphosphates of the nucleoside analogues d4T and AZT are disclosed. The synthesis was achieved by using a phosphoramidite/oxidation route. In chemical hydrolysis studies, most of the compounds formed a nucleoside diphosphate. This was confirmed in CEM cell extracts, although the prodrug stability in extracts was lower than in phosphate buffer. Furthermore, the stability and the amount of nucleoside diphosphate formed were dependent on the substituent in the benzoyl moiety. Some of the compounds were more active against HIV in thymidine kinase‐deficient CEM/TK^? cells than were d4T or AZT.     

High Versatility of IPP and DMAPP Methyltransferases Enables Synthesis of C6, C7 and C8 Terpenoid Building Blocks

The natural substance class of terpenoids covers an extremely wide range of different structures, although their building block repertoire is limited to the C₅ compounds DMAPP and IPP. This study aims at the characterization of methyltransferases (MTases) that modify these terpene precursors and the demonstration of their suitability for biotechnological purposes. All seven enzymes tested accepted IPP as substrate and altogether five C₆ compounds and six C₇ compounds were formed within the reactions. A high selectivity for the deprotonation site as well as high stereoselectivity could be observed for most of the biocatalysts. Only the enzyme from Micromonospora humi also accepted DMAPP as substrate, converting it into (2R)-2-methyl-IPP in vitro. In vivo studies demonstrated the production of a C₈ compound and a hydride shift step within the MTase-catalyzed reaction. Our study presents IPP/DMAPP MTases with very different catalytic properties, which provide biosynthetic access to many novel terpene-derived structures.     

Tryptophan C5‐, C6‐ and C7‐Prenylating Enzymes Displaying a Preference for C‐6 of the Indole Ring in the Presence of Unnatural Dimethylallyl Diphosphate Analogues

The behavior of four dimethylallyltryptophan synthases (DMATSs) (5‐DMATS and 5‐DMATS_Sc as tryptophan C5‐prenyltransferases, and 6‐DMATS_Sa and 6‐DMATS_Sv as C6‐prenyltransferases) and one L ‐tyrosine prenyltransferase with a tryptophan C7‐prenyltransferase activity was investigated in the presence of two unnatural alkyl donors (methylallyl and 2‐pentenyl diphosphate) and one benzyl donor (benzyl diphosphate). Detailed biochemical investigations revealed the acceptance of these dimethylallyl diphosphate (DMAPP) analogues by all tested enzymes with different relative activities. Enzyme products with the allyl or benzyl moiety attached to different positions were identified in the reaction mixtures, whereby C‐6 alkylated or benzylated L ‐tryptophan was found as one of the main products. This observation demonstrates a preference of the five prenyltransferases toward C‐6 of the indole ring in the presence of unnatural DMAPP derivatives. Molecular dynamics simulation experiments with a homologous model of 5‐DMATS explained well its reactions with methylallyl and 2‐pentenyl diphosphate. Furthermore this study expands significantly the potential usage of tryptophan prenylating enzymes as biocatalysts for Friedel–Crafts alkylation.

     

CdpNPT, an N-prenyltransferase from Aspergillus fumigatus: overproduction, purification and biochemical characterisation

A putative prenyltransferase gene, cdpNPT, was identified in the genome sequence of Aspergillus fumigatus by a homology search by using known prenyltransferases and sequence analysis. CdpNPT consists of 440 amino acids and has a molecular mass of about 50 kDa. The coding sequence of cdpNPT was cloned in pQE60 and overexpressed in E. coli. The soluble His(6)-fusion CdpNPT was purified to near homogeneity and characterised biochemically. The enzyme showed broad substrate specificity towards aromatic substrates and was found to catalyse the prenylation of tryptophan-containing cyclic dipeptides at N1 of the indole moieties in the presence of dimethylallyl diphosphate (DMAPP); geranyl diphosphate was not accepted as prenyl donor. The structures of the enzymatic products were elucidated by NMR and MS analysis. The K(m) value for DMAPP was determined to be 650 microM. Due to substrate inhibition, K(m) values could not be obtained for the aromatic substrates. CdpNPT does not need divalent metal ions for its enzymatic reaction, although Ca(2+) enhances the reaction velocity by up to the threefold. CdpNPT is the first N-prenyltransferase that has been purified and characterised in a homogenous form after heterologous overproduction. Interestingly, it shows significant sequence similarity to other indole prenyltransferases that catalyse the formation of C--C bonds.     

An enzyme catalyzing O-prenylation of the glucose moiety of fusicoccin A, a diterpene glucoside produced by the fungus Phomopsis amygdali

Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane‐bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant‐pathogenic fungus Phomopsis amygdali and has a unique O‐prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6′‐hydroxy group of the glucose moiety of FC A to yield an O‐prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. K_m values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a k_cat of 55.3±3.3×10^?3 s. The enzyme did not act on representative substrates of the above‐mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P.     

Identification of the Verruculogen Prenyltransferase FtmPT3 by a Combination of Chemical,Bioinformatic and Biochemical Approaches

Previous studies showed that verruculogen is the end product of a biosynthetic gene cluster for fumitremorgin‐type alkaloids in Aspergillus fumigatus and Neosartorya fischeri. In this study, we isolated fumitremorgin A from N. fischeri. This led to the identification of the responsible gene, ftmPT3, for O‐prenylation of an aliphatic hydroxy group in verruculogen. This gene was found at a different location in the genome of N. fischeri than the identified cluster. The coding sequence of ftmPT3 was amplified by fusion PCR and overexpressed in Escherichia coli. The enzyme product of the soluble His₈‐FtmPT3 with verruculogen and dimethylallyl diphosphate (DMAPP) was identified unequivocally as fumitremorgin A by NMR and MS analyses. K_M values of FtmPT3 were determined for verruculogen and DMAPP at 5.7 and 61.5 μM , respectively. Average turnover number (k_cat) was calculated from kinetic parameters of verruculogen and DMAPP to be 0.069 s^?1. FtmPT3 also accepted biosynthetic precursors of fumitremorgin A, for example, fumitremorgin B and 12,13‐dihydroxyfumitremorgin C, as substrates and catalyses their prenylation.     

New Insights into the Biosynthesis of Prenylated Xanthones: Xptb from Aspergillus nidulans Catalyses an O‐Prenylation of Xanthones

Gene‐inactivation experiments have indicated that the putative prenyltransferase XptB from Aspergillus nidulans was likely to be responsible for the prenylation of 1,7‐dihydroxy‐6‐methyl‐8‐hydroxymethylxanthone. Recently, it was suggested that this enzyme might also accept as substrate the benzophenone arugosin H, which is assumed to be a precursor of prenylated xanthones. In this study, five benzophenones and ten xanthones were incubated with purified recombinant XptB in the presence of dimethylallyl diphosphate (DMAPP). XptB accepted four xanthones as substrates, including the proposed natural substrate, and catalysed regiospecific O‐prenylations at C‐7 of the xanthone core. K_m values in the range of 0.081–1.1 mM and turnover numbers (k_cat) between 0.02 and 0.5 s^?1 were determined for the accepted xanthones. The kinetic parameters for DMAPP were found to be 0.024 mM (K_m) and 0.13 s^?1 (k_cat). Arugosin H was not accepted by XptB under the tested conditions. XptB was relatively specific towards its prenyl donor and did not accept geranyl or farnesyl diphosphate as substrate. Mn²⁺ and Co²⁺ strongly enhanced XptB activity (up to eightfold); this has not been reported before for prenyltransferases of the DMATS superfamily.     

Geranylation of Cyclic Dipeptides by the Dimethylallyl Transferase AnaPT Resulting in a Shift of Prenylation Position on the Indole Ring

The dimethylallyl transferase AnaPT from Neosartorya fischeri is involved in the biosynthesis of acetylaszonalenin and catalyses the regioselective and stereospecific C3α‐prenylation of (R)‐benzodiazepinedione in the presence of dimethylallyl diphosphate. This enzyme also converts several tryptophan‐containing cyclic dipeptides to C3α‐prenylated indolines. In this study, we demonstrate the geranylation of (R)‐benzodiazepinedione and five other cyclic dipeptides by AnaPT in the presence of geranyl diphosphate (GPP). Interestingly, structure elucidation by NMR and MS analyses revealed that, with GPP, the geranyl moiety is attached to C‐6 or C‐7 rather than C‐3 of the indole ring of the enzyme products. For (R)‐benzodiazepinedione, one dominant C6‐geranylated derivative was obtained, whereas the other five substrates yielded both C6‐ and C7‐geranylated products. Neither acceptance of GPP by a dimethylallyl transferase from the dimethylallyltryptophan synthase superfamily, nor the alkylation shift from C‐3 to the benzene ring of the indole nucleus has been reported previously.     

Reverse prenyltransferase in the biosynthesis of fumigaclavine C in Aspergillus fumigatus: gene expression, purification, and characterization of fumigaclavine C synthase FGAPT1

A putative prenyltransferase gene-fgaPT1-has been identified in the biosynthetic gene cluster of fumigaclavines in Aspergillus fumigatus AF293. The gene was cloned and overexpressed in Escherichia coli, and the His6-fusion FgaPT1 was purified to near homogeneity and characterized biochemically. The enzyme was found to convert fumigaclavine A into fumigaclavine C by attaching a dimethylallyl moiety to C-2 of the indole nucleus in a "reverse" manner, that is, by connection of C-3 of the dimethylallyl moiety to an aromatic nucleus. FgaPT1 is a soluble, dimeric protein with a subunit size of 50 kDa. K m(app) values for fumigaclavine A and dimethylallyl diphosphate were determined to be 6 and 13 microM, respectively, while the turnover number was 0.8 s(-1). Metal ions such as Mg2+ and Ca2+ are not essential for the enzymatic activity. FgaPT1 showed relatively strict substrate specificity towards fumigaclavine A, with only dimethylallyl diphosphate being accepted as a donor under our conditions. FgaPT1 is the first reverse prenyltransferase from fungi to have been purified and characterized in homogenous form after heterologous overproduction. Surprisingly, it shows very low sequence similarity to the recently identified prenyltransferase LtxC from cyanobacteria, which also catalyzes the reverse prenylation of an indole nucleus.     

The mechanism of phosphorylation of natural nucleosides and anti-HIV analogues by nucleoside diphosphate kinase is independent of their sugar substituents

The reaction mechanism of the phosphoryl transfer catalyzed by dinucleoside diphosphate kinase from Dictyostelium discoideum is investigated by semiempirical AM1 molecular orbital computation of an active site model system on the basis of various X-ray crystallographic structures. The computational results suggest that the phosphoryl transfer from adenosine triphosphate to the His122 residue is accompanied by the simultaneous shift of a proton from the histidine residue to one of the oxygen atoms of the gamma phosphate group. This involves a doubly protonated His122 residue whilst this residue is neutral in its ternary complex with ADP and the transition state analogue AlF(3). The proposed mechanism is thus analogous to that of phosphoryl transfer by cyclic adenosine monophosphate dependent protein kinase and uridine/cytidine monophosphate kinase as found in our earlier work and clarifies the role of the ribose 3'-OH group. Furthermore, the energetics of phosphoryl transfer onto other nucleoside analogues such as 3'-azido-3'-deoxythymidine-diphosphate and 2',3'-dideoxy-2',3'-didehydro-thymidine-diphosphate are investigated. The calculated reaction barriers for the phosphorylation of the diphosphates by the enzyme are all within a range of 13.1 kJ mol(-1), which suggests that variations in the activation energies alone cannot account for the experimentally observed differences in enzymatic activity. Consequences for the design of new anti-HIV nucleoside analogues are discussed. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2268/2002/f360_s.pdf or from the author.     

Functional Characterization and Screening of Promiscuous Kinases and Isopentenyl Phosphate Kinases for the Synthesis of DMAPP via a One-Pot Enzymatic Cascade

Dimethylallyl diphosphate (DMAPP) is a key intermediate metabolite in the synthesis of isoprenoids and is also the prenyl donor for biosynthesizing prenylated flavonoids. However, it is difficult to prepare DMAPP via chemical and enzymatic methods. In this study, three promiscuous kinases from Shigella flexneri (SfPK), Escherichia coli (EcPK), and Saccharomyces cerevisiae (ScPK) and three isopentenyl phosphate kinases from Methanolobus tindarius (MtIPK), Methanothermobacter thermautotrophicus str. Delta H (MthIPK), and Arabidopsis thaliana (AtIPK) were cloned and expressed in Escherichia coli. The enzymatic properties of recombinant enzymes were determined. The Kcat/Km value of SfPK for DMA was 6875 s⁻¹ M⁻¹, which was significantly higher than those of EcPK and ScPK. The Kcat/Km value of MtIPK for DMAP was 402.9 s⁻¹ M⁻¹, which was ~400% of that of MthIPK. SfPK was stable at pH 7.0–9.5 and had a 1 h half-life at 65 °C. MtIPK was stable at pH 6.0–8.5 and had a 1 h half-life at 50 °C. The stability of SfPK and MtIPK was better than that of the other enzymes. Thus, SfPK and MtIPK were chosen to develop a one-pot enzymatic cascade for producing DMAPP from DMA because of their catalytic efficiency and stability. The optimal ratio between SfPK and MtIPK was 1:8. The optimal pH and temperature for the one-pot enzymatic cascade were 7.0 and 35 °C, respectively. The optimal concentrations of ATP and DMA were 10 and 80 mM, respectively. Finally, maximum DMAPP production reached 1.23 mM at 1 h under optimal conditions. Therefore, the enzymatic method described herein for the biosynthesis of DMAPP from DMA can be widely used for the synthesis of isoprenoids and prenylated flavonoids.     

A proposed mechanism for the reductive ring opening of the cyclodiphosphate MEcPP, a crucial transformation in the new DXP/MEP pathway to isoprenoids based on modeling studies and feeding experiments

Experimental and theoretical investigations concerning the second-to-last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4-oxo-DMAPP (12) and 4-hydroxy-DMAPP (11) were synthesized in deuterium- or tritium-labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP (7), 11, and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three-dimensional model of the fragment L271-A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe(4)S(4) cluster, and MEcPP (7) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP (7), hydroxyl abstraction, and formation of HMBPP (8). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe(4)S(4) cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP (8) to IPP (9) and DMAPP (10) in the last step.     

Evidence for the combined participation of a C10 and a C15 precursor in the biosynthesis of moenocinol, the lipid part of the moenomycin antibiotics

Upon feeding of [2-(13)C,4-(2)H]-1-deoxy-D-xylulose to Streptomyces ghanaensis, the deuterium label was retained exclusively at positions C-7 and C-17 in the moenocinol part of the moenomycin antibiotics. This result vindicates the hypothesis that the C(25) structure of moenocinol is assembled from a C(10) and a C(15) precursor, each of which requires for its formation the involvement of a dimethylallyl diphosphate starter unit.     

Deploying Fluorescent Nucleoside Analogues for High-Throughput Inhibitor Screening

High-throughput small-molecule screening in drug discovery processes commonly rely on fluorescence-based methods including fluorescent polarization and fluorescence/Förster resonance energy transfer. These techniques use highly accessible instrumentation; however, they can suffer from high false-negative rates and background signals, or might involve complex schemes for the introduction of fluorophore pairs. Herein we present the synthesis and application of fluorescent nucleoside analogues as the foundation for directed approaches for competitive binding analyses. The general approach describes selective fluorescent environment-sensitive (ES) nucleoside analogues that are adaptable to diverse enzymes that act on nucleoside-based substrates. We demonstrate screening a set of uridine analogues and development of an assay for fragment-based lead discovery with the TcdB glycosyltransferase (GT), an enzyme associated with virulence in Clostridium difficile. The uridine-based probe used for this high-throughput screen has a K_D value of 7.2 μm with the TcdB GT and shows a >30-fold increase in fluorescence intensity upon binding. The ES-based probe assay is benchmarked against two other screening approaches.     

Design,Synthesis, and Biological Evaluation of Simplified Side Chain Hybrids of the Potent Actin Binding Polyketides Rhizopodin and Bistramide

The natural products rhizopodin and bistramide belong to an elite class of highly potent actin binding agents. They show powerful antiproliferative activities against a range of tumor cell lines, with IC₅₀ values in the low‐nanomolar range. At the molecular level they disrupt the actin cytoskeleton by binding specifically to a few critical sites of G‐actin, resulting in actin filament stabilization. The important biological properties of rhizopodin and bistramide, coupled with their unique and intriguing molecular architectures, render them attractive compounds for further development. However, this is severely hampered by the structural complexity of these metabolites. We initiated an interdisciplinary approach at the interface between molecular modeling, organic synthesis, and chemical biology to support further biological applications. We also wanted to expand structure–activity relationship studies with the goal of accessing simplified analogues with potent biological properties. We report computational analyses of actin–inhibitor interactions involving molecular docking, validated on known actin binding ligands, that show a close match between the crystal and modeled structures. Based on these results, the ligand shape was simplified, and more readily accessible rhizopodin–bistramide mimetics were designed. A flexible and modular strategy was applied for the synthesis of these compounds, enabling diverse access to dramatically simplified rhizopodin–bistramide hybrids. This novel analogue class was analyzed for its antiproliferative and actin binding properties.