Library Screening, In Vivo Confirmation, and Structural and Bioinformatic Analysis of Pentapeptide Sequences as Substrates for Protein Farnesyltransferase.

Protein farnesylation is a post-translational modification where a 15-carbon farnesyl isoprenoid is appended to the C-terminal end of a protein by farnesyltransferase (FTase). This process often causes proteins to associate with the membrane and participate in signal transduction pathways. The most common substrates of FTase are proteins that have C-terminal tetrapeptide CaaX box sequences where the cysteine is the site of modification. However, recent work has shown that five amino acid sequences can also be recognized, including the pentapeptides CMIIM and CSLMQ. In this work, peptide libraries were initially used to systematically vary the residues in those two parental sequences using an assay based on Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). In addition, 192 pentapeptide sequences from the human proteome were screened using that assay to discover additional extended CaaaX-box motifs. Selected hits from that screening effort were rescreened using an in vivo yeast reporter protein assay. The X-ray crystal structure of CMIIM bound to FTase was also solved, showing that the C-terminal tripeptide of that sequence interacted with the enzyme in a similar manner as the C-terminal tripeptide of CVVM, suggesting that the tripeptide comprises a common structural element for substrate recognition in both tetrapeptide and pentapeptide sequences. Molecular dynamics simulation of CMIIM bound to FTase further shed light on the molecular interactions involved, showing that a putative catalytically competent Zn(II)-thiolate species was able to form. Bioinformatic predictions of tetrapeptide (CaaX-box) reactivity correlated well with the reactivity of pentapeptides obtained from in vivo analysis, reinforcing the importance of the C-terminal tripeptide motif. This analysis provides a structural framework for understanding the reactivity of extended CaaaX-box motifs and a method that may be useful for predicting the reactivity of additional FTase substrates bearing CaaaX-box sequences.

Structural Insight into Geranylgeranyl Diphosphate Synthase (GGDPS) for Cancer Therapy.

Geranylgeranyl diphosphate synthase (GGDPS), the source of the isoprenoid donor in protein geranylgeranylation reactions, has become an attractive target for anticancer therapy due to the reliance of cancers on geranylgeranylated proteins. Current GGDPS inhibitor development focuses on optimizing the drug-target enzyme interactions of nitrogen-containing bisphosphonate-based drugs. To advance GGDPS inhibitor development, understanding the enzyme structure, active site, and ligand/product interactions is essential. Here we provide a comprehensive structure-focused review of GGDPS. We reviewed available yeast and human GGDPS structures and then used AlphaFold modeling to complete unsolved structural aspects of these models. We delineate the elements of higher-order structure formation, product-substrate binding, the electrostatic surface, and small-molecule inhibitor binding. With the rise of structure-based drug design, the information provided here will serve as a valuable tool for rationally optimizing inhibitor selectivity and effectiveness.

Mechanistic insight into impact of phosphorylation on the enzymatic steps of farnesyltransferase.

Farnesyltransferase (FTase) is a heterodimeric enzyme, which catalyzes covalent attachment of the farnesyl group to target proteins, thus coordinating their trafficking in the cell. FTase has been demonstrated to be highly expressed in cancer and neurological diseases; hence considered as a hot target for therapeutic purposes. However, due to the nonspecific inhibition, there has been only one inhibitor that could be translated into the clinic. Importantly, it has been shown that phosphorylation of the α-subunit of FTase increases the activity of the enzyme in certain diseases. As such, understanding the impact of phosphorylation on dynamics of FTase provides a basis for targeting a specific state of the enzyme that emerges under pathological conditions. To this end, we performed 18 µs molecular dynamics (MD) simulations using complexes of (non)-phosphorylated FTase that are representatives of the farnesylation reaction. We demonstrated that phosphorylation modulated the catalytic site by rearranging interactions between farnesyl pyrophosphate (FPP)/peptide substrate, catalytic Zn2+ ion/coordinating residues and hot-spot residues at the interface of the subunits, all of which led to the stabilization of the substrate and facilitation of the release of the product, thus collectively expediting the reaction rate. Importantly, we also identified a likely allosteric pocket on the phosphorylated FTase, which might be used for specific targeting of the enzyme. To the best of our knowledge, this is the first study that systematically examines the impact of phosphorylation on the enzymatic reaction steps, hence opens up new avenues for drug discovery studies that focus on targeting phosphorylated FTase.

Indolizine-phenothiazine hybrids as the first dual inhibitors of tubulin polymerization and farnesyltransferase with synergistic antitumor activity.

In the incessant search for innovative cancer control strategies, this study was devoted to the design, synthesis and pharmacological evaluation of dual inhibitors of farnesyltransferase and tubulin polymerization (FTI/MTIs). A series of indolizine-phenothiazine hybrids 16 (amides) and 17 (ketones) has been obtained in a 4-step procedure. The combination of the two heterocycles provided potent tubulin polymerization inhibitors with similar efficiency as the reference phenstatin and (-)-desoxypodophyllotoxin. Ketones 17 were also able to inhibit human farnesyltransferase (FTase) in vitro. Interestingly, three molecules 17c, 17d and 17f were very effective against both considered biological targets. Next, nine indolizine-phenothiazine hybrids 16c, 16f, 17a-f and 22b were evaluated for their cell growth inhibition potential on the NCI-60 cancer cell lines panel. Ketones 17a-f were the most active and displayed promising cellular activities. Not only they arrested the cell growth of almost all tested cancer cells, but they displayed cytotoxicity potential with GI50 values in the low nanomolar range. The most sensitive cell lines upon treatment with indolizine-phenothiazine hybrids were NCI-H522 (lung cancer), COLO-205 and HT29 (colon cancer), SF-539 (human glioblastoma), OVCAR-3 (ovarian cancer), A498 (renal cancer) and especially MDA-MB-435 (melanoma). Demonstrating the preclinical effectiveness of these dual inhibitors can be crucial. A single dual molecule could induce a synergy of antitumor activity, while increasing the effectiveness and reducing the toxicity of the classical combo treatments currently used in chemotherapy.

Protein Farnesyltransferase Catalyzes Unanticipated Farnesylation and Geranylgeranylation of Shortened Target Sequences.

Protein prenylation is a posttranslational modification involving the attachment of a C15 or C20 isoprenoid group to a cysteine residue near the C-terminus of the target substrate by protein farnesyltransferase (FTase) or protein geranylgeranyltransferase type I (GGTase-I), respectively. Both of these protein prenyltransferases recognize a C-terminal "CaaX" sequence in their protein substrates, but recent studies in yeast- and mammalian-based systems have demonstrated FTase can also accept sequences that diverge in length from the canonical four-amino acid motif, such as the recently reported five-amino acid C(x)3X motif. In this work, we further expand the substrate scope of FTase by demonstrating sequence-dependent farnesylation of shorter three-amino acid "Cxx" C-terminal sequences using both genetic and biochemical assays. Strikingly, biochemical assays utilizing purified mammalian FTase and Cxx substrates reveal prenyl donor promiscuity leading to both farnesylation and geranylgeranylation of these sequences. These findings expand the substrate pool of sequences that can be potentially prenylated, further refine our understanding of substrate recognition by FTase and GGTase-I, and suggest the possibility of a new class of prenylated proteins within proteomes.

ω-Hydroxy isoprenoid bisphosphonates as linkable GGDPS inhibitors.

The enzyme geranylgeranyl diphosphate synthase (GGDPS) is a potential therapeutic target for multiple myeloma. Malignant plasma cells produce and secrete large amounts of monoclonal protein, and inhibition of GGDPS results in disruption of protein geranylgeranylation which in turn impairs intracellular protein trafficking. Our previous work has demonstrated that some isoprenoid triazole bisphosphonates are potent and selective inhibitors of GGDPS. To explore the possibility of selective delivery of such compounds to plasma cells, new analogues with an ω-hydroxy group have been synthesized and examined for their enzymatic and cellular activity. These studies demonstrate that incorporation of the ω-hydroxy group minimally impairs GGDPS inhibitory activity. Furthermore conjugation of one of the novel ω-hydroxy GGDPS inhibitors to hyaluronic acid resulted in enhanced cellular activity. These results will allow future studies to focus on the in vivo biodistribution of HA-conjugated GGDPS inhibitors.

Metal Coordination Is Crucial for Geranylgeranyl Diphosphate Synthase-Bisphosphonate Interactions: A Crystallographic and Computational Analysis.

Geranylgeranyl diphosphate synthase (GGPPS) is a central metalloenzyme in the mevalonate pathway, crucial for the prenylation of small GTPases. As small GTPases are pivotal for cellular survival, GGPPS was highlighted as a potential target for treating human diseases, including solid and hematologic malignancies and parasitic infections. Most available GGPPS inhibitors are bisphosphonates, but the clinically available compounds demonstrate poor pharmacokinetic properties. Although the design of novel bisphosphonates with improved physicochemical properties is highly desirable, the structure of wild-type human GGPPS (hGGPPS) bound to a bisphosphonate has not been resolved. Moreover, various metal-bisphosphonate-binding stoichiometries were previously reported in structures of yeast GGPPS (yGGPPS), hampering computational drug design with metal-binding pharmacophores (MBP). In this study, we report the 2.2 Å crystal structure of hGGPPS in complex with ibandronate, clearly depicting the involvement of three Mg2+ ions in bisphosphonate-protein interactions. Using drug-binding assays and computational docking, we show that the assignment of three Mg2+ ions to the binding site of both hGGPPS and yGGPPS greatly improves the correlation between calculated binding energies and experimentally measured affinities. This work provides a structural basis for future rational design of additional MBP-harboring drugs targeting hGGPPS. SIGNIFICANCE STATEMENT: Bisphosphonates are inhibitors of geranylgeranyl diphosphate synthase (GGPPS), a metalloenzyme crucial for cell survival. Bisphosphonate binding depends on coordination by Mg2+ ions, but various Mg2+-bisphosphonate-binding stoichiometries were previously reported. In this study, we show that three Mg2+ ions are vital for drug binding and provide a structural basis for future computational design of GGPPS inhibitors.

Farnesyl Transferase Inhibitors as Potential Anticancer Agents.

Farnesyl Transferase is a hetero-dimer transferase that targets Ras proteins and attaches a farnesyl group to it. This Ras protein, on localization to the cell membrane, has the ability to induce activation of various growth and proliferation pathways of the cell. Over-activation of mutated Ras may lead to the development of cancer. Farnesyl Transferase catalyses the initial step in the posttranslational modification of normal as well as mutated Ras gene, thus facilitating its tethering to the cell membrane. Inhibition of Farnesyl Transferase is the main step in restricting the activity of mutant Ras protein. Thus the above enzyme has emerged as a novel target for anti-cancer agents. Here we review the role of Farnesyl Transferase in tumorigenesis and various compounds of synthetic and natural origin acting as Farnesyl Transferase inhibitors as potential anti-cancer agents.

Olefin Isomers of a Triazole Bisphosphonate Synergistically Inhibit Geranylgeranyl Diphosphate Synthase.

The isoprenoid donor for protein geranylgeranylation reactions, geranylgeranyl diphosphate (GGDP), is the product of the enzyme GGDP synthase (GGDPS) that condenses farnesyl diphosphate (FDP) and isopentenyl pyrophosphate. GGDPS inhibition is of interest from a therapeutic perspective for multiple myeloma because we have shown that targeting Rab GTPase geranylgeranylation impairs monoclonal protein trafficking, leading to endoplasmic reticulum stress and apoptosis. We reported a series of triazole bisphosphonate GGDPS inhibitors, of which the most potent was a 3:1 mixture of homogeranyl (HG) and homoneryl (HN) isomers. Here we determined the activity of the individual olefin isomers. Enzymatic and cellular assays revealed that although HN is approximately threefold more potent than HG, HN is not more potent than the original mixture. Studies in which cells were treated with varying concentrations of each isomer alone and in different combinations revealed that the two isomers potentiate the induced-inhibition of protein geranylgeranylation when used in a 3:1 HG:HN combination. A synergistic interaction was observed between the two isomers in the GGDPS enzyme assay. These results suggested that the two isomers bind simultaneously to the enzyme but within different domains. Computational modeling studies revealed that HN is preferred at the FDP site, that HG is preferred at the GGDP site, and that both isomers may bind to the enzyme simultaneously. These studies are the first to report a set of olefin isomers that synergistically inhibit GGDPS, thus establishing a new paradigm for the future development of GGDPS inhibitors.

Mammalian farnesyltransferase α subunit regulates vacuolar protein sorting-associated protein 4A (Vps4A)--dependent intracellular trafficking through recycling endosomes.

The protein farnesyltransferase (FTase) mediates posttranslational modification of proteins with isoprenoid lipids. FTase is a heterodimer and although the ß subunit harbors the active site, it requires the α subunit for its activity. Here we explore the other functions of the FTase α subunit in addition to its established role in protein prenylation. We found that in the absence of the ß subunit, the α subunit of FTase forms a stable autonomous dimeric structure in solution. We identify interactors of FTase α using mass spectrometry, followed by rapid in vitro analysis using the Leishmania tarentolae cell - free system. Vps4A was validated for direct binding to the FTase α subunit both in vitro and in vivo. Analysis of the interaction with Vps4A in Hek 293 cells demonstrated that FTase α controls trafficking of transferrin receptor upstream of this protein. These results point to the existence of previously undetected biological functions of the FTase α subunit that includes control of intracellular membrane trafficking.

Characterization and Functional Identification of a Gene Encoding Geranylgeranyl Diphosphate Synthase from Dunaliella bardawil.

Geranylgeranyl diphosphate synthase (GGPS) catalyzes the biosynthesis of geranylgeranyl diphosphate, a key precursor for carotenoid biosynthesis. In this study, a full-length cDNA encoding GGPS from Dunaliella bardawil (DbGGPS) was isolated by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of DbGGPS was 1814 bp, containing a 1074 bp ORF encoding 357 amino acids with a calculated mass of 38.88 kDa. Analysis of DbGGPS genomic DNA revealed that it contained 10 exons and 9 introns. It was predicted that DbGGPS possessed a 48 amino acid transit peptide at its N terminus. Bioinformatic analysis revealed that DbGGPS was a member of a group of polyprenyltransferases with five conserved domains and two highly conserved aspartate-rich motifs. Using heterologous expression, carotenoid complementation assay, and gene deletion analysis, it was shown that the coding region of DbGGPS encodes a functional GGPS. This provides new gene sources for carotenoid genetic engineering.

Farnesyltransferase and geranylgeranyltransferase I: structures, mechanism, inhibitors and molecular modeling.

Farnesyltransferase (FTase) and geranylgeranyltransferase type I (GGTase-I) have crucial roles in the post-translational modifications of Ras proteins and, therefore, they are promising therapeutic targets for the treatment of various Ras-induced cancers and several other kinds of diseases. In this review, we provide an overview of the structures and biological functions of FTase and GGTase-I. Then, we summarize the typical inhibitors of FTase and GGTase-I, and highlight the drug candidates in clinical trials. In addition, we survey some recent advances in computer-aided drug design (CADD) and molecular modeling studies of FTase and GGTase-I.

Three types of geranylgeranyl diphosphate synthases from the medicinal caterpillar fungus, Cordyceps militaris (Ascomycetes).

Geranylgeranyl diphosphate synthase (GGPPS) is a key enzyme in the carotenoid biosynthetic pathway, catalyzing the synthesis of its C20 precursor. In the present study, three types of ggpps genes were cloned and analyzed from the Caterpillar Medicinal Fungus Cordyceps militaris, a valued carotenoid-producing species. The sequences were named as ggpps727, ggpps191, and ggpps595. The open reading frame codes for predicted polypeptides of 464, 550, and 431 aa. Three predicted GGPPSs had a high similarity to that from Beauveria bassiana ARSEF 2860 with identity of 73%, 71%, and 56%, respectively. Homology comparison of the deduced peptide sequences of the various GGPPSs revealed highly conserved domains. Both GGPPS727 and GGPPS191 from C. militaris contained all five domains highly conserved among prenyltransferases as well as two aspartate-rich DDXX(XX)D motifs in domains II and V, which have been proven essential for prenyltransferase activity. By constructing the phylogenetic tree of fungal GGPPSs, it was found that fungi-derived GGPPSs could be divided into three clusters, suggesting there were three types of GGPPSs in fungi. Each type may be responsible for a different metabolism. Three types of GGPPSs from C. militaris belonged to the different clusters separately. Expression analysis of three ggpps genes during the fruit body cultivation of C. militaris by real-time polymerase chain reaction (PCR) suggested the ggpps 191 gene may be involved in the synthesis of carotenoids and ggpps 727 may be responsible for primary metabolism. This is the first report of the GGPPS from C. militaris, a valued edible and medicinal fungus.

Theoretical studies on binding and specificity mechanisms of farnesyltransferase (FTase) and geranylgeranyltransferase type-I (GGTase-I) inhibitors by molecular modeling.

Farnesyltransferase (FTase) and geranylgeranyltransferase type-I (GGTase-I) are two members of protein prenyltransferases, which play critical roles in lipid post-translational modifications. Potent inhibitors of FTase and GGTase-I have been confirmed to show favorable influence on the therapies of various diseases, such as cancers, malaria and Toxoplasmosis. However, designing highly specific inhibitors toward FTase or GGTase-I without influencing their binding affinity remains a big challenge. In this work, molecular docking, molecular dynamics (MD) simulations and MM/GBSA free energy calculations were employed to study the bindings of two highly selective inhibitors (lonafarnib and GGTI-2133) towards FTase or GGTase-I. The specificities of the studied inhibitors derived from the predicted binding free energies are consistent with the experimental data. The analysis of the energetic components illustrates that both the non-polar and polar interactions play critical roles in determining the specificity between FTase and GGTase-I. Moreover, the protein-inhibitor interaction spectra for the studied inhibitors were determined through the decomposition of the binding free energies, and the important residues for binding and specificity were highlighted. Our study provides useful information for the rational design of selective FTase or GGTase-I inhibitors.

Crystal structures of the fungal pathogen Aspergillus fumigatus protein farnesyltransferase complexed with substrates and inhibitors reveal features for antifungal drug design.

Species of the fungal genus Aspergillus are significant human and agricultural pathogens that are often refractory to existing antifungal treatments. Protein farnesyltransferase (FTase), a critical enzyme in eukaryotes, is an attractive potential target for antifungal drug discovery. We report high-resolution structures of A. fumigatus FTase (AfFTase) in complex with substrates and inhibitors. Comparison of structures with farnesyldiphosphate (FPP) bound in the absence or presence of peptide substrate, corresponding to successive steps in ordered substrate binding, revealed that the second substrate-binding step is accompanied by motions of a loop in the catalytic site. Re-examination of other FTase structures showed that this motion is conserved. The substrate- and product-binding clefts in the AfFTase active site are wider than in human FTase (hFTase). Widening is a consequence of small shifts in the α-helices that comprise the majority of the FTase structure, which in turn arise from sequence variation in the hydrophobic core of the protein. These structural effects are key features that distinguish fungal FTases from hFTase. Their variation results in differences in steady-state enzyme kinetics and inhibitor interactions and presents opportunities for developing selective anti-fungal drugs by exploiting size differences in the active sites. We illustrate the latter by comparing the interaction of ED5 and Tipifarnib with hFTase and AfFTase. In AfFTase, the wider groove enables ED5 to bind in the presence of FPP, whereas in hFTase it binds only in the absence of substrate. Tipifarnib binds similarly to both enzymes but makes less extensive contacts in AfFTase with consequently weaker binding.

Moiety-linkage map reveals selective nonbisphosphonate inhibitors of human geranylgeranyl diphosphate synthase.

Bisphosphonates are potent inhibitors of farnesyl pyrophosphate synthase (FPPS) and geranylgeranyl diphosphate synthase (GGPPS). Current bisphosphonate drugs (e.g., Fosamax and Zometa) are highly efficacious in the treatment of bone diseases such as osteoporosis, Paget's disease, and tumor-induced osteolysis, but they are often less potent in blood and soft-tissue due to their phosphate moieties. The discovery of nonbisphosphonate inhibitors of FPPS and/or GGPPS for the treatment of bone diseases and cancers is, therefore, a current goal. Here, we propose a moiety-linkage-based method, combining a site-moiety map with chemical structure rules (CSRs), to discover nonbisphosphonate inhibitors from thousands of commercially available compounds and known crystal structures. Our moiety-linkage map reveals the binding mechanisms and inhibitory efficacies of 51 human GGPPS (hGGPPS) inhibitors. To the best of our knowledge, we are the first team to discover two novel selective nonbisphosphonate inhibitors, which bind to the inhibitory site of hGGPPS, using CSRs and site-moiety maps. These two compounds can be considered as a novel lead for the potent inhibitors of hGGPPS for the treatment of cancers and mevalonate-pathway diseases. Moreover, based on our moiety-linkage map, we identified two key residues of hGGPPS, K202, and K212, which play an important role for the inhibitory effect of zoledronate (IC50 = 3.4 µM and 2.4 µM, respectively). This result suggests that our method can discover specific hGGPPS inhibitors across multiple prenyltransferases. These results show that the compounds that highly fit our moiety-linkage map often inhibit hGGPPS activity and induce tumor cell apoptosis. We believe that our method is useful for discovering potential inhibitors and binding mechanisms for pharmaceutical targets.

Peptidomimetic modification improves cell permeation of bivalent farnesyltransferase inhibitors.

Bivalent enzyme inhibitors, in which a surface binding module is linked to an active site binding module through a spacer, are a robust approach for site-selectively delivering a minimally-sized agent to a protein surface to regulate its functions, such as protein-protein interactions (PPIs). Previous research revealed that these agents effectively disrupt the interaction between farnesyltransferase (FTase) and the C-terminal region of K-Ras4B protein. However, the whole cell activity of these peptide-based agents is limited due to their low membrane permeability. In this study, we tested a peptidomimetic modification of these bivalent agents using a previously developed inhibitor, FTI-249, and evaluated their cell permeability and biological activity in cells. Confocal cell imaging using fluorescently-labeled agents showed that the peptidomimetic 3-BODIPY penetrated cells, while the peptide-based 1-BODIPY did not. Cell-based evaluation demonstrated that peptidomimetic 3 at a concentration of 100µM inhibited HDJ-2 processing in cells, indicating that this peptidomimetic modification improves cell permeability, thus leading to enhanced whole cell activity of the bivalent compounds.

Molecular dynamics analysis of a series of 22 potential farnesyltransferase substrates containing a CaaX-motif.

Protein farnesyltransferase (FTase) is an important target in many research fields, more markedly so in cancer investigation since several proteins known to be involved in human cancer development are thought to serve as substrates for FTase and to require farnesylation for proper biological activity. Several FTase inhibitors (FTIs) have advanced into clinical testing. Nevertheless, despite the progress in the field several functional and mechanistic doubts on the FTase catalytic activity have persisted. This work provides some crucial information on this important enzyme by describing the application of molecular dynamics simulations using specifically designed molecular mechanical parameters for a variety of 22 CaaX peptides known to work as natural substrates or inhibitors for this enzyme. The study involves a comparative analysis of several important molecular aspects, at the mechanistic level, of the behavior of substrates and inhibitors at the dynamic level, including the behavior of the enzyme and peptides, as well as their interaction, together with the effect of the solvent. Properties evaluated include the radial distribution function of the water molecules around the catalytically important zinc metal atom and cysteine sulfur of CaaX, the conformations of the substrate and inhibitor and the corresponding RMSF values, critical hydrogen bonds, and several catalytically relevant distances. These results are discussed in light of recent experimental and computational evidence that provides new insights into the activity of this enzyme.

Dapson in heterocyclic chemistry part VI: synthesis and molecular docking of some novel sulfonebiscompounds of expected anticancer activity.

To discover new bioactive lead compounds for medicinal purposes, herein, sulfone biscompounds bearing dihydrothiazoles (3-9, 14, 15), acrylamide (11), thiazolidinones (12, 13, 20), thiophenes (16, 17) and benzothiophene (19) were prepared and tested for their anticancer activity. The structures of the products were confirmed from elemental analysis as well as spectral data. All the synthesized compounds showed remarkable anticancer activity against human breast cancer cell line especially, compound (3) with IC50 value 23.02 µM which was better than that of Doxorubicin by three folds. In order to elucidate the mechanism of action of their cytotoxic activity molecular docking on the active sites of farnesyl transferase and arginine methyl transferase was performed for all synthesized compounds and good results were obtained.

Dapson in heterocyclic chemistry, part V: synthesis, molecular docking and anticancer activity of some novel sulfonylbiscompounds carrying biologically active dihydropyridine, dihydroisoquinoline, 1,3-dithiolan, 1,3-dithian, acrylamide, pyrazole, pyrazolopyrimidine and benzochromenemoieties.

N,N'-(4,4'-Sulfonylbis(4,1-phenylene))bis(2-cyanoacetamid) 2 was utilized as a key intermediate for the synthesis of novel dihydropyridines 3, 4, 8, dihydroisoquinolines 5-7, dithiolan 10, dithian 11, acrylamide 12, benzochromenes 17 and 18 and chromenopyridones 19 and 20. Compound 2 was the starting material in the synthesis of the acrylamide derivative 14, the pyrazole derivative 15 and the pyrazolopyrimidine derivative 16. All the synthesized compounds were evaluated for their in vitro anticancer activity against human breast cancer cell line (MCF7). Compound 19 showed the best cytotoxic activity with IC(50) value 19.36 µM. In addition, molecular docking study of the synthesized compounds on the active sites of farnesyltransferase and arginine methyltransferase was performed in order to give a suggestion about the mechanism of action of their cytotoxic activity.