Substrate-Based Design of Human Farnesyltransferase Peptide-like Pain Antagonists

Human protein farnesyltransferase is a key enzyme for the lipid modification of a large and important number of proteins, which has been recognized as the promising therapeutic target of pain disorder and other diseases such as inflammation and cancer. In this study, we systematically investigated the binding behavior of existing peptide substrates and antagonists to farnesyltransferase at structural level and revealed that peptide’s C-terminus is primarily responsible for the binding, while exposing N-terminus to solvent. The amino acid property preference profile at each of the six core N-terminal residue positions of a cocrystallized chimera peptide substrate was defined, based on which a combinatorial library that contains more than twenty thousands of peptide-like compounds (PLCs) was generated using sixteen structurally diverse non-natural amino acids as building blocks. Subsequently, a systematic protocol was exhaustively carried out to perform virtual screening against the library, in order to discover those PLCs that match well the property preference profile and simultaneously exhibit high binding potency to farnesyltransferase. Consequently, ninety hits were identified from the library, in which five structurally diverse PLCs with high consensus scores were determined to have potent or moderate affinity to the active site of farnesyltransferase through nonbonded/coordination interactions. These identified PLCs can be considered as promising lead molecular entities to further develop peptidomimetic farnesyltransferase antagonists combating pain, inflammation and cancer.

     

New farnesyltransferase inhibitors in the phenothiazine series

The biological screening of the chemical library of our Organic Chemistry Department, carried out on an automated fluorescence-based FTase assay, allowed us to discover that a phenothiazine derivative (1d) was an inhibitor of farnesyltransferase. Three new series of human farnesyltransferase inhibitors, based on a phenothiazine scaffold, were synthesized with protein farnesyltransferase inhibition potencies in the low micromolar range. Ester derivative 9d was the most active compound in these series. Four synthesized compounds were evaluated for their antiproliferative activity on a NCI-60 cancer cell line panel. The modest results obtained in this preliminary investigation showed that mixing the phenothiazine and the 1,2,3-triazole motif in the structure of a single compound can lead to new scaffolds in the field of farnesyltransferase inhibitors.     

Identification of Novel Peptide Substrates for Protein Farnesyltransferase Reveals Two Substrate Classes with Distinct Sequence Selectivities

Prenylation is a posttranslational modification essential for the proper localization and function of many proteins. Farnesylation, the attachment of a 15-carbon farnesyl group near the C-terminus of protein substrates, is catalyzed by protein farnesyltransferase (FTase). Farnesylation has received significant interest as a target for pharmaceutical development, and farnesyltransferase inhibitors are in clinical trials as cancer therapeutics. However, as the total complement of prenylated proteins is unknown, the FTase substrates responsible for farnesyltransferase inhibitor efficacy are not yet understood. Identifying novel prenylated proteins within the human proteome constitutes an important step towards understanding prenylation-dependent cellular processes. Based on sequence preferences for FTase derived from analysis of known farnesylated proteins, we selected and screened a library of small peptides representing the C-termini of 213 human proteins for activity with FTase. We identified 77 novel FTase substrates that exhibit multiple-turnover (MTO) reactivity within this library; our library also contained 85 peptides that can be farnesylated by FTase only under single-turnover (STO) conditions. Based on these results, a second library was designed that yielded an additional 29 novel MTO FTase substrates and 45 STO substrates. The two classes of substrates exhibit different specificity requirements. Efficient MTO reactivity correlates with the presence of a nonpolar amino acid at the a₂ position and a Phe, Met, or Gln at the terminal X residue, consistent with the proposed Ca₁a₂X sequence model. In contrast, the sequences of the STO substrates vary significantly more at both the a₂ and the X residues and are not well described by current farnesylation algorithms. These results improve the definition of prenyltransferase substrate specificity, test the efficacy of substrate algorithms, and provide valuable information about therapeutic targets. Finally, these data illuminate the potential for in vivo regulation of prenylation through modulation of STO versus MTO peptide reactivity with FTase.     

Solid-phase synthesis of a pepticinnamin E library

Pepticinnamin E is a naturally occurring bisubstrate inhibitor of farnesyltransferase. Based on the structure of the natural product, a compound library was synthesized by variation of eight structural parameters. Following three different routes, a total of 51 analogues was synthesized on the polymeric support in 6-11-step parallel syntheses. Overall yields ranged from 3 to 63%, and the compounds were obtained with >90% purity.     

Identification of mono- and bisubstrate inhibitors of protein farnesyltransferase and inducers of apoptosis from a pepticinnamin E library

A library of 51 analogues of the naturally occurring protein farnesyltransferase inhibitor pepticinnamin E was investigated biologically. Several compounds with pronounced inhibitory activity were discovered with the lowest IC(50) value reaching 1 microM. The library contains inhibitors which are competitive to either farnesylpyrophosphate or the peptide substrate and a bisubstrate inhibitor. This activity is supported and rationalized by molecular modelling experiments and different binding modes of the inhibitors deduced from them. Several compounds induced apoptosis in a Ras-transformed tumour cell line, and in one case this correlated with farnesyltransferase-inhibiting activity.     

Discovery and preliminary structure–activity relationship studies on tecomaquinone I and tectol as novel farnesyltransferase and plasmodial inhibitors

Biological screening of a library of synthesized benzo[c]chromene-7,10-dione natural products against human farnesyltransferase (FTase) has identified tecomaquinone I (IC₅₀ of 0.065 ± 0.004 μM) as being one of the more potent natural product inhibitors identified to date. Anti-plasmodial screening of the same library against a drug-resistant strain of Plasmodium falciparum identified the structurally-related dichromenol tectol as a moderately active growth inhibitor with an IC₅₀ 3.44 ± 0.20 μM. Two novel series of analogues, based on the benzo[c]chromene-7,10-dione scaffold, were subsequently synthesized, with one analogue exhibiting farnesyltransferase inhibitory activity in the low micromolar range. A preliminary structure–activity relationship (SAR) study has identified different structural requirements for anti-malarial activity in comparison to FTase activities for these classes of natural products. Our results identify tecomaquinone I as a novel scaffold from which more potent inhibitors of human and parasitic FTase could be developed.     

Probing the hydrophobic pocket of farnesyltransferase: aromatic substitution of CAAX peptidomimetics leads to highly potent inhibitors

Cysteine farnesylation at the carboxylate terminal tetrapeptide CAAX of Ras protein is catalyzed by farnesyltransferase. This lipid modification is necessary for regulatory function of both normal and oncogenic Ras. The high frequency of Ras mutation in human cancers has prompted an intensive study on finding ways of controlling oncogenic Ras function. Inhibition of farnesyltransferase is among the most sought after targets for cancer chemotherapy. We report here the design, synthesis and biological characterization of a series of peptidomimetics as farnesyltransferase inhibitors. These compounds are extremely potent towards farnesyltransferase with IC₅₀ values ranging from subnanomolar to low nanomolar concentrations. They have a high selectivity for farnesyltransferase over the closely related geranylgeranyltransferase-I. Structure–activity relationship studies demonstrated that a properly positioned hydrophobic group significantly enhanced inhibition potency, reflecting an improved complementarity to the large hydrophobic pocket in the CAAX binding site.     

Identification and characterisation of Toxoplasma gondii protein farnesyltransferase

Prenylated proteins are involved in the regulation of DNA replication and cell cycling and have important roles in the regulation of cell proliferation. Protein farnesyltransferase and protein geranylgeranyltransferase are the two enzymes responsible for catalysing isoprene lipid modifications. Recently these enzymes have been targets for the development of cancer chemotherapeutics. Using metabolic labelling we identified isoprenylated proteins which suggests the presence of protein farnesyltransferase in Toxoplasma gondii. T. gondii protein farnesyltransferase is heat-labile and requires Mg(2+) and Zn(2+) ions for full activity. Peptidomimetic analogues as well as short synthetic peptides were tested in vitro as possible competitors for farnesyltransferase substrates. We found that the synthetic peptide (KTSCVIA) specifically inhibited T. gondiiprotein farnesyltransferase but not mammalian (HeLa cells) farnesyltransferase. Therefore this study suggests the possible development of specific inhibitors of T. gondiiprotein farnesyltransferase as an approach to parasitic protozoa therapy.     

A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group

Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.     

4-methyl-1,2,4-triazol-3-yl heterocycle as an alternative to the 1-methylimidazol-5-yl moiety in the farnesyltransferase inhibitor ZARNESTRA

Replacement of the 1-methylimidazol-5-yl moiety in the farnesyltransferase inhibitor ZARNESTRA^™ series by a 4-methyl-1,2,4-triazol-3-yl group gave us compounds with similar structure–activity relationship profiles showing that this triazole is potentially a good surrogate to imidazole for farnesyltransferase inhibition.     

Non-thiol farnesyltransferase inhibitors: N-(4-acylamino-3-benzoylphenyl)-3-[5-(4-nitrophenyl)-2-furyl]acrylic acid amides

We have designed the nitrophenylfurylacryl-substituted benzophenone 4f as a non-thiol farnesyltransferase inhibitor utilizing a novel aryl binding site of farnesyltransferase. Variation of the 2-acylamino substituent at the benzophenone core structure of our initial lead 4f yielded several non-thiol farnesyltransferase inhibitors with improved activity. These compounds display activity in the low nanomolar range.     

Non-thiol farnesyltransferase inhibitors: structure-activity relationships of benzophenone-based bisubstrate analogue farnesyltransferase inhibitors

Investigations on the structure-activity relationships of benzophenone-based bisubstrate analogue farnesyltransferase inhibitors yielded a bisubstrate analogue farnesyltransferase inhibitor lacking any prenylic or peptidic substructures with nanomolar activity. This represents a considerable progress in comparison to those non-prenylic, non-peptidic bisubstrate analogue farnesyltransferase inhibitors we have described before which utilized AAX-peptidomimetic substructures different from the benzophenone since those inhibitors displayed activity only in the micromolar range.     

Peptide chemistry applied to a new family of phenothiazine-containing inhibitors of human farnesyltransferase

Novel phenothiazine derivatives bearing an amino acid residue were synthesized via peptide chemistry, and evaluated for their inhibitory potential on human farnesyltransferase. The phenothiazine unit proved to be an important bulky unit in the structure of the synthesized inhibitors. Propargyl ester 20 bearing a tyrosine residue exhibited the best biological potential in vitro in the present study. Further syntheses and biological evaluation of phenothiazine derivatives are necessary in order to gain a full view of SAR in this family of farnesyltransferase inhibitors.     

Non-thiol farnesyltransferase inhibitors: N-(4-tolylacetylamino-3-benzoylphenyl)-3-arylfurylacrylic acid amides

We have designed arylfurylacryl-substituted benzophenones as non-thiol farnesyltransferase inhibitors utilizing a novel aryl binding site of farnesyltransferase. These compounds display activity in the low nanomolar range.     

Farnesyl-diphosphate farnesyltransferase 1 regulates hepatitis C virus propagation

To identify the novel genes involved in lipid metabolism and lipid droplet formation that may play important roles in Hepatitis C virus (HCV) propagation, we have screened the small interfering RNA library using cell culture derived HCV (HCVcc)-infected cells. We selected and characterized the gene encoding farnesyl-diphosphate farnesyltransferase 1 (FDFT1). siRNA-mediated knockdown of FDFT1 impaired HCV replication in both subgenomic replicon and HCVcc infected cells. Moreover, YM-53601, an inhibitor of FDFT1 enzyme activity, abrogated HCV propagation. HCV infection increased FDFT1 protein level but not FDFT1 mRNA level. These results suggest that HCV may modulate FDFT1 protein level to facilitate its own propagation.     

Development of tripeptidyl farnesyltransferase inhibitors

The first example of tripeptide inhibitors of farnesyltransferase with sub-micromolar inhibition activity was developed based on the fact that CVFM is not a substrate for farnesyltransferase.     

2-Oxo-tetrahydro-1,8-naphthyridines as selective inhibitors of malarial protein farnesyltransferase and as anti-malarials

A new class of 2-oxo-tetrahydro-1,8-naphthyridine-based protein farnesyltransferase inhibitors were synthesized and found to inhibit protein farnesyltransferase from the malaria parasite with potencies in the low nanomolar range. The compounds were much less potent on mammalian protein prenyltransferases. Two of the compounds block the growth of malaria in culture with potencies in the sub-micromolar range. Some of the compounds were found to be much more metabolically stable than previously described tetrahydroquinoline-based protein farnesyltransferase inhibitors.     

Farnesyltransferase inhibitors: antineoplastic mechanism and clinical prospects

Recent work suggests that farnesyltransferase inhibitors suppress cancer cell proliferation through mechanisms other than inhibiting Ras isoprenylation, which is not a crucial event. Recent evidence also suggests that the antineoplastic properties of farnesyltransferase inhibitors are due to alterations in the isoprenylation of RhoB, an endosomal Rho protein that functions in receptor trafficking. A shift in conceptual focus from Ras to Rho to understand how farnesyltransferase inhibitors act provides a new vantage to address old questions in the field and suggests strategies to improve and potentially widen clinical applications.     

3-Imidazolylmethylaminophenylsulfonyltetrahydroquinolines, a novel series of farnesyltransferase inhibitors

Design, synthesis and structure-activity relationship of a series of 3-imidazolylmethylaminophenylsulfonyltetrahydroquinolines as farnesyltransferase inhibitors are presented. A working pharmacophore of inhibiting farnesyltransferase by this series of inhibitors is proposed.     

Protein farnesyltransferase and geranylgeranyltransferase share a common alpha subunit

Mammalian farnesyltransferase, which attaches a 15 carbon isoprenoid, farnesyl, to a cysteine in p21ras proteins, contains two subunits, alpha and beta. The beta subunit is known to bind p21ras proteins. We show here that the alpha subunit is shared with another prenyltransferase that attaches 20 carbon geranylgeranyl to Ras-related proteins. Farnesyltransferase and geranylgeranyltransferase have similar molecular weights on gel filtration, but are separated by ion exchange chromatography. Both enzymes are precipitated and immunoblotted by multiple antibodies directed against the alpha subunit of farnesyltransferase. The two transferases have different specificities for the protein acceptor; farnesyltransferase prefers methionine or serine at the COOH-terminus and geranylgeranyltransferase prefers leucine. The current data indicate that both prenyltransferases are heterodimers that share a common alpha subunit with different beta subunits.