Caged protein prenyltransferase substrates: tools for understanding protein prenylation

Originally designed to block the prenylation of oncogenic Ras, inhibitors of protein farnesyltransferase currently in preclinical and clinical trials are showing efficacy in cancers with normal Ras. Blocking protein prenylation has also shown promise in the treatment of malaria, Chagas disease and progeria syndrome. A better understanding of the mechanism, targets and in vivo consequences of protein prenylation are needed to elucidate the mode of action of current PFTase (Protein Farnesyltransferase) inhibitors and to create more potent and selective compounds. Caged enzyme substrates are useful tools for understanding enzyme mechanism and biological function. Reported here is the synthesis and characterization of caged substrates of PFTase. The caged isoprenoid diphosphates are poor substrates prior to photolysis. The caged CAAX peptide is a true catalytically caged substrate of PFTase in that it is to not a substrate, yet is able to bind to the enzyme as established by inhibition studies and X-ray crystallography. Irradiation of the caged molecules with 350 nm light readily releases their cognate substrate and their photolysis products are benign. These properties highlight the utility of those analogs towards a variety of in vitro and in vivo applications.     

Patent Update Oncologic,Endocrine & Metabolic: Oncologic,Endocrine & Metabolic: Inhibitors of protein farnesylation

In this article, we update the progress reported towards developing an inhibitor of protein farmesyltransferase (PFTase) as a new type of cancer therapy. For the rationale behind this undertaking, please refer to the review published in this journal in December 1995 [1], and other reviews [2,3]. A few experiments bearing on the utility of PFTase inhibitors as antitumour agents are considered first, including biological results obtained with compounds described in our earlier review. Finally, new compounds described in the scientific and patent literature in the past year are reported. As in the previous review, these are grouped according to their kinetic mechanism of inhibition of PFTase.     

Inhibitors of protein farnesyltransferase as novel anticancer agents

This paper describes recent progress in the design, synthesis and biological evaluation of inhibitors for the enzyme protein farnesyltransferase (PFTase). This enzyme plays a critical role in the post-translational modification of a range of different intracellular proteins. In particular, PFTase attaches a farnesyl group to the GTPase Ras whose oncogenically mutated form is found in over 30% of human cancers. As a result PFTase inhibitors have been developed as potential cancer therapeutic drugs either by rational design based on the structure of the CAAX carboxyl terminus of Ras or random screening of chemical libraries or natural products. Some of these inhibitors show remarkable inhibition potency against PFTase at subnanomolar concentrations and >1000-fold selectivity compared to the related enzyme geranylgeranyltransferase-I. Certain of these compounds are highly effective at blocking the growth of human tumors in animal models and are now undergoing clinical trials. However, several issues in the research remain unsolved, including the mechanism by which PFTase inhibitors suppress tumor growth. Although it has been established that PFTase inhibitors block prenylation of Ras in vitro, the results in wholecells and animal studies suggest the possibility that proteins other than Ras are affected.     

Site‐Specific Labeling of Proteins and Peptides with Trans‐cyclooctene Containing Handles Capable of Tetrazine Ligation

There is a growing library of functionalized non‐natural substrates for the enzyme protein farnesyltransferase (PFTase). PFTase covalently attaches these functionalized non‐natural substrates to proteins ending in the sequence CAAX, where C is a cysteine that becomes alkylated, A represents an aliphatic amino acid, and X is Ser, Met, Ala, or Gln. Reported substrates include a variety of functionalities that allow modified proteins to undergo subsequent bioconjugation reactions. To date the most common strategy used in this approach has been copper catalyzed azide‐alkyne cycloaddition (CuAAC). While being fast and bioorthogonal CuAAC has limited use in live cell experiments due to copper's toxicity.¹ Here, we report the synthesis of trans‐cyclooctene geranyl diphosphate. This substrate can be synthesized from geraniol in six steps and be enzymatically transferred to peptides and proteins that end in a CAAX sequence. Proteins and peptides site‐specially modified with trans‐cyclooctene geranyl diphosphate were subsequently targeted for further modification via tetrazine ligation. As tetrazine ligation is bioorthogonal, fast, and is contingent on ring strain rather than the addition of a copper catalyst, this labeling strategy should prove useful for labeling proteins where the presence of copper may hinder solubility or biological reactivity.     

Search for protein farnesyltransferase inhibitors of microbial origin: our strategy and results as well as the results obtained by other groups

Mutant ras oncogenes are associated with various human tumors, being found in approximately 25% of all human cancers. Since its identification, the enzyme Ras protein farnesyltransferase (PFTase), which catalyzes the initial step of Ras-processing, has been viewed as a most promising target for cancer therapy. Consequently, a number of synthetic and natural small molecules have been searched and developed according to this concept during the 1990s. Among these, microbial metabolites have provided diverse structural classes of compounds which exhibit PFTase inhibitory activity. This article reviews our work on PFTase inhibitors originating from microbial metabolites, and the results of similar works carried out by several other research groups.     

Inhibitors of protein prenylation 2000

The development of farnesyltransferase inhibitors (FTIs) was conceived of as a rational approach to the design of new cancer chemotherapeutics and the first examples of this class are currently being evaluated in Phase II and Phase III clinical trials. This review summarises the scientific and patent literature published in the 14 month period from March 1999, since the previous review [1]. In addition to a survey of novel prenyl transferase inhibitors, recent clinical data and developments in related biochemistry and biology are described.     

Clinical activity of tipifarnib in hematologic malignancies

Farnesyltransferase inhibitors are a novel class of anticancer agents that competitively inhibit farnesyltransferase. Initially developed to inhibit the farnesylation that is necessary for Ras activation, their mechanism of action seems to be more complex, involving other proteins unrelated to Ras. Of the four classes of farnesyltransferase inhibitors, at least three agents have been investigated in hematologic malignancies. Tipifarnib (R-115777), an orally administered non-peptidomimetic farnesyltransferase inhibitor, has shown promising clinical activity. Preliminary results from clinical trials demonstrate enzyme target inhibition, an acceptable toxicity profile and promising evidence of clinical activity. Ongoing studies will better determine the mechanism of action of tipifarnib and the role of combination with other agents, defining its place in the therapeutic arsenal of hematologic disorders.     

Synthesis and reactivity of 6,7-dihydrogeranylazides: reagents for primary azide incorporation into peptides and subsequent staudinger ligation

Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide moiety to a peptide substrate, N-dansyl-GCVIA. Because geranylazide is actually a mixture of isomeric, interconverting primary and secondary azides, incorporation of this isoprenoid into peptides can potentially result in a corresponding mixture of prenylated peptides. Here, we first examined the reactivity of geranyl azide in a model Staudinger reaction and determined that a mixture of products is formed. We then describe the synthesis of 6,7-dihydrogeranylazide diphosphate and demonstrate that this compound allows exclusive incorporation of a primary azide into a peptide. The resulting azide-containing peptide was derivatized with a triphenylphosphine-based reagent to generate an O-alkyl imidate-linked product. Finally, we show, using a series of model reactions, that the Staudinger ligation frequently produces small amounts of O-alkyl imidate products in addition to the major amide-linked products. Thus, the alkoxyimidates we have observed as the exclusive products in the reactions of peptides containing prenylated azides also appear to be a common type of product formed using other azide-containing reactants, although at greatly reduced levels. This method for chemical modification of the C-terminus of a protein should be useful for a variety of applications in protein chemistry.     

Potent, highly selective, and non-thiol inhibitors of protein geranylgeranyltransferase-I

The design, synthesis, and biological evaluation of a family of peptidomimetic inhibitors of protein geranylgeranyltransferase-I (PGGTase-I) are reported. The inhibitors are based on the C-terminal CAAL sequence of many geranylgeranylated proteins. Using 2-aryl-4-aminobenzoic acid derivatives as mimetics for the central dipeptide (AA), we have attached a series of imidazole and pyridine derivatives to the N-terminus as cysteine replacements. These non-thiol-containing peptidomimetics show exceptional selectivity for PGGTase-I over the closely related enzyme protein farnesyltransferase (PFTase). This selectivity is retained in whole cells where the inhibitors were shown to block the geranylgeranylation of Rap-1A without affecting the farnesylation of small GTP-binding proteins such as Ras.     

Evaluation of geranylazide and farnesylazide diphosphate for incorporation of prenylazides into a CAAX box‐containing peptide using protein farnesyltransferase*

Abstract: Protein farnesyltransferase (PFTase) catalyzes the attachment of a geranylazide (C10) or farnesylazide (C15) moiety from the corresponding prenyldiphosphates to a model peptide substrate, N‐dansyl‐Gly‐Cys‐Val‐Ile‐Ala‐OH. The rates of incorporation for these two substrate analogs are comparable and approximately twofold lower than that using the natural substrate farnesyl diphosphate (FPP). Reaction of N‐dansyl‐Gly‐Cys(S‐farnesylazide)‐Val‐Ile‐Ala‐OH with 2‐diphenylphosphanylbenzoic acid methyl ester then gives a stable alkoxy‐imidate linked product. This result suggests future generations whereby azide groups introduced using this enzymatic approach are functionalized using a broad range of azide‐reactive reagents. Thus, chemistry has been developed that could be used to achieve highly specific peptide and protein modification. The farnesylazide analog may be useful in certain biological studies, whereas the geranylazide group may be more useful for general protein modification and immobilization.     

抑制ras法尼基转移酶:一种新的、安全的肿瘤化疗方法(英文)

The 21-kDa Ras proteins are well known for their regulatory role in oncogenic, mitogenic, and developmental signaling pathways. GTP activated Ras interacts directly with the Raf protein to recruit the MAP kinases and their subordinates. Attachment of Ras protein to the plasma membrane that requires farnesylation by farnesyl pyrophosphate at its C-terminus, is essential for its biological activity. Ras oncogenes are associated with a wide variety of solid tumors and leukemias for which existing chemotherapeutics have limited utility. A . promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyl transferase. These inhibitors may be useful in blocking the action of Ras onco-proteins.     

Evaluation of alkyne-modified isoprenoids as chemical reporters of protein prenylation

Protein prenyltransferases catalyze the attachment of C15 (farnesyl) and C20 (geranylgeranyl) groups to proteins at specific sequences localized at or near the C-termini of specific proteins. Determination of the specific protein prenyltransferase substrates affected by the inhibition of these enzymes is critical for enhancing knowledge of the mechanism of such potential drugs. Here, we investigate the utility of alkyne-containing isoprenoid analogs for chemical proteomics experiments by showing that these compounds readily penetrate mammalian cells in culture and become incorporated into proteins that are normally prenylated. Derivatization via Cu(I) catalyzed click reaction with a fluorescent azide reagent allows the proteins to be visualized and their relative levels to be analyzed. Simultaneous treatment of cells with these probes and inhibitors of prenylation reveals decreases in the levels of some but not all of the labeled proteins. Two-dimensional electrophoretic separation of these labeled proteins followed by mass spectrometric analysis allowed several labeled proteins to be unambiguously identified. Docking experiments and density functional theory calculations suggest that the substrate specificity of protein farnesyl transferase may vary depending on whether azide- or alkyne-based isoprenoid analogs is employed. These results demonstrate the utility of alkyne-containing analogs for chemical proteomic applications.     

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC₅₀ ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC₅₀ = 61.4 μM) to those required to inhibit [³H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [³H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.     

Isothiazole dioxide derivative 6n inhibits vascular smooth muscle cell proliferation and protein farnesylation

Isothiazole dioxides have been shown to inhibit Trypanosoma brucei protein farnesyltransferase (PFTase) in isolated enzyme, but elicited only a minor effect on mammalian PFTase. In the present study we have evaluated the effect of 3-diethylamino-4-(4-methoxyphenyl)-isothiazole 1,1-dioxides with different substituents at C5, on rat PFTase and protein geranylgeranyltransferase-I (PGGTase-I) with the final aims to improve the potency against mammalian PFTase and to identify new compounds with antiproliferative properties. For these purposes, in vitro and cell culture models have been utilized. The results showed that isothiazole dioxides with C4–C5 double bond and sulfaryl substituted at the C5 position but none of the dihydro-derivatives, were able to inhibit in vitro PFTase in a concentration dependent manner (IC₅₀ ranging from 8.56 to 1015 μM). Among those, compound 6n (C5; methyl-S) displayed 500-fold higher inhibitory potency on PFTase than PGGTase-I. Compound 6n was shown to affect rat smooth muscle cell (SMC) proliferation at concentrations similar (IC₅₀ = 61.4 μM) to those required to inhibit [³H]-farnesol incorporation into cellular proteins (−44.1% at 100 μM). Finally, compound 6n interferes with rat SMC proliferation by blocking the progression of G0/G1 phase without inducing apoptosis, as assessed by [³H]-thymidine incorporation assay and flow cytometry analysis. Taken together, we described a new PFTase inhibitor containing the isothiazole dioxide moiety that affects mammalian protein farnesylation and SMC proliferation by inhibiting G0/G1 phase of the cell cycle.     

7-(咪唑-4-烷基酰胺基)-1,3-二氢-1-羧基烷基-5-苯基-2H-1,4-苯并二氮杂-2-酮一类新的法呢基蛋白转移酶抑制剂

目的设计并合成新结构类型的法呢基蛋白转移酶抑制剂。方法本文结合法呢基蛋白转移酶(FTase)的作用机理和已有FTase抑制剂结构特征,设计了一类以苯并二氮杂为分子骨架,一端连接有可与锌离子配位结合的咪唑基,另一端连接不同长度的末端含羧基的侧链的化合物。此类化合物模拟了FTase配体之一CAAX四肽片段,共合成10个此类新化合物(6～12,16～18),并对其进行体外生物活性测定。结果 所有新目的化合物均经1HNMR和HRMS方法确证结构。结论对FTase抑制活性测定结果表明其中5个化合物(9,10,16～18)有较强的抑制活性。     

Farnesyltransferase inhibitors: recent advances

Farnesyltransferase inhibitors (FTIs) represent a promising new approach for the treatment of cancer. Although the exact mechanism of action of these compounds is not fully understood, several compounds have progressed into clinical trials with proven efficacy. This review will describe the recent patents (2000 to present) that cover FTIs in the clinic, together with other classes that are still under development.     

Update on patented cholesterol absorption inhibitors

Background: Atherosclerosis is one of the most life-threatening diseases primarily associated with hypercholesterolemia and is characterized by increased serum cholesterol level. Cholesterol originates from both its de novo synthesis within the hepatic cells and its absorption into the intestine in the form of dietary or bile cholesterol. Interventions influencing both of these processes are promising therapeutic options to lower the cholesterol level. Hydroxymethyl glutaryl-CoA reductase inhibitors, commonly known as statins, effectively block the rate determining step in the biosynthesis of cholesterol. Ezetimibe is the first new class of drugs used to treat hypercholesterolemia by inhibition of cholesterol absorption through Niemann Pick C1 Like 1 membrane of enterocytes. Therefore, combination therapy of ezetimibe and statins offers an efficacious new approach for the prevention and treatment of hypercholesterolemia. Objectives: The present review focuses on updates on ezetimibe and patented profile of novel cholesterol absorption inhibitors followed by critical analysis of different targets such as cholesterol esterase inhibitors, bile acid transport inhibitors or phospholipase-A₂ inhibitors, etc.which play an important role in the lipid absorption. Conclusion: The discovery of ezetimibe has opened a new door for the management of hyper-cholesterolemia in combination with statins. There are newer analogues that are under clinical trials, among which darapladib, FM-VP4 and A-002 are promising compounds.