Daniele Marciano

Selective Inhibition of Ras-dependent Cell Growth by Farnesylthiosalisylic Acid

S-trans,trans-Farnesylthiosalicylic acid (FTS) is a novel farnesylated rigid carboxylic acid derivative. In cell-free systems, it acts as a potent competitive inhibitor (K = 2.6 μM) of the enzyme prenylated protein methyltransferase (PPMTase), which methylates the carboxyl-terminal S-prenylcysteine in a large number of prenylated proteins including Ras. In such systems, FTS inhibits Ras methylation but not Ras farnesylation. Inhibition of the PPMTase by FTS in homogenates or membranes of a variety of tissues and cell lines is inferred from a block in the methylation of exogenously added substrates such as N-acetyl-S-trans,trans-farnesyl-L-cysteine and of endogenous substrates including small GTP-binding proteins. FTS can also inhibit methylation of these proteins in intact cells (e.g. in Rat-1 fibroblasts, Ras-transformed Rat-1, and B16 melanoma cells). Unlike in cell-free systems, however, relatively high concentrations of FTS (50-100 μM) are required for partial blocking (10-40%) of protein methylation in the intact cells. Thus, FTS is a weak inhibitor of methylation in intact cells. Because methylation is the last step in the processing of Ras and related proteins, FTS is not likely to affect steps that precede it, e.g. protein prenylation. This may explain why the growth and gross morphology of a variety of cultured cell types (including Chinese hamster ovary, NIH3T3, Rat1, B16 melanoma, and PC12) is not affected by up to 25 μM FTS and is consistent with the observed lack of FTS-induced cytotoxicity. Nevertheless, FTS reduces the levels of Ras in cell membranes and can inhibit Ras-dependent cell growth in vitro, independently of methylation. It inhibits the growth of human Ha-ras-transformed cells (EJ cells) and reverses their transformed morphology in a dose-dependent manner (0.1-10 μM). The drug does not interfere with the growth of cells transformed by v-Raf or T-antigen but inhibits the growth of ErbB2-transformed cells and blocks the mitogenic effects of epidermal and basic fibroblast growth factors, thus implying its selectivity toward Ras growth signaling, possibly via modulation of Ras-Raf communication. Taken together, the results raise the possibility that FTS may specifically interfere with the interaction of Ras with a farnesylcysteine recognition domain in the cell membrane. This drug, and perhaps other farnesylated rigid carboxylic acid analogs, may be used for in vitro characterization of the PPMTase and for the identification of a putative Ras farnesylcysteine recognition domain in cell membranes.

Mitogen-Activated Protein Kinase-Dependent and Protein Kinase C-Dependent Pathways Link the m1 Muscarinic Receptor to β-Amyloid Precursor Protein Secretion

Abstract: Full and functionally selective M1 muscarinic agonists (carbachol and AF102B, respectively) activate secretion of the soluble form of amyloid precursor protein (APPs) in PC12 cells expressing the m1 muscarinic receptor (PC12M1 cells). This activation is further augmented by neurotrophins such as nerve growth factor and basic fibroblast growth factor. Muscarinic stimulation activates two transduction pathways that lead to APPs secretion: protein kinase C (PKC)‐dependent and mitogen‐activated protein kinase (MAPK)‐dependent pathways. These pathways operate in parallel and converge with transduction pathways of neurotrophins, resulting in enhancement of APPs secretion when both muscarinic agonist and neurotrophins stimulate PC12M1 cells. These conclusions are supported by the following findings: (a) Only partial blockade of APPs secretion is observed when PKC, p21ras, or MAPK is fully inhibited by their respective specific inhibitors, GF109203X, S‐trans,trans‐farnesylthiosalicylic acid, and PD98059. (b) K252a, which blocks PKC and phorbol 12‐myristate 13‐acetate‐induced APPs secretion, enhances both muscarinic‐stimulated MAPK activation and APPs secretion. (c) Activation of MAPK in PC12M1 cells by muscarinic agonists is Ras‐dependent but PKC‐independent and is enhanced synergistically by neurotrophins. These results suggest that muscarinic stimulation of APPs secretion is mediated by at least two independent pathways that converge and enhance the signal for APPs secretion at the convergence point.

Targeting of K-Ras 4B by S-trans,trans-farnesyl thiosalicylic acid

Ras proteins regulate cell growth, differentiation and apoptosis. Their activities depend on their anchorage to the inner surface of the plasma membrane, which is promoted by their common carboxy-terminal S-farnesylcysteine and either a stretch of lysine residues (K-Ras 4B) or S-palmitoyl moieties (H-Ras, N-Ras and K-Ras 4A). We previously demonstrated dislodgment of H-Ras from EJ cell membranes by S-trans,trans-farnesylthiosalicylic acid (FTS), and proposed that FTS disrupts the interactions between the S-prenyl moiety of Ras and the membrane anchorage domains. In support of this hypothesis, we now show that FTS, which is not a farnesyltransferase inhibitor, inhibits growth of NIH3T3 cells transformed by the non-palmitoylated K-Ras 4B(12V) or by its farnesylated, but unmethylated, K-Ras 4B(12) CVYM mutant. The growth-inhibitory effects of FTS followed the dislodgment and accelerated degradation of K-Ras 4B(12V), leading in turn to a decrease in its amount in the cells and inhibition of MAPK activity. FTS did not affect the rate of degradation of the K-Ras 4B, SVIM mutant which is not modified post-translationally, suggesting that only farnesylated Ras isoforms are substrates for facilitated degradation. The putative Ras-recognition sites (within domains in the cell membrane) appear to tolerate both C(15) and C(20) S-prenyl moeities, since geranylgeranyl thiosalicylic acid mimicked the growth-inhibitory effects of FTS in K-Ras 4B(12V)-transformed cells and FTS inhibited the growth of cells transformed by the geranylgeranylated K-Ras 4B(12V) CVIL isoform. The results suggest that FTS acts as a domain-targeted compound that disrupts Ras-membrane interactions. The fact that FTS can target K-Ras 4B(12V), which is insensitive to inhibition by farnesyltransfarase inhibitors, suggests that FTS may target Ras (and other prenylated proteins important for transformed cell growth) in an efficient manner that speaks well for its potential as an anticancer therapeutic agent.

M1 Agonists for the Treatment of Alzheimer's Disease.

The AF series compounds, AF102B and congeners of AF150(S), are functionally selective agonists for m1 muscarinic receptors (m1AChRs). This is shown in stable transfected CHO and PC12 cells (PC12M1) with m1‐m5AChRs and m1AChRs, respectively. AF102B and AF150(S) are partial agonists, but AF150, AF151, and AF151(S) are full agonists in stimulating phosphoinositides hydrolysis or arachidonic acid release in these cells. Yet, all these compounds behave as antagonists when compared with carbachol in elevating cAMP levels. In PC12M1 cells, unlike carbachol, the AF series compounds induce only minimal to moderate neurite outgrowth. Yet, these agonists synergize strongly with NGF, which by itself mediates only a mild response. Stimulation of m1AChRs by AF102B, AF150(S) and AF151(S) in PC12M1 cells enhances secretion of β/A4 amyloid precursor protein derivatives (APPs). The enhanced APPs secretion induced by AF102B is potentiated by NGF. AF102B also stimulates APPs secretion from rat cortical slices. Stimulation of m1AChR in PC12M1 cells with carbachol or AF102B decreases tau phosphorylation as indicated by specific tau‐1 mAb and alkaline phosphatase treatment. Due to the above mentioned properties m1 agonists may be of unique value in delaying the progression of Alzheimers disease (AD). The AF series compounds show a wide safety margin and improve memory and learning deficits in animal models for AD. There is a dearth of clinical reports on m1 agonists. These include studies on AF102B and xanomeline, another m1 selective agonist. We tested AF102B in escalating doses of 20, 40, 60 mg, tid, po, (each dose for 2 weeks) for a total of 10 weeks. This was a single‐blind placebo‐controlled, parallel‐group study in patients with probable AD. AFl02B was signiticantly effective at 40 and 60 mg, tid in the ADAS, ADAS‐cognitive and ADAS‐word recognition scales.

Stringent structural requirements for anti-Ras activity of S-prenyl analogues

The carboxy terminal S-farnesylcysteine of Ras oncoproteins is required for their membrane anchorage and transforming activities. We showed previously that S-farnesylthiosalicylic acid (FTS) affects the membrane anchorage of activated H-Ras in EJ cells and inhibits their growth. We report here on structural elements in S-prenyl derivatives that specifically inhibit the growth of EJ cells, but not of untransformed Rat-1 cells. Inhibition of the Ras-dependent extracellular signal-regulated protein kinase (ERK), of DNA synthesis and of EJ cell growth were apparent after treatment with FTS or its 5-fluoro, 5-chloro and 4-fluoro derivatives or with the C20 S-geranylgeranyl derivative of thiosalicylic acid. The 4-Cl-FTS analogue was a weak inhibitor of EJ cell growth. The 3-Cl-FTS analogue and the FTS carboxyl methyl ester were inactive, as were the C10 S-geranyl derivative of thiosalicylic acid, farnesoic acid, N-acetyl-S-farnesyl-L-cysteine and S-farne-sylthiopropionic acid. The structural requirements for anti-Ras activity of S-prenyl analogues thus appear to be rather stringent. With regard to chain length, the C15 farnesyl group linked to a rigid backbone seems to be necessary and sufficient. A free carboxyl group in an appropriately rigid orientation, as in thiosalicylic acid, is also required. Halogenic substitutents on the benzene ring of the thiosalicylic acid are tolerated only at position 5 or 4. This information may facilitate the design of potent Ras antagonists and deepen our understanding of the mode of association of Ras with the plasma membrane.

Selective Signaling via Unique Ml Muscarinic Agonistsa

Rigid analogs of acetylcholine (ACh) were designed for selective actions at muscarinic receptor (mAChR) subtypes and distinct second messenger systems. AF102B, AF150, and AF151 are such rigid analogs of ACh. AF102B, AF150 and AF151 are centrally active M1 agonists. AF102B has a unique agonistic profile showing, inter alia: only part of the M1 electrophysiology of ACh and unusual binding parameters to mAChRs. AF150 and AF151 are more efficacious agonists than AP102B for M1 AChRS in rat cortex and in CHO cells stably transfected with the ml AChR subtype. Notably, the selectivity of the new ml agonists is reflected also by activation of select second messenger systems via distinct G‐proteins. These compounds reflect a new pharmacological concept, tentatively defined as ligand‐selective signaling. Thus, agonist/m1AChR complexes may activate different combinations of signaling pathways, depending on the ligand used. Rigid agonists may activate a limited repertoire of signaling systems. In various animal models for Alzheimers disease (AD) the agonists AF102B, AF150 and AF151, exhibited positive effects on mnemomic processes and a wide safety margin. Such agonists, and especially AF102B, can be considered as a rational treatment strategy for AD.

Application of Fluorescent Nanocrystals (q-dots) for the Detection of Pathogenic Bacteria by Flow-Cytometry

Fluorescent semiconductor nanocrystals (q-dots) benefit from practical features such as high fluorescence intensity, broad excitation band and emission diameter dependency. These unique spectroscopic characterizations make q-dots excellent candidates for new fluorescent labels in multi-chromatic analysis, such as Flow-Cytometry (FCM). In this work we shall present new possibilities of multi-labeling and multiplex analysis of pathogenic bacteria, by Flow-Cytometry (FCM) analysis and new specific IgG—q-dots conjugates. We have prepared specific conjugates against B. anthracis spores (q-dots585-IgGαB. anthracis and q-dots655-IgGαB.anthracis). These conjugates enabled us to achieve double staining of B. anthracis spores which improve the FCM analysis specificity versus control Bacillus spores. Moreover, multiplexed analysis of B. anthracis spores and Y. pestis bacteria was achieved by using specific antibodies labeled with different q-dots to obtain: q-dots585-IgGαB. anthracis and q-dots655-IgGαY.pestis, each characterized by its own emission peak as a marker. Specific and sensitive multiplex analysis for both pathogens has been achieved, down to 103 bacteria per ml in the sample.

Novel m1 muscarinic agonists in treatment and delaying the progression of Alzheimer's disease: An unifying hypothesis

M1 selective agonists from the AF series (e.g. AF102B, AF150(S)), via m1 muscarinic receptors, activate distinct signal transductions, enhance amyloid precursors proteins secretion from transfected cells and primary cell cultures, show neurotrophic effects and are beneficial in a variety of animal models for Alzheimers disease. Such m1 agonists may be effective in the treatment and therapy of Alzheimers disease.

Novel inhibitors of the prenylated protein methyltransferase reveal distinctive structural requirements

Abstract Inhibitors of a prenylated protein methyltransferase were synthesized and evaluated. S-farnesyl-5-fluorothiosalicylic acid and the 5-chloro analog (but not the 4-fluoro, 4-chloro or 3-chloro analogs) were potent inhibitors, as was the parent compound S-farnesyl thiosalicylic acid (FTS), whose methyl ester was far less active. S-geranyl and S-geranylgeranyl thiosalicylic acids were more than ten times less potent than FTS.

Rigid analogs of acetylcholine can be m1-selective agonists: implications for a rational treatment strategy in Alzheimer's disease

Abstract Rigid analogs of acetylcholine offer an opportunity for selective actions at muscarinic receptor subtypes, since restricted conformational mobility alters the capacity of ligands to adapt to subtle differences in receptor structure. AF102B , a highly rigid analog of acetylcholine, is a centrally active M1 agonist and is evaluated in light of some currently available therapeutic strategies in Alzheimers disease.