Localization of retinoic acid-binding protein in nuclei and the nuclear uptake of retinoic acid.

Retinoic acid-binding protein (RABP) has been detected in the nuclei of chick embryo skin and Lewis lung tumor. The nuclear binding component showed the same ligand specificity and sedimentation value as the cytosol RABP. Whereas pronase completely digested the nuclear binding component, DNase showed 40%, and RNase showed negligible digestive action. Retinoic acid binding to the nuclear RABP was completely inhibited by a mercurial, and the inhibition was reversed by dithiothreitol. The nonspecific uptake of retinoic acid by Lewis drug nuclei and chick embryo skin nuclei was inhibited up to 50% by cytosol RABP. The maximal inhibitory effect produced by cytosol RABP was after 45-min incubation. Incubation of Lewis lung tumor with [3H]retinoic acid resulted in the appearance of nuclear RABP: [3H]retinoic acid in the nuclei. The complex formed was weak, and most of the bound retinoic acid could be removed by dialysis.

A retinoic acid-binding protein from chick embryo skin.

Previous studies by others have indicated that retinoic acid reverses metaplastic changes caused by chemical caricinogens in vivo and in organ culture. The present study deals with the detection of a specific retinoic acid-binding protein from chick embryo metatarsal skin that may be mediating these biological effects. The protein has an S20 value of 2 and an isoelectric pH of 4.6. Competition experiments with labeled retinoic acid and 200-fold molar excesses of unlabeled retinoic acid, retinol, retinal, methyl retinoate, diethylretinamide, synthetic analogs of retinoic acid, and gamma-linolenic acid reveal that only retinoic acid and its analogs with a free carboxyl group bind to this protein. Among the analogs of retinoic acid, a cyclopentenyl analog, a trimethylmethoxyphenyl analog, 13-cis-retinoic acid, and alpha-retinoic acid compete for the binding site on the protein, with the cyclopentenyl analog having greater affinity than retinoic acid does. Phenyl and pyridyl analogs of retinoic acid are poor binders. In general, the ability of the various analgos to bind to this protein correlates with their biological activity in the reversal of keratinization and in the production of mucous metaplasia by chick embryo metatarsal skin as reported by others.

Retinoic acid-binding protein in experimental tumors and in tissues with metastatic tumor foci.

Screening for retinoic acid-binding protein (RABP) in experimental tumors revealed the presence of this protein in three mammary tumors, two metastatic colon tumors, B16 melanoma. Lewis lung carcinoma, Ridgway osteogenic sarcoma, and keratoacanthoma. RABP was below the limits of detection in two weakly metastatic colon tumors and in Sarcoma 180. After s.c. implantation of RABP-containing tumors into mice, this protein could be traced in the lungs due to pulmonary metastasis. Following implantation of Lewis lung tumors, RABP was detected in the lung on the 6th day. On the 15th day after implantation, RABP was present in lung and brain, but not in other tissues where this protein was normally lacking. In primary cultures of Lewis lung carcinoma, the lower limit for detection of RABP by sucrose gradient sedimentation technique corresponded to 0.12 mg protein that was extractable from 3 X 10(5) cells. Both chick embryo skin and rabbit ear skin extracts contained RABP; the level of cellular retinol-binding protein was high in chick embryo skin but only marginal in rabbit ear. The amounts of these proteins on chick embryo skin and rabbit ear skin correlate with the biological potency of retinol and retinoic acid, as observed by others.

Relationship between binding affinities to cellular retinoic acid-binding protein and biological potency of a new series of retinoids.

Binding affinities of a new and unusual series of retinoic acid analogues to cellular retinoic acid-binding protein, a possible mediator of their biological function in the control of differentiation and tumorigenesis, and to serum albumin, their plasma transport protein, were determined. Also, biological activity of these retinoids in the reversal of keratinization in hamster tracheal organ cultures was assessed and compared with their binding affinities. Analogues that possessed high biological activity showed high binding efficiency to cellular retinoic acid-binding protein. Those that were biologically less active were poor binders to the binding protein. Three retinoids, 4657-57, 3920-59, and 4445-75, which showed 90 to 100% binding efficiency of that of retinoic acid for cellular retinoic acid-binding protein expressed high biological activity detectable in the range of 10(-10) M as against 10(-11) M for retinoic acid. The correlation noticed in these two activities not only enhances the confidence in the two assay procedures but also paves the way for design and development of potential chemopreventive agents. No apparent differences were observed in the binding affinities of the retinoids to binding proteins of a normal tissue or a tumor tissue. No correlation existed between the binding affinities of these retinoids to serum albumin and their biological activity. Structure-activity relationships of the retinoids in relation to their binding affinities and biological activities have been discussed.

Purification and properties of retinol- and retinoic acid-binding proteins from a transplantable mouse colon tumor.

Cellular retinol-binding protein and retinoic acid-binding protein, the possible mediators of the action of retinoids in epithelial differentiation and control of tumorigenesis, have been reproducibly purified from mouse colon tumor 26, and some of their properties were studied. The main steps of purification involved acid-precipitation, DEAE-Sephadex, CM-cellulose and Sephadex G-100 chromatography. About 2 mg of the binding proteins were isolated from 60 g tumor. The purified preparations showed only two protein bands on polyacrylamide gel electrophoresis. The two binding proteins were partially resolved by sedimentation equilibrium technique; but was completely separable by preparative electrophoresis in the presence of sodium dodecyl sulfate. The retinol- and retinoic acid-binding proteins are presumably monomers with molecular weights of 15,500 and 14,600, respectively, as determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. On gel filtration however, both the binding proteins retarded to the same molecular size of 17,800. On preparative columns, both the proteins expressed the same isoelectric pH, 4.5. Both proteins of the tumor possessed functional thiol groups. The mercurial inhibition of the binding capacity of the proteins for their ligands was reversible upon treatment with thiol compounds.

5'-Haloacetamido-5'-deoxythymidines: novel inhibitors of thymidylate synthase.

5'-Bromoacetamido-5'-deoxythymidine (BAT), 5'-iodoacetamido-5'-deoxythymidine (IAT), 5'-chloroacetamido-5'-deoxythymidine (CAT) and [14C]BAT were synthesized and their interactions with thymidylate synthase purified from L1210 cells were investigated. The inhibitory effects of these compounds on thymidylate synthase were in the order BAT greater than IAT greater than CAT, which is in agreement with their cytotoxic effects in L1210 cells. In the presence of substrate during preincubation, the concentration required for 50% inhibition of the enzyme activity by these inhibitors was 4-8-fold higher than it was in the absence of dUMP. The I50 values for BAT were 1 X 10(-5) M and 1.2 X 10(-6) M in the presence and absence, respectively, of dUMP during preincubation. These results were in agreement with the observed inhibition of thymidylate synthase by BAT in intact L1210 cells. A Lineweaver-Burk plot revealed that BAT behaved as a competitive inhibitor. The Km for the enzyme was 9.2 microM, and the Ki determined for competitive inhibition by BAT was 5.4 microM. Formation of a tight, irreversible complex is inferred from the finding that BAT-inactivation of thymidylate synthase was not reversible on prolonged dialysis and that the enzyme-BAT complex was nondissociable by gel filtration through a Sephadex G-25 column or by TSK-125 column chromatography. Incubation of thymidylate synthase with BAT resulted in time-dependent, irreversible loss of enzyme activity by first-order kinetics. The rate constant for inactivation was 0.4 min-1, and the steady-state constant of inactivation, Ki, was estimated to be 6.6 microM. The 5'-haloacetamido-5'-deoxythymidines provide specific inhibitors of thymidylate synthase that may also serve as reagents for studying the enzyme mechanism.

Terminal bifunctional retinoids. Synthesis and evaluations related to cancer chemopreventive activity.

Retinoids that have two functional groups at the side-chain terminus have been synthesized. The two terminal functional groups are combinations of the carboxyl, carbethoxy, and N-(ethylamino)carbonyl groups. The synthesis route is based on the sodium amide catalyzed condensation of (E,E)-beta-ionylideneacetaldehyde with diethyl isopropylidenemalonate. Ethyl 14-carboxyretinoate (6), the initial bifunctional analogue, undergoes isomerization in unbuffered aqueous ethanol and reaches a state of equilibrium with ethyl 14-carboxy-13-cis-retinoate. Both of the possible amide-esters and amide-acids were obtained. The structures of the isomeric bifunctional analogues were established by studies of nuclear Overhauser effects. The bifunctional analogues induce differentiation of mouse embryonal carcinoma cells, and those analogues that have a free carboxyl group bind to cellular retinoic acid binding protein.

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

Effect of structural modifications in the C7-C11 region of the retinoid skeleton on biological activity in a series of aromatic retinoids.

A series of conformationally restricted analogues of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl ] benzoic acid--(E)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 - propenyl]benzoic acid, (E)-4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-bu ten- 2-yl]benzoic acid, trans-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) cyclopropyl]benzoic acid, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid, 6-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2- naphthalenecarboxylic acid--were synthesized and screened for retinoid biological activity. Comparison of the conformers of these analogues generated by molecular mechanics calculations with the biological activity profiles of these compounds indicates that geometric constraints required for high biological activity are imposed on the bridge joining the two aromatic ring systems by the retinoid receptor.

Conformationally defined 6-s-trans-retinoic acid analogs. 3. Structure-activity relationships for nuclear receptor binding, transcriptional activity, and cancer chemopreventive activity.

We recently demonstrated that conformationally defined 6-s-trans-retinoic acid (RA) analogs were effective in the prevention of skin papillomas (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) and selective agonists for nuclear receptor binding and activation (Alam et al. J. Med. Chem. 1995, 38, 2302-2310). In order to probe important structure-activity relationships, we evaluated a homologous series of four 6-s-trans-retinoids that are 8-(2'-cyclohexen-1'-ylidene)-3,7-dimethyl-2,4,6-octatrienoic acids with different substituents at 2' (R2) and 3' (R1) positions on the cyclohexene ring. UAB1 (R1 = R2 = H), UAB4 (R1 = R2 = Me), UAB7 (R1 = Me, R2 = iPr), and UAB8 (R1 = Et, R2 = iPr) contain alkyl R groups that mimic, to different extents, portions of the trimethylcyclohexenyl ring of RA. Both 9Z- and all-E-isomers of these retinoids were evaluated in binding assays for cellular retinoic acid-binding proteins (CRABP-I and CRABP-II), a nuclear retinoic acid receptor (RAR alpha), and a nuclear retinoid X receptor (RXR alpha). The all-E-isomers of UAB retinoids bound tightly to CRABPs and RAR alpha, the binding affinity of the all-E-isomer increased systematically from UAB1 to UAB8, and binding for the latter was comparable to that of all-E-RA. In contrast to RA, the (9Z)-UAB retinoids were at least 200-fold less active than the all-E-isomers in binding to RAR alpha. The (9Z)-UAB isomers exhibited increasingly stronger binding to RXR alpha, and (9Z)-UAB8 was nearly as effective as (9Z)-RA in binding affinity. The retinoids were also evaluated in gene expression assays mediated by RAR alpha and RXR alpha homodimers or RAR alpha/RXR alpha heterodimers. Consistent with the binding affinities, the (all-E)-UAB retinoids activated gene transciption mediated by RAR alpha homodimers or RAR alpha/RXR alpha heterodimers, while the (9Z)-UAB isomers activated only the RXR alpha homodimer-mediated transcription. The all-E- and 9Z-isomers of the UAB retinoids were further evaluated for their capacity to prevent the induction of mouse skin papillomas. When compared to RA, only the (all-E)-UAB retinoids containing bulky R1 and R2 groups were effective in this chemoprevention assay. (9Z)-RA displayed equal capacity as RA to prevent papillomas, while the 9Z-isomers of the UAB retinoids were much less effective. Taken together, these studies demonstrate that the cyclohexenyl ring substituents of 6-s-trans-UAB retinoids are important for their biological activities and that the chemopreventive effect of the all-E-isomers of these retinoids correlates well with their capacity to bind to RARs and activate RAR/RXR-mediated transcription.

A conformationally defined 6-s-trans-retinoic acid isomer: synthesis, chemopreventive activity, and toxicity.

A conformationally defined retinoic acid analog (1) which contains a dimethylene bridge to maintain the 6-s-trans orientation for two terminal double bonds in the polyene chain was synthesized. A Reformatsky reaction was utilized to extend the polyene chain of the starting enone, which provided exclusively the 9Z-configuration for the intermediate aldehyde. A Horners-Emmons condensation with this aldehyde then produced retinoic acid analogs with both 9Z- and 9Z,13Z-configurations. An I2-catalyzed isomerization of the intermediate 9Z-aldehyde yielded the all-E-aldehyde, which was olefinated as above to yield the (all-E)- and (13Z)-retinoic acid analogs of 1. Each configurational isomer of 1 was evaluated for its ability to inhibit the binding of retinoic acid to CRABP (chick skin) and to inhibit the chemical induction of ornithine decarboxylase in mouse skin. In each assay (all-E)-1 was the most active isomer, and this activity was comparable to or better than that for (all-E)-retinoic acid. (all-E)-1 and (13Z)-1 were both shown to be equally effective as (13Z)-retinoic acid in suppressing the proliferation of human sebaceous cells in vitro. (all-E)-1 was further evaluated for its ability to prevent the induction of mouse skin papillomas and to induce signs of vitamin A toxicity in mice. The cancer chemopreventive activity of (all-E)-1 was comparable to that of (all-E)-retinoic acid, and the toxicity was comparable to or slightly better than that of the natural vitamin.

Conformationally defined retinoic acid analogues. 4. Potential new agents for acute promyelocytic and juvenile myelomonocytic leukemias.

We recently synthesized several conformationally constrained retinoic acid (RA) analogues [8-(2'-cyclohexen-1'-ylidene)-3, 7-dimethyl-2,4,6-octatrienoic acids with different alkyl substituents at 2' (R1) and 3' (R2) positions on the cyclohexene ring] (Muccio et al. J. Med. Chem. 1996, 39, 3625) as cancer chemopreventive agents. UAB8 (R1 = Et; R2 = iPr), which contains sufficient steric bulk at the terminal end of the polyene chain to mimic the trimethylcyclohexenyl ring of RA, displayed biological properties similar to those of RA. To explore the efficacy of this retinoid in acute promyelocytic leukemia (APL) and juvenile myelomonocytic leukemia (JMML), we evaluated UAB8 isomers in in vitro assays which measure the capacity of retinoids to inhibit aberrant myeloid colony growth from blood or bone marrow cells obtained from human JMML patients and in assays measuring the potential of retinoids to differentiate NB4 cells (an APL cell line). Both (all-E)- and (13Z)-UAB8 were 2-fold more active than RA in the NB4 cell differentiation assay; however, only (all-E)-UAB8 had comparable activity to the natural retinoids in the JMML cell assays. These results were compared to the biological effectiveness of a new retinoid, UAB30 [8-(3', 4'-dihydro-1'(2'H)-naphthalen-1'-ylidene)-3,7-dimethyl-2,4, 6-octatrienoic acid], which had different nuclear receptor binding and transactivational properties than UAB8. Relative to (all-E)-RA and (all-E)-UAB8, (all-E)-UAB30 bound well to RARalpha but did not activate transcription-mediated RARalpha homodimers, even though it was effective in RARbeta- and RARgamma-mediated transactivational assays. In APL assays, this retinoid had much reduced activity and was only moderately effective in JMML assays and in cancer chemoprevention assays.

Didehydroretinoic acid: retinoid receptor-mediated transcriptional activation and binding properties.

All-trans-3,4-Didehydroretinoic acid (vitamin A2 acid; DDRA) is one of the retinoids present in human skin, the most responsive tissue to retinoid treatment. To understand the mechanism of action of DDRA in the control of differentiation and tumorigenesis, we studied its interaction with cellular retinoic acid-binding proteins (CRABPs) and nuclear all-trans-retinoic acid (RA) receptors (RARs), and 9-cis-retinoic acid receptors (RXRs). The IC50 plots of DDRA for inhibition of [3H]RA binding to CRABP I and II and to RAR alpha, beta and gamma illustrate that this retinoid binds with the same affinity as RA to these proteins. DDRA, however, showed higher affinity than RA for RXR alpha. Evaluation of the transcriptional activation potential of DDRA in CV-1 cells showed that this retinoid induced RAR alpha-mediated transcription to the same magnitude as RA in the 10(-9) to 10(-6) M concentration range. However, in comparison to RA, DDRA produced a 2- to 3-fold higher activation of the transcription mediated by RXR alpha homodimers, as well as RAR beta-RXR alpha heterodimers. These results suggest that the biological activity of retinoids in the skin may be attained through the joint potential of both RA and DDRA.

Retinoyl beta-glucuronide: lack of binding to receptor proteins of retinoic acid as related to biological activity.

Retinoid beta-glucuronides have emerged as biologically active, water-soluble, natural retinoids with relatively few toxic and teratogenic effects. The mechanism of action of these glucuronides in the control of epithelial differentiation, growth, and tumorigenesis is unknown. Since retinoyl beta-glucuronide (RAG) contains a free carboxyl group, we studied the interactions of RAG with cellular retinoic acid-binding protein (CRABP) and nuclear receptors of retinoic acid (RARs), the possible mediators of the biological action of retinoic acid (RA). RAG did not exhibit any significant affinity to bind either CRABP or RARs. During 24- and 48-hr incubations of RAG in chick cytosol, detectable amounts of RA were generated which interacted with the RA receptors. In chick skin, the biological activity of RAG may be due to this slowly released RA. Other possible modes of action of RAG are suggested.

Anhydroretinol, a retinoid active in preventing mammary cancer induced in rats by N-methyl-N-nitrosourea.

As determined by in vitro tests, anhydroretinol, a metabolic product of retinol, was bound specifically by serum retinol-binding protein and by cellular retinol-binding protein but not by cellular retinoic acid-binding protein or the nuclear receptors, RARs and RXRs. For rats dosed with the mammary carcinogen, N-methyl-N-nitrosourea (45 mg/kg body weight) and given diets containing either the retinoid vehicle, anhydroretinol (67, 134, 268, or 536 mg/kg of diet), or retinyl acetate (328 mg/kg of diet), there were, over a 90-day observation period, no significant differences in body weights. The compound did not accumulate in liver tissue or cause an increase in hepatic levels of retinyl palmitate (potential problems observed with other retinoids). The numbers of mammary cancers were as follows: no retinoid, 4.5/rat; retinyl acetate, 2.1/rat; and increasing doses of anhydroretinol, 2.9, 3.3, 3.0, and 1.7/rat, respectively. Thus, anhydroretinol, at non-toxic levels, was effective as a preventive agent in this experimental model of breast cancer.

Biochemical characteristics and differentiating activity of 4-oxo analogs of retinoic acid.

3-Methyl-4-oxoretinoic acid and 3-cinnamyl-4-oxoretinoic acid bind to a cellular retinoic acid-binding protein (CRABP-II) and to a retinoic acid-receptor protein (RARa). These analogs of 4-oxoretinoic acid, as well as the parent compound, have less binding affinity than retinoic acid. Cotransfection assays in CV-1 cells with plasmids containing cDNAs for RAR alpha, RAR beta and RAR gamma (homodimers) and RAR alpha-RXR alpha and RAR beta-RXR alpha (heterodimers), indicate that 3-cinnamyl-4-oxoretinoic acid induces relatively less transcriptional activity than 4-oxoretinoic acid and its 3-methyl analog, both of which are less effective than retinoic acid. In differentiating mouse F9 embryocarcarcinoma cells, the order of effectiveness is retinoic acid > 4-oxoretinoic acid = 3-methyl-4-oxoretinoic acid > 3-cinnamyl-4-oxoretinoic acid. This order of potency is similar to that for inhibition of induction of ornithine decarboxylase (ODC) activity and for prevention of papillomas on the skin of mice. Binding to CRABP-II and activation of RARs appear to be important factors for expression of differentiating activity, inhibition of induction of ODC activity and prevention of papillomas on the skin of mice.

Biologically active aromatic retinoids bearing azido photoaffinity-labeling groups and their binding to cellular retinoic acid-binding protein.

Retinoids bearing azido photoaffinity-labeling groups (azidoretinoids) have potential as probes for investigating the molecular mechanisms of action of all-trans-retinoic acid (RA) as mediated by its cellular retinoic acid-binding protein (CRABP) and nuclear receptor proteins. Two new azidoretinoids, 3-azido-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1E- propen-1-yl]-benzonic acid and 4-(4-azido-5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)be nzoic acid were synthesized, and evaluated for their in vitro biological potency, and binding affinity for CRABP. Like RA, these aromatic azides had significant activity in modulating cell differentiation in retinoid-deficient hamster tracheal organ culture (ED500.02 nM and 0.03 nM, respectively) and in the inhibition of the induction of ornithine decarboxylase in mouse epidermis (ED50 7.0 nmol and 0.5 nmol, respectively). They also possessed high binding affinity for CRABP (ID50 0.9 microM and 0.85 microM, respectively). The tritiated aromatic azides were further evaluated for their ability to bind covalently to CRABP after photolysis. On photolysis at -78 degrees C, the two radiolabeled azidoretinoids formed stable adducts with CRABP. Treatment of the adducts with either RA or p-chloromercuriphenylsulfonic acid (CMPS) and subsequent dialysis did not cause any dissociation, indicating the formation of a covalent bond. In contrast, treatment of the unirradiated complexes with RA or CMPS led to dissociation of the complex. Synthesis of affinity labels and characterization of CRABP-retinoid complexes should provide useful information on the ligand-binding regions and insights into the mechanism of action of RA.