The carcinogenicity of fluorenylhydroxamic acids and N-acetoxy-N-fluorenylacetamides for the rat as related to the reactivity of the esters toward nucleophiles.

In extension of previous work indicating that the carcinogenicity of isomeric fluorenylhydroxamic acids depends on the point of attachment of the nitrogen atom on the fluorene system, the carcinogenicities of N-hydroxy-3-fluorenylacetamide and of N-hydroxy-4-fluorenylacetamide were evaluated in male and female Sprague-Dawley rats by several routes of administration and were compared with the carcinogenicity of N-hydroxy-2-fluorenylacetamide. The earlier observation that N-hydroxy-3-fluorenylacetamide is a specific mamary carcinogen was confirmed. N-Hydroxy-4-fluorenylacetamide was only marginally carcinogenic. Neither isomer gave tumors at the site after i.m. administration of the compounds into the hind leg of the rat. A comparison of the carcinogenicity of the isomers indicated the following order of activity: N-Hydroxy-2-fluorenylacetamide greater than N-hydroxy-3-fluorenylacetamide greater than N-hydroxy-4-fluorenylacetamide. Because of the current concept that arylhydroxamic acids are further acitvated to electrophilic reactants capable of interacting covalently with cellular nucleophiles and because esters of N-hydroxy-2-fluorenylacetamide give rise to an electrophilic reactant, the acetate esters of N-hydroxy-3-fluorenylacetamide and N-hydroxy-4-fluorenylacetamide were prepared and tested for their carcinogenicity in male and female Spaguw-Dawley rats by i.p. and i.m. administration. The order of carcinogenicity of the isomeric esters followed that of the parent hydroxamic acids (N-acetoxy-2-fluorenylacetamide greater than N-acetoxy-3-fluorenylacetamide greater than N-acetoxy-4-fluorenylacetamide). In order to correlate the carcinogeniciyt of the isomeric esters with their reactivity toward nucleophiles, the esters were reacted with methionine, transfer RNA, and the nucleosides, guanosine and adenosine. Under identical conditions, the reactivity of N-acetoxy-2-fluorenylacetamide towards methionine was at least tenfold greater than that of N-acetoxy-4-fluorenylacetamide. In addition to o-methylthio-2-fluorenylacemide, a new adduct, o-methylsulfoxo-2-fluorenylacetamide, was isolated from the reaction of N-acetoxy-2-fluorenylacetamide with methionine. Reaction of N-acetoxy-4-fluorenylacetamide and 1-methylthio-4-fluorenylacetamide. N-Acetoxy-3-fluorenylacetamide did not react with methionine. Continued.

Comparative effects of strain, species, and sex on the acyltransferase- and sulfotransferase-catalyzed activations of N-hydroxy-N-2-fluorenylacetamide.

The arylhydroxamic acid acyltransferase, an enzyme that promotes the introduction of arylamine groups into nucleic acids, is greater in the stomach, small intestine, colon, and lung of the Sprague-Dawley rat than in comparable tissues of Fischer animals. The enzyme is distributed relatively evenly from the glandular stomach to the distal portion of the colon. No consistent differences in acyltransferase activities of the liver, kidney, brain, or spleen of these two strains were noted. Acyltransferase activity was readily demonstrable in the livers of guinea pigs, hamsters, rabbits, and monkeys; in the kidneys of guinea pigs and hamsters; in the stomachs of guinea pigs and hamsters; in the small intestines of guinea pigs, hamsters, rabbits, and monkeys; in the colons of guinea pigs and hamsters; and in lungs of hamsters. Mouse, dog, and goat tissues were essentia-ly devoid of acyltransferase activity. The transformation of N-hydroxy-N-2-fluorenylacetamide into a reactive species by conjugation with sulfate was carried out with 105,000 times g supernatants of liver from Sprague-Dawley and Fischer rats and their Flhydrids. The abilities of liver extracts from the hybrids to carry out this activation were intermediate between those from animals of the same sex of the two parental strains.

A convenient preparation of aldonohydroxamic acids in water and crystal structure of L-erythronohydroxamic acid.

Hydroxamic acids derived from aldonic acids, namely aldonohydroxamic acids, have become an increasingly important class of inhibitors of enzymes involved in the metabolism of carbohydrates. We now report the straightforward preparation of various types of aldonohydroxamic acids by a new methodology involving the use of commercial 50% aqueous hydroxylamine as the source of the free base hydroxylamine that reacts directly with the corresponding aldonolactone dissolved in water. The reaction proceeds almost instantaneously in water at room temperature, yielding generally pure products in quantitative yield. To date, this methodology is probably the most facile and efficient way to synthesize aldonohydroxamic acids. We also determined by X-ray diffraction analysis the first crystal structure of a free aldonohydroxamic acid reported to date. Crystals of L-erythronohydroxamic acid belonged to the monoclinic system, space group P2(1), a=5.511(3), b=7.556(1), c=8.071(3) A, beta=109.10 degrees, and Z=2.

Combination of the histone deacetylase inhibitor LBH589 and the hsp90 inhibitor 17-AAG is highly active against human CML-BC cells and AML cells with activating mutation of FLT-3.

Present studies show that LBH589, a novel cinnamic hydroxamic acid analog histone deacetylase inhibitor, induces acetylation of histone H3 and H4 and of heat shock protein 90 (hsp90), increases p21 levels, as well as induces cell-cycle G(1) phase accumulation and apoptosis of the human chronic myeloid leukemia blast crisis (CML-BC) K562 cells and acute leukemia MV4-11 cells with the activating length mutation of FLT-3. In MV4-11 cells, this was associated with marked attenuation of the protein levels of p-FLT-3, FLT-3, p-AKT, and p-ERK1/2. In K562 cells, exposure to LBH589 attenuated Bcr-Abl, p-AKT, and p-ERK1/2. Treatment with LBH589 inhibited the DNA binding activity of signal transducers and activators of transcription 5 (STAT5) in both K562 and MV4-11 cells. The hsp90 inhibitor 17-allyl-amino-demethoxy geldanamycin (17-AAG) also induced polyubiquitylation and proteasomal degradation of FLT-3 and Bcr-Abl by reducing their chaperone association with hsp90. Cotreatment with LBH589 and 17-AAG exerted synergistic apoptosis of MV4-11 and K562 cells. In the imatinib mesylate (IM)-refractory leukemia cells expressing Bcr-Abl with the T315I mutation, treatment with the combination attenuated the levels of the mutant Bcr-Abl and induced apoptosis. Finally, cotreatment with LBH589 and 17-AAG also induced more apoptosis of IM-resistant primary CML-BC and acute myeloid leukemia (AML) cells (with activating mutation of FLT-3) than treatment with either agent alone.

Complete assignment of carbon signals in a stereospecific peptide via selective and single off-resonance proton decoupling experiments. Analysis of the carbon-13 nuclear magnetic resonance spectrum of alumichrome at 67.88 MHz.

Polypeptides and proteins in native conformation exhibit 13C NMR spectra which are highly nondegenerate. Assignment of resonances to carbons in particular residues is hence a prerequisite for a structural analysis of the spectroscopic data. For nonprotonated carbonyl carbons, the assignment can be achieved by selective (1H alpha)13C' 2J decoupling. Using this method, we have assigned the Orn1 and Gly2 carbonyl resonances in alumichrome at 67.9 MHz. We show that a single off-resonance experiment with the decoupling frequency centered in the aliphatic proton spectrum is sufficient to assign unequivocally all the protonated carbon resonances via analysis of the reduced 1J heteronuclear splittings. Alumichrome thus becomes the first complex polypeptide spin system whose 1H, 15N, and now 13C nuclear resonances have been fully identified to date. 13C chemical shifts and 1H--13C spin--spin couplings are discussed in terms of structural strain leading to specific orbital hybridizations and on the basis of polarization effects due to electron density shifts toward hydrogen-bonding and metal-binding sites. A number of 3J(13C--C--C--1H) coupling constants measured on selected multiplets after resolution enhancement were used to derive the x-related Karplus relationship 3J(theta) = (10.2 cos2 theta -- 1.3 cos theta + 0.2) Hz.

Amide proton spin-lattice relaxation in polypeptides. A field-dependence study of the proton and nitrogen dipolar interactions in alumichrome.

The proton nuclear magnetic resonance (NMR) spin-lattice relaxation of all six amides of deferriferrichrome and of various alumichromes dissolved in hexadeutero-dimethylsulfoxide have been investigated at 100, 220, and 360 MHz. We find that, depending on the type of residue (glycyl or ornithyl), the amide proton relaxation rates are rather uniform in the metal-free cyclohexapeptide. In contrast, the (1)H spinlattice relaxation times (T(1)'s) are distinct in the Al(3+)-coordination derivative. Similar patterns are observed in a number of isomorphic alumichrome homologues that differ in single-site residue substitutions, indicating that the spin-lattice relaxation rate is mainly determined by dipole-dipole interactions within a rigid molecular framework rather than by the specific primary structures. Analysis of the data in terms of (1)H-(1)H distances (r) calculated from X-ray coordinates yields a satisfactory linear fit between T(1) (-1) and Sigmar(-6) at the three magnetic fields. Considering the very sensitive r-dependence of T(1), the agreement gives confidence, at a quantitative level, both on the fitness of the crystallographic model to represent the alumichromes' solution conformation and on the validity of assuming isotropic rotational motion for the globular metallopeptides. An extra contribution to the amide proton T(1) (-1) is proposed to mainly originate from the (1)H-(14)N dipolar interaction: this was supported by comparison with measurements on an (15)N-enriched peptide. The nitrogen dipolar contribution to the peptide proton relaxation is discussed in the context of {(1)H}-(1)H nuclear Overhauser enhancement (NOE) studies because, especially at high fields, it can be dominant in determining the amide proton relaxation rates and hence result in a decreased effectiveness for the (1)H-(1)H dipolar mechanism to cause NOE's. From the slope and intersect values of T(1) (-1) vs. Sigmar(-6) linear plots, a number of independent estimates of tau(r), the rotational correlation time, were derived. These and the field-dependence of the T(1)'s yield a best estimate approximately 0.37 ns, in good agreement with 0.38 ns [unk] [unk] 0.41 ns, previously determined from (13)C and (15)N spin-lattice relaxation data.

Comparison of the lipopretein profiles and the effect of N-phenylpropyl-N-benzyloxy acetamide in primates (38473).

Analytical ultracentrifugation showed Cebus and Rhesus monkeys had two low density components while only one was present in Squirrel monkeys. In untreated or W1372 treated monkeys, neither chylomicrons nor very low density lipoproteins were detected on analytical ultracentrifugation. Chylomicrons were not observed on agarose gel electrophoresis. Ultracentrifugal analysis showed W1372 treatment decreased the amount of LDL in all animals and also the HDL in Cebus monkeys on an atherogenic diet. Both untreated and W1372 treated Cebus monkeys on an atherogenic diet had abnormal amounts of LDL and HDL, while the LDL in treated animals occurred as multiple peaks. This was also evident on agarose gel electrophoresis. Accumulation of lipds in the liver and decrease of serum lipids indicated W1372 prevented release of lipoproteins from the liver.

Role of urease in pyelonephritis resulting from urinary tract infection with Proteus.

The role of urease in induction of pyelonephritis was studied by treatment of proteus-infected rats with acetohydroxamic acid, a potent inhibitor of urease. Infection was produced by introduction of Proteus mirabilis into the bladder along with a zinc disk. Controls were treated identically but received no acetohydroxamic acid. The number of bacteria per milliliter of urine was the same in both groups. The number of bacteria in the kidneys and the extent of renal damage was much greater in controls. Common enterobacteraceal antigen was not detected in the renal parenchyma of rats treated with acetohydroxamic acid. Treatment with acetohydroxamic acid thus prevented invasion of and damage to kidney tissue without reduction of urinary infection. Thus new evidence was found that the invasive properties of Proteus in the urinary tract are dependent on alkalinization of urine by urease and the resulting damage to the renal epithelium.

Benurestat, a urease inhibitor for the therapy of infected ureolysis.

A single oral administration of the urease inhibitor benurestat (2-(p-chlorobenz-amido)acetohydroxamic acid) to the human at 15 or 25 mg per kg produced, for 4 hr, mean urinary levels of inhibitory activity that were 700 to 1900 times that equivalent concentration of benurestat required to inhibit Proteus mirabilis urease by 90 per cent. In the rat these same dosage levels produced urinary inhibitory activity equivalent to 16 to 140 fold that required for 90 per cent urease inhibition. Benurestat administration, 25, 50, or 100 mg per kg, caused a decrease in the urinary excretion of ammonia from rats with experimental P. mirabilis genitourinary tract infection. The formation of struvite calculi was inhibited under these conditions. Nitrofurantoin, sulfamethoxazole, and ampicillin also slowed the formation of struvite calculi in infected rats and together with benurestat a potentiation of the inhibition of calculi formation was secured. Some combination therapies composed of benurestat plus an antibacterial agent, sulfamethoxazole or ampicillin, were effective in promoting the net dissolution of formed calculi. The number of viable bacteria present in the bladders of infected rats was significantly less after the administration of benurestat plus nitrofurantoin, sulfamethoxazole, or ampicillin than the respective numbers that were obtained from control infected rats or from rats administered either component of the combination separately.

Evidence for common binding sites for ferrichrome compounds and bacteriophage phi 80 in the cell envelope of Escherichia coli.

Mutants ton A and ton B of Escherichia coli K12, known to be resistant to bacteriophage phi80, were found to be insensitive as well to albomycin, an analogue of the specific siderochrome ferrichrome. Ferrichrome at micromolar concentrations strongly inhibited plaque production by phi80. Preincubation with ferrichrome did not inactivate the phage. At a concentration at which ferrichrome allowed 90% inhibition of plaque formation, the chromium analogue of ferrichrome showed no detectable activity. Similarly, ethylenediaminetetraacetic acid, ferrichrome A, and certain siderochromes structurally distinct from ferrichrome, such as ferrioxamine B, schizokinen, citrate, and enterobactin, did not show detectable inhibitory activity. However, rhodotorulic acid showed moderate activity. A host range mutant of phi80, phi80h, was also inhibited by ferrichrome, as was a hybrid of phage lambda possessing the host range of phi80. However, phage lambdacI- and a hybrid of phi80 possessing the host range of lambda were not affected by ferrichrome. Finally, ferrichrome and chromic deferriferrichrome were shown to inhibit adsorption of phi80 to sensitive cells, ferrichrome giving 50% inhibition of adsorption at a minimal concentration of 8 nM. It is suggested that a component of the ferrichrome uptake system may reside in the outer membrane of E. coli K12 and may also function as a component of the receptor site for bacteriophage phi80, and that ferrichrome inhibition of the phage represents a competition for this common site.