[show abstract][hide abstract] ABSTRACT: Arginase is a binuclear manganese metalloenzyme that hydrolyzes L-arginine to form L-ornithine and urea, and aberrant arginase activity is implicated in various diseases such as erectile dysfunction, asthma, atherosclerosis, and cerebral malaria. Accordingly, arginase inhibitors may be therapeutically useful. Continuing our efforts to expand the chemical space of arginase inhibitor design and inspired by the binding of 2-(difluoromethyl)-L-ornithine to human arginase I, we now report the first study of the binding of α,α-disubstituted amino acids to arginase. Specifically, we report the design, synthesis, and assay of racemic 2-amino-6-borono-2-methylhexanoic acid and racemic 2-amino-6-borono-2-(difluoromethyl)hexanoic acid. X-ray crystal structures of human arginase I and Plasmodium falciparum arginase complexed with these inhibitors reveal the exclusive binding of the L-stereoisomer; the additional α-substituent of each inhibitor is readily accommodated and makes new intermolecular interactions in the outer active site of each enzyme. Therefore, this work highlights a new region of the protein surface that can be targeted for additional affinity interactions, as well as the first comparative structural insights on inhibitor discrimination between a human and a parasitic arginase.
Journal of Medicinal Chemistry 08/2011; 54(15):5432-43. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: As part of our continuing search for new amino acid inhibitors of metalloenzymes, we now report the synthesis and biological evaluation of the trifluoromethylketone analogue of L-arginine, (S)-2-amino-8,8,8-trifluoro-7-oxo-octanoic acid (10). While this novel amino acid was initially designed as a potential inhibitor of human arginase I, it exhibits no measurable inhibitory activity against this enzyme. Surprisingly, however, 10 is a potent inhibitor of human histone deacetylase 8, with IC(50)=1.5 ± 0.2 μM. Additionally, 10 weakly inhibits the related bacterial enzyme, acetylpolyamine amidohydrolase, with IC(50)=110 ± 30 μM. The lack of inhibitory activity against human arginase I may result from unfavorable interactions of the bulky trifluoromethyl group of 10 in the constricted active site. Since the active site of histone deacetylase 8 is less constricted, we hypothesize that it accommodates 10 as the gem-diol, which mimics the tetrahedral intermediate and its flanking transition states in catalysis. Therefore, we suggest that 10 represents a new lead in the design of an amino acid or peptide-based inhibitor of histone deacetylases with simpler structure than previously studied trifluoromethylketones.
[show abstract][hide abstract] ABSTRACT: The 2.15 A resolution crystal structure of arginase from Plasmodium falciparum, the parasite that causes cerebral malaria, is reported in complex with the boronic acid inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) (K(d) = 11 microM). This is the first crystal structure of a parasitic arginase. Various protein constructs were explored to identify an optimally active enzyme form for inhibition and structural studies and to probe the structure and function of two polypeptide insertions unique to malarial arginase: a 74-residue low-complexity region contained in loop L2 and an 11-residue segment contained in loop L8. Structural studies indicate that the low-complexity region is largely disordered and is oriented away from the trimer interface; its deletion does not significantly compromise enzyme activity. The loop L8 insertion is located at the trimer interface and makes several intra- and intermolecular interactions important for enzyme function. In addition, we also demonstrate that arg- Plasmodium berghei sporozoites show significantly decreased liver infectivity in vivo. Therefore, inhibition of malarial arginase may serve as a possible candidate for antimalarial therapy against liver-stage infection, and ABH may serve as a lead for the development of inhibitors.
[show abstract][hide abstract] ABSTRACT: Alternatively activated macrophages prevent lethal intestinal pathology caused by worm ova in mice infected with the human parasite Schistosoma mansoni through mechanisms that are currently unclear. This study demonstrates that arginase I (Arg I), a major product of IL-4- and IL-13-induced alternatively activated macrophages, prevents cachexia, neutrophilia, and endotoxemia during acute schistosomiasis. Specifically, Arg I-positive macrophages promote TGF-beta production and Foxp3 expression, suppress Ag-specific T cell proliferation, and limit Th17 differentiation. S. mansoni-infected Arg I-deficient bone marrow chimeras develop a marked accumulation of worm ova within the ileum but impaired fecal egg excretion compared with infected wild-type bone marrow chimeras. Worm ova accumulation in the intestines of Arg I-deficient bone marrow chimeras was associated with intestinal hemorrhage and production of molecules associated with classical macrophage activation (increased production of IL-6, NO, and IL-12/IL-23p40), but whereas inhibition of NO synthase-2 has marginal effects, IL-12/IL-23p40 neutralization abrogates both cachexia and intestinal inflammation and reduces the number of ova within the gut. Thus, macrophage-derived Arg I protects hosts against excessive tissue injury caused by worm eggs during acute schistosomiasis by suppressing IL-12/IL-23p40 production and maintaining the Treg/Th17 balance within the intestinal mucosa.
The Journal of Immunology 06/2010; 184(11):6438-46. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Arginase, a key metalloenzyme of the urea cycle that converts L-arginine into L-ornithine and urea, is presently considered a pharmaceutical target for the management of diseases associated with aberrant l-arginine homeostasis, such as asthma, cardiovascular diseases, and erectile dysfunction. We now report the design, synthesis, and evaluation of a series of 2-aminoimidazole amino acid inhibitors in which the 2-aminoimidazole moiety serves as a guanidine mimetic. These compounds represent a new class of arginase inhibitors. The most potent inhibitor identified in this study, 2-(S)-amino-5-(2-aminoimidazol-1-yl)pentanoic acid (A1P, 10), binds to human arginase I with K(d) = 2 microM and significantly attenuates airways hyperresponsiveness in a murine model of allergic airways inflammation. These findings suggest that 2-aminoimidazole amino acids represent new leads for the development of arginase inhibitors with promising pharmacological profiles.
Journal of Medicinal Chemistry 05/2010; 53(10):4266-76. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Human arginase I is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. We demonstrate that N-hydroxy-L-arginine (NOHA) binds to this enzyme with K(d)=3.6 microM, and nor-N-hydroxy-L-arginine (nor-NOHA) binds with K(d)=517 nM (surface plasmon resonance) or K(d) approximately 50 nM (isothermal titration calorimetry). Crystals of human arginase I complexed with NOHA and nor-NOHA afford 2.04 and 1.55 A resolution structures, respectively, which are significantly improved in comparison with previously-determined structures of the corresponding complexes with rat arginase I. Higher resolution structures clarify the binding interactions of the inhibitors. Finally, the crystal structure of the complex with L-lysine (K(d)=13 microM) is reported at 1.90 A resolution. This structure confirms the importance of hydrogen bond interactions with inhibitor alpha-carboxylate and alpha-amino groups as key specificity determinants of amino acid recognition in the arginase active site.
Archives of Biochemistry and Biophysics 02/2010; 496(2):101-8. · 3.37 Impact Factor
[show abstract][hide abstract] ABSTRACT: There is increasing evidence that upregulation of arginase contributes to impaired endothelial function in aging. In this study, we demonstrate that arginase upregulation leads to endothelial nitric oxide synthase (eNOS) uncoupling and that in vivo chronic inhibition of arginase restores nitroso-redox balance, improves endothelial function, and increases vascular compliance in old rats. Arginase activity in old rats was significantly increased compared with that shown in young rats. Old rats had significantly lower nitric oxide (NO) and higher superoxide (O2(-)) production than young. Acute inhibition of both NOS, with N(G)-nitro-l-arginine methyl ester, and arginase, with 2S-amino- 6-boronohexanoic acid (ABH), significantly reduced O2(-) production in old rats but not in young. In addition, the ratio of eNOS dimer to monomer in old rats was significantly decreased compared with that shown in young rats. These results suggest that eNOS was uncoupled in old rats. Although the expression of arginase 1 and eNOS was similar in young and old rats, inducible NOS (iNOS) was significantly upregulated. Furthermore, S-nitrosylation of arginase 1 was significantly elevated in old rats. These findings support our previously published finding that iNOS nitrosylates and activates arginase 1 (Santhanam et al., Circ Res 101: 692-702, 2007). Chronic arginase inhibition in old rats preserved eNOS dimer-to-monomer ratio and significantly reduced O2(-) production and enhanced endothelial-dependent vasorelaxation to ACh. In addition, ABH significantly reduced vascular stiffness in old rats. These data indicate that iNOS-dependent S-nitrosylation of arginase 1 and the increase in arginase activity lead to eNOS uncoupling, contributing to the nitroso-redox imbalance, endothelial dysfunction, and vascular stiffness observed in vascular aging. We suggest that arginase is a viable target for therapy in age-dependent vascular stiffness.
Journal of Applied Physiology 09/2009; 107(4):1249-57. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: Oxidized low-density lipoproteins increase arginase activity and reciprocally decrease endothelial NO in human aortic endothelial cells. Here, we demonstrate that vascular endothelial arginase activity is increased in atherogenic-prone apolipoprotein E-null (ApoE(-/-)) and wild-type mice fed a high cholesterol diet. In ApoE(-/-) mice, selective arginase II inhibition or deletion of the arginase II gene (Arg II(-/-) mice) prevents high-cholesterol diet-dependent decreases in vascular NO production, decreases endothelial reactive oxygen species production, restores endothelial function, and prevents oxidized low-density lipoprotein-dependent increases in vascular stiffness. Furthermore, arginase inhibition significantly decreases plaque burden. These data indicate that arginase II plays a critical role in the pathophysiology of cholesterol-mediated endothelial dysfunction and represents a novel target for therapy in atherosclerosis.
Circulation Research 05/2008; 102(8):923-32. · 11.86 Impact Factor
[show abstract][hide abstract] ABSTRACT: The synthesis of dendritic dipeptides (4-3,4-3,5-4)12G2-CH(2)-Boc-L-Tyr-L-Ala-OMe and (4-3, 4-3,5-4)12G2-CH(2)-Boc-D-Tyr-D-Ala-OMe is described. These dendritic dipeptides self-assemble into porous elliptical and circular columns that in turn self-organize into centered rectangular columnar and hexagonal columnar periodic arrays. The transition from porous elliptical to porous circular columns is mediated in a reversible or irreversible way by the thermal history of the sample. A method to determine the dimensions of hollow elliptical and circular columns by the reconstruction of the small-angle powder X-ray diffractograms of the centered rectangular or hexagonal columnar lattices was elaborated. This technique together with wide-angle X-ray experiments performed on aligned fibers provided access to the structural and retrostructural analysis of elliptical supramolecular pores. This procedure is general and can be adapted for the determination of the dimensions of pores of any columnar shape.
Journal of the American Chemical Society 06/2006; 128(20):6713-20. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: The self-assembly of the dendritic dipeptides (4-3,4-3,5)nG2-CH2-Boc-L-Tyr-L-Ala-OMe and their achiral dendritic alcohol (4-3,4-3,5)nG2-CH2OH precursors, both with n = 1-16, where n represents the number of methylenic units in the alkyl groups of the dendron, are reported. All chiral dendritic dipeptides and achiral dendritic alcohols self-assemble into helical porous columns that are stable in both solution and solid state. The pore diameter (D(pore)) of the columns self-assembled from dendritic dipeptides is approximately 10 A larger than that of structures assembled from dendritic alcohols. The increase of the D(pore) at the transition from dendritic alcohol to dendritic dipeptide is accompanied by a decreased solid angle of the building block. This trend is in agreement with previous pore size-solid angle dependences observed with different protective groups of the dipeptide and primary structures of the dendron. However, within the series of dendritic alcohols and dendritic dipeptides with various n, the D(pore) increases when the solid angle increases. The results of these investigations together with those of previous studies on the role of dipeptide stereochemistry and protective groups on this self-assembly process provide the molecular principles required to program the construction of supramolecular helical pores with diameter controlled at the A level from a single dendritic dipeptide architecture. These principles are expected to be valid for libraries of dendritic dipeptides based on dendrons and dipeptides with various primary structures.
Proceedings of the National Academy of Sciences 02/2006; 103(8):2518-23. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: The synthesis of dendritic dipeptides (4-3,4-3,5)12G2-CH2-X-L-Tyr-L-Ala-OMe with X = Boc, Moc, and Ac; their self-assembly in bulk and in solution; and the structural and retrostructural analysis of their supramolecular helical porous assemblies are reported. The dimensions, structure, internal order, thermal stability of the supramolecular helical pores, and conformations of the dendron and supramolecular dendrimer are programmed by the nature of the protective groups of the dipeptide. The ability of the protective groups to program the structure of the helical pore reveals the simplest design strategy that complements the more complex strategies based on the architecture of the dendron, the stereochemistry, and the structure of the dipeptide.
Journal of the American Chemical Society 01/2006; 127(50):17902-9. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Polyfluorinated carbonic anhydrase inhibitors (CAIs) show very good inhibitory properties against carbonic anhydrase (CA) and excellent in vivo antiglaucoma properties after topical administration in rabbits. Still, the pentafluorinated compounds reported previously by this group (Scozzafava et al. J. Med. Chem. 2000, 43, 4542-4551) showed high reactivity with thiol groups of cysteine, glutathione, and presumably other proteins containing such moieties, which may lead to severe ocular side effects. Here, we report an approach for obtaining fluorinated CA inhibitors without the undesired enhanced reactivity. Thus, new compounds have been obtained by attaching moieties with reduced reactivity toward aromatic substitution reactions to the molecules of aromatic/heterocyclic sulfonamides possessing derivatizable amino moieties. The employed tails of the 2,3,5,6-tetrafluorobenzoyl, 2,3,5,6-tetrafluophenylsulfonyl, and pentafluorophenylureido types induced excellent CA inhibitory properties in the new reported sulfonamides, mainly against the isozymes involved in aqueous humor secretion, CA II and CA IV, whereas affinity for CA I was lower. Several low-nanomolar CA II inhibitors were detected, which did not react with cysteine or glutathione, in contrast to the corresponding perfluorinated compounds previously reported. These derivatives also showed a potent reduction of the intraocular pressure (IOP) in hypertensive rabbits, amounting to 13-21 mmHg at 1 h postadministration (compared to 5 mmHg obtained with dorzolamide, a clinically used drug), and the decreased IOP was maintained for 4-5 h after the administration. These compounds constitute valuable candidates for obtaining topically acting antiglaucoma CA inhibitors of a new generation, with enhanced efficacy, prolonged duration of action, and reduced side effects.
Journal of Medicinal Chemistry 06/2004; 47(11):2796-804. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Two series of halogenated sulfonamides have been prepared. The first consists of mono/dihalogenated sulfanilamides, whereas the second one consists of the mono/dihalogenated aminobenzolamides, incorporating equal or different halogens (F, Cl, Br, and I). These sulfonamides have been synthesized from the corresponding anilines by acetylation (protection of the amino group), chlorosulfonylation, followed either by amidation, or reaction with 5-amino-1,3,4-thiadiazole-2-sulfonamide (and eventually deacetylation). All these compounds, together with the six clinically used sulfonamide inhibitors (acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, and brinzolamide) were investigated as inhibitors of the transmembrane, tumor-associated isozyme carbonic anhydrase (CA) IX. Inhibition data against the classical, physiologically relevant isozymes I, II, and IV were also obtained. CA IX shows an inhibition profile which is generally completely different from those of isozymes I, II, and IV, with potent inhibitors (inhibition constants in the range of 12-40 nM) among both simple aromatic (such as 3-fluoro-5-chloro-4-aminobenzenesulfonamide) as well as heterocyclic compounds (such as acetazolamide, methazolamide, 5-amino-1,3,4-thiadiazole-2-sulfonamide, aminobenzolamide, and dihalogenated aminobenzolamides). This first detailed CA IX inhibition study revealed many interesting leads, suggesting the possibility to design even more potent and eventually CA IX-selective inhibitors, with putative applications as antitumor agents.
Journal of Medicinal Chemistry 06/2003; 46(11):2187-96. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Novel matrix metalloproteinase (MMP)/bacterial collagenase inhibitors are reported, considering the sulfonylated amino acid hydroxamates as lead molecules. A series of compounds was prepared by reaction of arylsulfonyl isocyanates with N-(5H-dibenzo[a,d]cyclohepten-5-yl)- and N-(10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5-yl) methyl glycocolate, respectively, followed by the conversion of the COOMe to the carboxylate/hydroxamate moieties. The corresponding derivatives with methylene and ethylene spacers between the polycyclic moiety and the amino acid functionality were also obtained by related synthetic strategies. These new compounds were assayed as inhibitors of MMP-1, MMP-2, MMP-8 and MMP-9, and of the collagenase isolated from Clostridium histolyticum (ChC). Some of the new derivatives reported here proved to be powerful inhibitors of the four MMPs mentioned above and of ChC, with activities in the low nanomolar range for some of the target enzymes, depending on the substitution pattern at the sulfonylureido moiety and on the length of the spacer through which the dibenzosuberenyl/suberyl group is connected with the rest of the molecule. Several of these inhibitors also showed selectivity for the deep pocket enzymes (MMP-2, MMP-8 and MMP-9) over the shallow pocket ones MMP-1 and ChC.
[show abstract][hide abstract] ABSTRACT: A series of tight binding carbonic anhydrase (CA) activators was obtained by reaction of amino-azoles (3-amino-pyrazole, 2-amino-imidazole, and 5-amino-tetrazole) with tri- or tetrasubstituted pyrylium salts. Many of the new pyridinium salts incorporating azole moieties reported here proved to be efficient in vitro activators of three CA isozymes, CA I, II, and IV. Very good activity was detected against hCA I and bCA IV (h = human; b = bovine isozymes), for which some of the new compounds showed affinities in the low nanomolar range, whereas against hCA II, their affinities were in the range of 95-150 nM. Substitution patterns of the pyridinium ring leading to best activity included 4-phenyl-2,6-dialkyl moieties or 2,4,6-tri- and 2,3,4,6-tetraalkyl groups. Ex vivo experiments showed some of the new activators to strongly enhance CA activity after incubation with human erythrocytes. Furthermore, due to their cationic nature, some of these compounds (the imidazole and pyrazole derivatives) are membrane-impermeant, discriminating thus between cytosolic and membrane-bound CA isozymes. The present paper is the first report of membrane-impermeant CA activators. The pyridinium tetrazole derivatives on the other hand do penetrate through biological membranes. Such CA activators might lead to the development of drugs/diagnostic tools for the management of CA deficiency syndromes as well as for the pharmacological enhancement of synaptic efficacy, spatial learning, and memory. This may constitute a new approach for the treatment of Alzheimer disease and other conditions in need of achieving memory therapy.
Journal of Medicinal Chemistry 02/2002; 45(2):504-10. · 5.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Important physiological and physio-pathological functions are played by several carbonic anhydrase (CA, EC 220.127.116.11) isozymes, which are strongly inhibited by aromatic and heterocyclic sulfonamides. Here we report several new types of such sulfonamides, incorporating furan-, thiophene- and pyrrole-carboxamide moieties in their molecules. Some of these compounds showed very good CA II and CA IV inhibitory properties, with affinities for the enzymes in the low nanomolar range. Due to their relatively low water solubility, some of the most active CA II inhibitors reported here have been formulated as aqueous suspension for topical administration as antiglaucoma agents, in normotensive and glaucomatous rabbits. The derivatives incorporating furan- and pyrrole-carboxamide moieties (but not the corresponding thiophene-substituted derivatives), showed effective and long-lasting intraocular pressure (IOP) lowering both in normotensive as well as glaucomatous animals, with potencies superior to dorzolamide and brinzolamide, the two available topically acting sulfonamide drugs. This is the first example of non-water soluble sulfonamides that significantly lower IOP, being thus similar with the recently introduced drug brinzolamide, which belongs to a completely different chemical family of antiglaucoma sulfonamides.