Molecular identification of β-citrylglutamate hydrolase as glutamate carboxypeptidase 3

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.     

Effect of varying chain length between P1 and P1′ position of tripeptidomimics on activity of angiotensin-converting enzyme inhibitors

One of the efficient modes of treatments of chronic hypertension and cardiovascular disorders has been to restrain the formation of angiotensin-II by inhibiting the action of angiotensin-converting enzyme (ACE) on angiotensin-I. The efforts continue towards achieving superior molecules or drugs with improved affinities, better bioavailability and thus to achieve long duration of action with minimum side effects. Previously, we reported a library of tripeptidomimics of Ornithyl–Proline (Orn–Pro) conjugated with various unnatural amino acids and carboxylic acid derived heterocyclics based on the SAR studies of existing ACE inhibitors. Their synthesis and screening for possible inhibitors of angiotensin-converting enzyme (ACE) revealed that increase in the backbone chain length by one carbon atom results in a sudden decrease in their activity. Therefore, in the present study heterocycles with different chain length were introduced to interact with the hydrophobic S₂ sub-site of ACE and screened for their in vitro ACE inhibition activity. Further, their binding interaction with C-domain of somatic ACE was also determined. Docking and consequent LUDI scores showed good correlation with binding of these molecules in the active site of ACE. Results suggest that heterocycles with C₃ chain length are more appropriate for the effective binding of the tripeptidomimics within the active site of ACE.     

Synthesis and activity of quinolinylmethyl P1′ α-sulfone piperidine hydroxamate inhibitors of TACE

A series of α-sulfone piperidine hydroxamate TACE inhibitors 11a–n bearing a quinolinyl methyl P1′ group was prepared, and their activity was compared to analogous α- and β-sulfone piperidine hydroxamates with a butynyloxy P1′ group. The quinolinyl methyl P1′ group affords increased inhibitory enzyme activity relative to the corresponding butynyloxy P1′ analogs in the α-sulfone piperidine hydroxamate series, and greater selectivity than the corresponding butynyloxy P1′ analogs in the β-sulfone piperidine hydroxamate series.     

Kinetic characterization of 4,4′-biphenylsulfonamides as selective non-zinc binding MMP inhibitors

Abstract

We describe the characterisation of a series of 4,4′-biphenylsulfonamides as selective inhibitors of matrix metalloproteases MMP-2 and -13, two enzymes involved in cell invasion and angiogenesis. Double-inhibitor studies in the presence of acetohydroxamic acid show that these molecules do not bind the catalytic zinc. Moreover, two of the characterised inhibitors (11 and 19) act as non-competitive inhibitors, whereas the para-methyl ester derivative 13 behaves as a competitive inhibitor. This finding suggests that this class of molecules binds to a catalytic subsite, possibly the S1′-pocket. Moreover, since these compounds also act as inhibitors of carbonic anhydrases (CAs), another family of enzymes involved in cell invasion, they could be potentially useful as CA/MMP dual target inhibitors with increased efficacy as anticancer agents.     

Thermodynamic and kinetic characterization of hydroxyethylamine β-secretase-1 inhibitors

Alzheimer’s disease (AD) is a devastating neurodegenerative disease affecting millions of people. β-Secretase-1 (BACE-1), an enzyme involved in the processing of the amyloid precursor protein (APP) to form Aβ, is a well validated target for AD. Herein, the authors characterize 10 randomly selected hydroxyethylamine (HEA) BACE-1 inhibitors in terms of their association and dissociation rate constants and thermodynamics of binding using surface plasmon resonance (SPR). Rate constants of association (k_a) measured at 25 °C ranged from a low of 2.42 × 10⁴ M⁻¹ s⁻¹ to the highest value of 8.3 × 10⁵ M⁻¹ s⁻¹. Rate constants of dissociation (k_d) ranged from 1.09 × 10⁻⁴ s⁻¹ (corresponding to a residence time of close to three hours), to the fastest of 0.028 s⁻¹. Three compounds were selected for further thermodynamic analysis where it was shown that equilibrium binding was enthalpy driven while unfavorable entropy of binding was observed. Structural analysis revealed that upon ligand binding, the BACE-1flap folds down over the bound ligand causing an induced fit. The maximal difference between alpha carbon positions in the open and closed conformations of the flap was over 5 Å. Thus the negative entropy of binding determined using SPR analysis was consistent with an induced fit observed by structural analysis.     

Bioisosterism of urea-based GCPII inhibitors: Synthesis and structure–activity relationship studies

We report a strategy based on bioisosterism to improve the physicochemical properties of existing hydrophilic, urea-based GCPII inhibitors. Comprehensive structure–activity relationship studies of the P1′ site of ZJ-43- and DCIBzL-based compounds identified several glutamate-free inhibitors with K_i values below 20 nM. Among them, compound 32d (K_i = 11 nM) exhibited selective uptake in GCPII-expressing tumors by SPECT-CT imaging in mice. A novel conformational change of amino acids in the S1′ pharmacophore pocket was observed in the X-ray crystal structure of GCPII complexed with 32d.     

3,4-Disubstituted benzofuran P1′ MMP-13 inhibitors: Optimization of selectivity and reduction of protein binding

Potent 3,4-disubstituted benzofuran P1′ MMP-13 inhibitors have been prepared. Selectivity over MMP-2 was achieved through a substituent at the C4 position of the benzofuran P1′ moiety of the molecule. By replacing a backbone benzene with a pyridine and valine with threonine, compounds (e.g., 44) with greatly reduced plasma protein binding were also obtained.     

Novel BACE1 inhibitors with a non-acidic heterocycle at the P1′ position

We have reported potent peptidic and non-peptidic BACE1 inhibitors with a hydroxymethylcarbonyl (HMC) isostere as a substrate transition-state mimic. However, our potent inhibitors possess a tetrazole ring at the P₁′ position. It is desirable that central nervous system (CNS) drugs do not possess an acidic moiety. In this study, we synthesized non-acidic BACE1 inhibitors with heterocyclic derivatives at the P₁′ position. KMI-1764 (27) exhibited potent inhibitory activity (IC₅₀ = 27 nM). Interestingly, these non-acidic inhibitors tended to follow the quantitative structure–activity relationship (QSAR) equation and interacted with BACE1-Arg235 in the binding model.     

Structural assessment and identification of 11β-hydroxysteroid dehydrogenase type 1 inhibitors

Communicated by Ramaswamy H. Sarma     

Improving the permeability of the hydroxyethylamine BACE-1 inhibitors: Structure–activity relationship of P2′ substituents

Herein we describe further evolution of hydroxyethylamine inhibitors of BACE-1 with enhanced permeability characteristics necessary for CNS penetration. Variation at the P2′ position of the inhibitor with more polar substituents led to compounds 19 and 32, which retained the potency of more lipophilic analog 1 but with much higher observed passive permeability in MDCK cellular assay.     

Design and biological evaluation of novel HIV-1 protease inhibitors with isopropanol as P1′ ligand to enhance binding with S1′ subsite

Newly designed HIV-1 protease inhibitors that maximize interactions with the protein backbone, especially in the form of hydrogen bonds, may enhance the antiviral potency of these compounds and minimize acquisition of drug-resistant mutations. Herein, we described a series of new HIV-1 PIs containing phenols as the P2 ligands and chiral isopropanol as the P1′ ligands, in combination with 4-trifluoromethylphenylsulfonamide or 4-nitrophenylsulfonamide as the P2′ ligands. And most of these compounds exhibited nanomolar inhibitory potency. In particular, inhibitors 13c and 13e with 4-trifluoromethylphenylsulfonamide as the P2′ ligand and (R) – isopropanol as the P1′ ligand, exhibited antiviral IC₅₀ values of 1.64 nM and 2.33 nM, respectively. Furthermore, they also showed remarkable activity against wild-type and DRV-resistant HIV-1 variants that raised the prospect of designing more effective PIs further.     

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.     

Structural characterization of cationic lipid–tRNA complexes

Despite considerable interest and investigations on cationic lipid–DNA complexes, reports on lipid–RNA interaction are very limited. In contrast to lipid–DNA complexes where lipid binding induces partial B to A and B to C conformational changes, lipid–tRNA complexation preserves tRNA folded state. This study is the first attempt to investigate the binding of cationic lipid with transfer RNA and the effect of lipid complexation on tRNA aggregation and condensation. We examine the interaction of tRNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant tRNA concentration and various lipid contents. FTIR, UV-visible, CD spectroscopic methods and atomic force microscopy (AFM) were used to analyze lipid binding site, the binding constant and the effects of lipid interaction on tRNA stability, conformation and condensation. Structural analysis showed lipid–tRNA interactions with G–C and A–U base pairs as well as the backbone phosphate group with overall binding constants of K_Chol = 5.94 (± 0.8) × 10⁴ M^–1, K_DDAB = 8.33 (± 0.90) × 10⁵ M^–1, K_DOTAP = 1.05 (± 0.30) × 10⁵ M^–1 and K_DOPE = 2.75 (± 0.50) × 10⁴ M^–1. The order of stability of lipid–tRNA complexation is DDAB > DOTAP > Chol > DOPE. Hydrophobic interactions between lipid aliphatic tails and tRNA were observed. RNA remains in A-family structure, while biopolymer aggregation and condensation occurred at high lipid concentrations.     

Design,synthesis, and X-ray structural studies of BACE-1 inhibitors containing substituted 2-oxopiperazines as P1′-P2′ ligands

We report the design and synthesis of a series of BACE1 inhibitors incorporating mono- and bicyclic 6-substituted 2-oxopiperazines as novel P1′ and P2′ ligands and isophthalamide derivative as P2-P3 ligands. Among mono-substituted 2-oxopiperazines, inhibitor 5a with N-benzyl-2-oxopiperazine and isophthalamide showed potent BACE1 inhibitory activity (K_i = 2 nM). Inhibitor 5g, with N-benzyl-2-oxopiperazine and substituted indole-derived P2-ligand showed a reduction in potency. The X-ray crystal structure of 5g-bound BACE1 was determined and used to design a set of disubstituted 2-oxopiperazines and bicyclic derivatives that were subsequently investigated. Inhibitor 6j with an oxazolidinone derivative showed a BACE1 inhibitory activity of 23 nM and cellular EC₅₀ of 80 nM.     

Novel Cu(II)-quinoline carboxamide complexes: Structural characterization, cytotoxicity and reactivity towards 5′-GMP

Three new ligands, N-(8-quinolyl)pyridine-2-carboxamide (HL₁), N-(8-quinolyl)glycine-N-Boc-carboxamide (HL₂), N-(8-quinolyl)-L-alanine-N-Boc-carboxamide (HL₃), and their Cu(II) complexes have been synthesized. Crystallographic data reveal that complex I, [Cu(L₁)(Ac)(H₂O)], is penta-coordinated with a square-pyramidal geometry while complexes V [Cu(L₂)(H₂O)] and VI [Cu(L₃)(H₂O)] are tetra-coordinated to give square planar geometry. In vitro tests showed that the Cu(II) complexes with L₁ (I-IV) exhibited cytotoxicity at a concentration of 10^–8 M against murine leukemia P-388 and human leukemia HL-60 cell lines, which is more potent than cisplatin. However, ligands HL₂ and HL₃ and their corresponding copper complexes demonstrated very weak in vitro activities towards the cell lines examined. ESMS data shows that complex I binds rapidly with 5-GMP to form 1:1 and 2:2 adduct.     

Discovery and characterization of synthetic 4′-hydroxyflavones—New CK2 inhibitors from flavone family

Human protein kinase CK2 is one of the most intriguing enzymes, which functional role still remains unclear despite of decades of studying. At present there is abundant evidence pointing to the fact that inhibitors of CK2 could be used as pharmaceutical agents to treat cancer, viral infections and inflammatory diseases. Here we report novel synthetic flavone inhibitors, 4′-hydroxyflavones, possessing high activity towards CK2. These compounds were identified with receptor-based virtual screening and then chemically optimized on the base of rationale derived from biochemical screening and molecular modeling. It has been demonstrated that synthetic flavone derivatives are much more potent CK2 inhibitors than the natural ones, and we believe that their further examination will be helpful for studying biological role of CK2 as well as for development of new kinase-oriented drugs.     

Functional characterization of 5′-regulatory region of flavonoid 3′,5′-hydroxylase-1 gene of banana plants

Protoplasma - Generation of crops with broad-spectrum tolerance to biotic and abiotic stress conditions depends upon availability of genetic elements suitable for varied situations and diverse...     

Structural characterization of cationic DODAB bilayers containing C24:1 β-glucosylceramide

The effect of 5 mol%, 9 mol%, and 16 mol% of C24:1 β-glucosylceramide (βGlcCer) on the structure of cationic DODAB bilayers was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR) spectroscopy and fluorescence microscopy. βGlcCer is completely miscible with DODAB at all fractions tested, since no domains were observed in fluorescence microscopy or ESR spectra. The latter showed that βGlcCer destabilized the gel phase of DODAB bilayers by decreasing the gel phase packing. As a consequence, βGlcCer induced a decrease in the phase transition temperature and cooperativity of DODAB bilayers, as seen in DSC thermograms. ESR spectra also showed that βGlcCer induced an increase in DODAB fluid phase order and/or rigidity. Despite their different structures, a similar effect of loosening the gel phase packing and turning the fluid phase more rigid/organized has also been observed when low molar fractions of cholesterol were incorporated in DODAB bilayers. The structural characterization of mixed membranes made of cationic lipids and glucosylceramides may be important for developing novel immunotherapeutic tools such as vaccine adjuvants.     

Design,synthesis, X-ray studies,and biological evaluation of novel macrocyclic HIV-1 protease inhibitors involving the P1′-P2′ ligands

Design, synthesis, and evaluation of a new class of HIV-1 protease inhibitors containing diverse flexible macrocyclic P1′-P2′ tethers are reported. Inhibitor 5a with a pyrrolidinone-derived macrocycle exhibited favorable enzyme inhibitory and antiviral activity (K_i = 13.2 nM, IC₅₀ = 22 nM). Further incorporation of heteroatoms in the macrocyclic skeleton provided macrocyclic inhibitors 5m and 5o. These compounds showed excellent HIV-1 protease inhibitory (K_i = 62 pM and 14 pM, respectively) and antiviral activity (IC₅₀ = 5.3 nM and 2.0 nM, respectively). Inhibitor 5o also remained highly potent against a DRV-resistant HIV-1 variant.     

Structural insights for producing CK2α1-specific inhibitors

Casein kinase 2 catalytic subunit (CK2α) is classified into two subtypes CK2α1 and CK2α2. CK2α1 is a drug discovery target, whereas CK2α2 is an off-target of CK2α1 inhibitors. High amino acid sequence homology between these subtypes hampers efforts to produce ATP competitive inhibitors that are highly selective to CK2α1. Hematein was identified previously as a non-ATP-competitive inhibitor for CK2α1, whereas this compound acts as an ATP competitive CK2α2 inhibitor. Crystal structures of CK2α1 and CK2α2 in complex with hematein revealed distinct binding features that provide structural insights for producing CK2α1-selective inhibitors.