C3′-endo-puckered pyrrolidine containing PNA has favorable geometry for RNA binding: Novel ethano locked PNA (ethano-PNA)

A novel peptide nucleic acid (PNA) analogue is designed with a constraint in the aminoethyl segment of the aegPNA backbone so that the dihedral angle β is restricted within 60–80°, compatible to form PNA:RNA duplexes. The designed monomer is further functionalized with positively charged amino-/guanidino-groups. The appropriately protected monomers were synthesized and incorporated into aegPNA oligomers at predetermined positions and their binding abilities with cDNA and RNA were investigated. A single incorporation of the modified PNA monomer into a 12-mer PNA sequence resulted in stronger binding with complementary RNA over cDNA. No significant changes in the CD signatures of the derived duplexes of modified PNA with complementary RNA were observed.     

Naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactones: antiproliferative activity and binding to bovine serum albumin and DNA

A naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactone (methyl 2-[7-hydroxy-2-oxonaphtho[1,2-b]furan-3(2H)-yliden]acetate) has been prepared as a representative compound of a potential class of cytotoxic agents. In vitro cytotoxicity has been evaluated against HCT-15 colon and MCF-7 breast cancer cells and IC(50) was 64-66 μM, causing morphological changes in cells, such as loss of adhesion, rounding, cell shrinkage, and detachment from the substratum. The binding constant K of the complex between the naphthyl lactone with bovine serum albumin (8 × 10(3) M(-1)) suggests a minor change in protein folding. The K of the binding with DNA (1.06 × 10(4) M(-1)) suggests nonspecific electrostatic interactions with DNA and this was confirmed by melting point data (Tm<0.6 °C). Therefore, naphthalene-fused (α-alkoxycarbonyl)methylene-γ-butyrolactone should not be able to intercalate with DNA but its interaction should occur at the level of DNA surface.     

Computational DNA binding studies of (–)-epigallocatechin-3-gallate

The catechin family of molecules that are present in the leaves of green tea has been under investigation since the antioxidant and anti-inflammatory properties of tea were discovered. Among multiple proposed therapeutic targets of these molecules, the direct interaction with nucleic acids has been proposed and experimentally observed but without clear knowledge about the potential binding modes between these ligands and DNA. One of these catechin structures, (–)-epigallocatechin gallate (EGCG), has three aromatic rings that could interact with double-stranded DNA via terminal base-pair stacking, intercalation, or through groove binding. Using enhanced sampling techniques and molecular dynamics simulations, we have found a stable complex between the EGCG ligand and DNA through intercalation of the trihydroxybenzoate aromatic ring and an ApC step. Moreover, we have calculated the absorption spectra of four possible binding modes and compared these to absorption profiles reported in the literature, and explored the possible DNA sequence preference for the EGCG ligand to bind. Our results suggest that an intercalative mode of interaction through the major groove is possible between the EGCG ligands and DNA with apparently very little DNA sequence selectivity.     

DNA binding by Ru(II)–bis(bipyridine)–pteridinyl complexes

The interactions of five bis(bipyridyl) Ru(II) complexes of pteridinyl-phenanthroline ligands with calf thymus DNA have been studied. The pteridinyl extensions were selected to provide hydrogen-bonding patterns complementary to the purine and pyrimidine bases of DNA and RNA. The study includes three new complexes [Ru(bpy)(2)(L-pterin)](2+), [Ru(bpy)(2)(L-amino)](2+), and [Ru(bpy)(2)(L-diamino)](2+) (bpy is 2,2'-bipyridine and L-pterin, L-amino, and L-diamino are phenanthroline fused to pterin, 4-aminopteridine, and 2,4-diaminopteridine), two previously reported complexes [Ru(bpy)(2)(L-allox)](2+) and [Ru(bpy)(2)(L-Me(2)allox)](2+) (L-allox and L-Me(2)allox are phenanthroline fused to alloxazine and 1,3-dimethyalloxazine), the well-known DNA intercalator [Ru(bpy)(2)(dppz)](2+) (dppz is dipyridophenazine), and the negative control [Ru(bpy)(3)](2+). Reported are the syntheses of the three new Ru-pteridinyl complexes and the results of calf thymus DNA binding experiments as probed by absorption and fluorescence spectroscopy, viscometry, and thermal denaturation titrations. All Ru-pteridine complexes bind to DNA via an intercalative mode of comparable strength. Two of these four complexes-[Ru(bpy)(2)(L-pterin)](2+) and [Ru(bpy)(2)(L-allox)](2+)-exhibit biphasic DNA melting curves interpreted as reflecting exceptionally stable surface binding. Three new complexes-[Ru(bpy)(2)(L-diamino)](2+), [Ru(bpy)(2)(L-amino)](2) and [Ru(bpy)(2)(L-pterin)](2+)-behave as DNA molecular "light switches."     

SAR analysis of innovative selective small molecule antagonists of sphingosine-1-phosphate 4 (S1P₄) receptor

Recent evidence suggests an innovative application of chemical modulators targeting the S1P(4) receptor as novel mechanism-based drugs for the treatment of influenza virus infection. Modulation of the S1P(4) receptor may also represent an alternative therapeutic approach for clinical conditions where reactive thrombocytosis is an undesired effect or increased megakaryopoiesis is required. With the exception of our recent research program disclosure, we are not aware of any selective S1P(4) antagonists reported in the literature to date. Herein, we describe complementary structure-activity relationships (SAR) of the high-throughput screening (HTS)-derived hit 5-(2,5-dichlorophenyl)-N-(2,6-dimethylphenyl)furan-2-carboxamide and its 2,5-dimethylphenyl analog. Systematic structural modifications of the furan ring showed that both steric and electronic factors in this region have a significant impact on the potency. The furan moiety was successfully replaced with a thiophene or phenyl ring maintaining potency in the low nanomolar range and high selectivity against the other S1P receptor subtypes. By expanding the molecular diversity within the hit-derived class, our SAR study provides innovative small molecule potent and selective S1P(4) antagonists suitable for in vivo pharmacological validation of the target receptor.     

Di-substituted pyridinyl aminohydantoins as potent and highly selective human β-secretase (BACE1) inhibitors

The identification of highly selective small molecule di-substituted pyridinyl aminohydantoins as β-secretase inhibitors is reported. The more potent and selective analogs demonstrate low nanomolar potency for the BACE1 enzyme as measured in a FRET assay, and exhibit comparable activity in a cell-based (ELISA) assay. In addition, these pyridine-aminohydantoins are highly selectivity (>500×) against the other structurally related aspartyl proteases BACE2, cathepsin D, pepsin and renin.Our design strategy followed a traditional SAR approach and was supported by molecular modeling studies based on the previously reported aminohydantoin 3a. We have taken advantage of the amino acid difference between the BACE1 and BACE2 at the S2′ pocket (BACE1 Pro₇₀ changed to BACE2 Lys₈₆) to build ligands with >500-fold selectivity against BACE2. The addition of large substituents on the targeted ligand at the vicinity of this aberration has generated a steric conflict between the ligand and these two proteins, thus impacting the ligand’s affinity and selectivity. These ligands have also shown an exceptional selectivity against cathepsin D (>5000-fold) as well as the other aspartyl proteases mentioned. One of the more potent compounds (S)-39 displayed an IC₅₀ value for BACE1 of 10 nM, and exhibited cellular activity with an EC₅₀ value of 130 nM in the ELISA assay.     

Calcium complexation with a highly calcium selective chelator: crystal structure of ca(CaFBAPTA) ·5H2O

The structure of the complex of calcium ions with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (FBAPTA) has been determined. The calcium complex, with stoichiometry Ca(CaFBAPTA) · 5H₂O crystallizes in a monoclinic space group P2₁/c with 4 formula units/unit cell with a(Å) = 9.767, b(Å) = 26.309,c(Å) = 11.769; β (deg) = 111.57. The structure may be considered as the calcium salt of a CaFBAPTA2- complex in which the octacoordinate calcium ion is complexed with the two ether oxygen atoms, the two nitrogens, and coordinated in a unidentate manner with the four acetate carboxyl groups. The geometry of the nitrogen atoms is nearly tetrahedral and provides a basis for the interpretation of the optical perturbations which result from calcium complexation with BAPTA type ligands. In addition, it is clear from the distance and relative orientation of the aromatic rings that ring current contributions to the fluorine chemical shifts are of negligible importance, so that such effects cannot be used in the design of NMR active indictators. Although BAPTA and analogs such as PBAPTA and quin2 have found extensive physiological applications as a consequence of the high degree of selectivity for calcium ions over magnesium ions which they exhibit, this is the first reported structural data for any of these complexes.     

β-PNA: peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.     

A selective inhibitor reveals PI3Kγ dependence of T(H)17 cell differentiation

We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.     

Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo? ) DNA polymerase

In our previous communication we reported the enzymatic recognition of unnatural imidazopyridopyrimidine:naphthyridine (Im:Na) base pairs, i.e. ImO^N:NaN^O and ImN^O:NaO^N, using the Klenow fragment exo⁻ [KF (exo⁻)]. We describe herein the successful results of (i) improved enzymatic recognition for ImN^O:NaO^N base pairs and (ii) further primer extension reactions after the Im:Na base pairs by Deep Vent DNA polymerase exo⁻ [Deep Vent (exo⁻)]. Since KF (exo⁻) did not catalyze primer extension reactions after the Im:Na base pair, we carried out a screening of DNA polymerases to promote the primer extension reaction as well as to improve the selectivity of base pair recognition. As a result, a family B DNA polymerase, especially Deep Vent (exo⁻), seemed most promising for this purpose. In the ImO^N:NaN^O base pair, incorporation of NaN^OTP against ImO^N in the template was preferable to that of the natural dNTPs, while incorporation of dATP as well as dGTP competed with that of ImO^NTP when NaN^O was placed in the template. Thus, the selectivity of base pair recognition by Deep Vent (exo⁻) was less than that by KF (exo⁻) in the case of the ImO^N:NaN^O base pair. On the other hand, incorporation of NaO^NTP against ImN^O in the template and that of ImN^OTP against NaO^N were both quite selective. Thus, the selectivity of base pair recognition was improved by Deep Vent (exo⁻) in the ImN^O:NaO^N base pair. Moreover, this enzyme catalyzed further primer extension reactions after the ImN^O:NaO^N base pair to afford a faithful replicate, which was confirmed by MALDI-TOF mass spectrometry as well as the kinetics data for extension fidelity next to the ImN^O:NaO^N base pair. The results presented in this paper revealed that the ImN^O:NaO^N base pair might be a third base pair beyond the Watson–Crick base pairs.     

A comprehensive study on the putative δ-opioid receptor (sub)types using the highly selective δ-antagonist, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH

The goal of our work was a throughout characterization of the pharmacology of the TIPP-analog, Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH and see if putative δ-opioid receptor subtypes can be distinguished. Analgesic latencies were assessed in mouse tail-flick assays after intrathecal administration. In vitro receptor autoradiography, binding and ligand-stimulated [³⁵S]GTPγS functional assays were performed in the presence of putative δ₁-(DPDPE: agonist, BNTX: antagonist), δ₂-(agonist: deltorphin II, Ile^5,6-deltorphin II, antagonist: naltriben) and μ-(DAMGO: agonist) opioid ligands. The examined antagonist inhibited the effect of DPDPE by 60%, but did not antagonize δ₂- and μ-agonist induced analgesia. The radiolabeled form identified binding sites with K_D = 0.18 nM and receptor densities of 102.7 fmol/mg protein in mouse brain membranes. The binding site distribution of the [³H]Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH agreed well with that of [³H]Ile^5,6-deltorphin II as revealed by receptor autoradiography. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH displayed 2.49 ± 0.06 and 0.30 ± 0.01 nM potency against DPDPE and deltorphin II in the [³⁵S]GTPγS functional assay, respectively. The rank order of potency of putative δ₁- and δ₂-antagonists against DPDPE and deltorphin was similar in brain and CHO cells expressing human δ-opioid receptors. Deletion of the DOR-1 gene resulted in no residual binding of the radioligand and no significant DPDPE effect on G-protein activation. Tyr-Tic-(2S,3R)-β-MePhe-Phe-OH is a highly potent and δ-opioid specific antagonist both in vivo and in vitro. However, the putative δ₁- and δ₂-opioid receptors could not be unequivocally distinguished in vitro.     

(Arene)Cl₂Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: interaction with sex hormone binding globulin and anticancer activity

(Arene)dichloridoruthenium(II) complexes with N-coordinated isonicotinates of androgens (6) and estrogens (9) were prepared and tested for affinity to the estrogen receptor (ERα) and sex hormone binding globulin (SHBG), as well as for cytotoxicity in cancer cells. None of the new complexes bound noticeably to the ER and most of them also bound less strongly to SHBG than the corresponding unmetallated steroids 7. In MTT assays the Ru(p-cymene) complexes 9 of 2-substituted estrones were equally or even more cytotoxic than the metal-free steroids against hormone-dependent (MCF-7 breast and KB-V1 cervix carcinomas) and hormone-independent (518A2 melanoma) cells. The addition of external SHBG to MTT assays lowered the cytotoxicities of the complexes 9 and distinctly more so those of some steroids 7, probably by the way of sequestration and reduction of the cellular uptake. In the absence of SHBG the estrogen complexes 9 were internalized by 518A2 melanoma cells and ruthenated their DNA as quantified by ICP-OES. They also ruthenated salmon sperm DNA but did not change the topology of plasmid DNA in EMSA experiments. In addition, the Ru(p-cymene) complex of 2-ethoxyestrone (9c) was shown to reduce the motility of 518A2 melanoma cells in a wound-healing assay.     

Analgesic activity of intracerebroventricular administration of morphiceptin and β-casomorphins: Correlation with the morphine (μ) receptor binding affinity

Analgesic activities of morphiceptin, β-casomorphins, [D-Ala², D-Leu⁵] enkephalin and Sandoz peptide, FK 33–824, were examined by intracerebroventricular administration in rats. Their relative potencies $\text{in vivo}$ were compared with their receptor binding activities. The receptor binding affinities were determined from the competition curves against [³H]naloxone binding in the absence and presence of sodium ions for morphine (μ) receptors and against ¹²⁵I-[D-Ala², D-Leu⁵] enkephalin binding for enkephalin (δ) receptors. A good correlation between analgesic activity and morphine (μ) receptor but not enkephin (δ) receptor binding affinity was obtained. These data extend the hypothesis that morphine (δ) receptors mediate the major portion of the analgesic activity of opioids.     

Peptide binding characteristics of the coeliac disease-associated DQ(α1*0501, β1*0201) molecule

Genetic susceptibility to coeliac disease (CD) is strongly associated with the expression of theHLA-DQ2 (α1^*0501, β1^*0201) allele. There is evidence that this DQ2 molecule plays a role in the pathogenesis of CD as a restriction element for gliadin-specific T cells in the gut. However, it remains largely unclear which fragments of gliadin can actually be presented by the disease-associated DQ dimer. With a view to identifying possible CD-inducing antigens, we studied the peptide binding properties of DQ2. For this purpose, peptides bound to HLA-DQ2 were isolated and characterized. Dominant peptides were found to be derived from two self-proteins: in addition to several sizevariants of the invariant chain (li)-derived CLIP peptide, a relatively large amount of an major histocompatibility complex (MHC) class I-derived peptide was found. Analogues of this naturally processed epitope (MHClα46–63) were tested in a cell-free peptide binding competition assay to investigate the requirements for binding to DQ2. First, a core sequence of 10 amino acids within the MHClα46–63 peptide was identified. By subsequent single amino acid substitution analysis of this core sequence, five putative anchor residues were identified at relative positions P1, P4, P6, P7, and P9. Replacement by the large, positively charged Lys at these positions resulted in a dramatic loss of binding. However, several other non-conservative substitutions had little or no discernable effect on the binding capacity of the peptides. Substitutions at P1 and P4 were most critical, suggesting a more prominent role as anchor residues. Structural features of the DQ2 molecule that may relate to the binding motif and to gluten sensitivity are discussed.     

A new class of non-thiazolidinedione, non-carboxylic-acid-based highly selective peroxisome proliferator-activated receptor (PPAR) γ agonists: design and synthesis of benzylpyrazole acylsulfonamides

Herein, we describe the design, synthesis, and structure-activity relationships of novel benzylpyrazole acylsulfonamides as non-thiazolidinedione (TZD), non-carboxylic-acid-based peroxisome proliferator-activated receptor (PPAR) γ agonists. Docking model analysis of in-house weak agonist 2 bound to the reported PPARγ ligand binding domain suggested that modification of the carboxylic acid of 2 would help strengthen the interaction of 2 with the TZD pocket and afford non-carboxylic-acid-based agonists. In this study, we used an acylsulfonamide group as the ring-opening analog of TZD as an isosteric replacement of carboxylic acid moiety of 2; further, preliminary modification of the terminal alkyl chain on the sulfonyl group gave the lead compound 3c. Subsequent optimization of the resulting compound gave the potent agonists 25c, 30b, and 30c with high metabolic stability and significant antidiabetic activity. Further, we have described the difference in binding mode of the carboxylic-acid-based agonist 1 and acylsulfonamide 3d.     

D21S210: A highly polymorphic (GT)n marker closely linked to the β-amyloid protein precursor (APP) gene

We describe a highly polymorphic (GT)n repeat with 14 alleles that is closely linked to the amyloid precursor protein (APP) gene on human chromosome 21. This marker, D21S210, will be useful for studies of linkage of disorders such as Alzheimer disease to the APP gene.