Exploration and optimization of substituted triazolothiadiazines and triazolopyridazines as PDE4 inhibitors

An expansion of structure–activity studies on a series of substituted 7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine PDE4 inhibitors and the introduction of a related [1,2,4]triazolo[4,3-b]pyridazine based inhibitor of PDE4 is presented. The development of SAR included strategic incorporation of known substituents on the critical catachol diether moiety of the 6-phenyl appendage on each heterocyclic core. From these studies, (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (10) and (R)-3-(2,5-dimethoxyphenyl)-6-(4-methoxy-3-(tetrahydrofuran-3-yloxy)phenyl)-[1,2,4]triazolo[4,3-b]pyridazine (18) were identified as highly potent PDE4A inhibitors. Each of these analogues was submitted across a panel of 21 PDE family members and was shown to be highly selective for PDE4 isoforms (PDE4A, PDE4B, PDE4C, PDE4D). Both 10 and 18 were then evaluated in divergent cell-based assays to assess their relevant use as probes of PDE4 activity. Finally, docking studies with selective ligands (including 10 and 18) were undertaken to better understand this chemotypes ability to bind and inhibit PDE4 selectively.     

Analysis of gene-derived SNP marker polymorphism in US wheat (Triticum aestivum L.) cultivars

In this study, we developed 359 detection primers for single nucleotide polymorphisms (SNPs) previously discovered within intron sequences of wheat genes and used them to evaluate SNP polymorphism in common wheat (Triticum aestivum L.). These SNPs showed an average polymorphism information content (PIC) of 0.18 among 20 US elite wheat cultivars, representing seven market classes. This value increased to 0.23 when SNPs were pre-selected for polymorphisms among a diverse set of 13 hexaploid wheat accessions (excluding synthetic wheats) used in the wheat SNP discovery project (). PIC values for SNP markers in the D genome were approximately half of those for the A and B genomes. D genome SNPs also showed a larger PIC reduction relative to the other genomes (P < 0.05) when US cultivars were compared with the more diverse set of 13 wheat accessions. Within those accessions, D genome SNPs show a higher proportion of alleles with low minor allele frequencies (<0.125) than found in the other two genomes. These data suggest that the reduction of PIC values in the D genome was caused by differential loss of low frequency alleles during the population size bottleneck that accompanied the development of modern commercial cultivars. Additional SNP discovery efforts targeted to the D genome in elite wheat germplasm will likely be required to offset the lower diversity of this genome. With increasing SNP discovery projects and the development of high-throughput SNP assay technologies, it is anticipated that SNP markers will play an increasingly important role in wheat genetics and breeding applications. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Structure and dynamic behaviour of lanthanide nitrate complexes with bis(diphenylphosphorylmethyl)mesitylene in solutions: The nature of unexpected P NMR spectra

The solution structures of the lanthanide complexes, [Ln(L)(NO₃)₃] and [Ln(L)₂(NO₃)₃], where L = bis(diphenylphosphorylmethyl)mesitylene and Ln = La, Ce, Nd, Er, were investigated by ³¹P NMR and IR spectroscopy, conductivity and sedimentation analysis. Variable-temperature ³¹P{¹H} NMR spectroscopy was used to identify species present in solution and to monitor their interconversions. The results indicate that equilibrium between molecular complexes [Ln(L)_n(NO₃)₃]⁰ and cationic species (as ion pairs [Ln(L)_n(NO₃)₂]⁺ · (NO₃)⁻ and as free ions [Ln(L)_n(NO₃)₂]⁺, throughout n = 1, 2) in solutions can be observed by ³¹P{¹H} NMR spectroscopy due to separate detection of the molecular complexes and cationic species. The chelate coordination of the ligand and nitrate ions is retained in all complex species at ambient temperature except for [Er(L)₂(NO₃)₃]. The crystal structure of [Nd(L)(NO₃)₃(MeCN)]MeCN was determined by X-ray diffraction.     

A new side opening on prolyl oligopeptidase revealed by electron microscopy

Prolyl oligopeptidase (POP) has gained importance as a target for the treatment of neuropsychiatric diseases and cognitive disturbances. Therefore, a variety of strategies are currently used to identify POP inhibitors. Here we performed electron microscopy (EM) studies of human POP. Our data reveal for the first time the presence of a new side opening in POP that was not observed in any of the crystallographic structures described to date. Finally, molecular dynamics, the relevant normal modes that contribute to the fluctuation of the catalytic triad residues and the algorithm CAVERN also support the existence of a new large side opening on POP.     

The Superagonistic Activity of Bovine Thyroid-stimulating Hormone (TSH) and the Human TR1401 TSH Analog Is Determined by Specific Amino Acids in the Hinge Region of the Human TSH Receptor

Bovine TSH (bTSH) has a higher affinity to the human TSHR (hTSHR) and a higher signaling activity than human TSH (hTSH). The molecular reasons for these phenomena are unknown. Distinct negatively charged residues (Glu²⁹⁷, Glu³⁰³, and Asp³⁸²) in the hinge region of the hTSHR are known to be important for bTSH binding and signaling. To investigate the potential relevance of these positions for differences between bTSH and hTSH in the interaction to the hTSHR, we determined bTSH- and hTSH-mediated cAMP production of several substitutions at these three hinge residues. To examine specific variations of hTSH, we also investigated the superagonistic hTSH analog TR1401 (TR1401), whose sequence differs from hTSH by four additional positively charged amino acids that are also present in bTSH. To characterize possible interactions between the acidic hTSHR positions Glu²⁹⁷, Glu³⁰³, or Asp³⁸² and the additional basic residues of TR1401, we investigated TR1401 binding and signaling properties. Our data reveal increased cAMP signaling of the hTSHR using TR1401 and bTSH compared with hTSH. Whereas Asp³⁸² seems to be important for bTSH- and TR1401-mediated but not for hTSH-mediated signaling, the substitution E297K exhibits a decreased signaling for all three TSH variants. Interestingly, bTSH and TR1401 showed only a slightly different binding pattern. These observations imply that specific residues of the hinge region are mediators of the superagonistic activity of bTSH and TR1401 in contrast to hTSH. Moreover, the simultaneous localization of binding components in the glycoprotein hormone molecule and the receptor hinge region permits important reevaluation of interacting hormone receptor domains.It is well known that bovine TSH (bTSH)² has a higher affinity to the human TSHR (hTSHR) and a 6–10-fold higher intrinsic signaling activity than human TSH (hTSH) (1–5). Human TSH and bTSH share high amino acid sequence identity in the α-subunit (74.1%) and β-subunit (88.4%) (6). Studies involving fusion of hTSH and bTSH α- and β-subunits indicate that the higher affinity and the superagonistic cAMP activity of bTSH at the hTSHR depend primarily on amino acid sequences of the bTSH α-subunit (6). The most noticeable sequence differences between bovine and human TSH consist of four positively charged residues located in the surface-exposed loops of the α-subunit and one positively charged residue in the β-subunit of bTSH (Fig. 1). Moreover, it has previously been shown that positively charged α loop 1 (α-L1) residues are important for the high bioactivity of bTSH, and they have been implicated in receptor binding. These specific characteristics led to the generation of superagonistic hTSH analogs (6). The human TSH analog TR1401 and bTSH differ from hTSH most importantly by four additional positively charged amino acids located in close spatial proximity at the α-L1, of which three are located at identical positions in bTSH and TR1401 (Fig. 1).Open in a separate windowFIGURE 1.Sequence differences between TSH variants used in the present study. A, alignment of the α- and β-subunit of the hTSH (SwissProt: GLHA_HUMAN {"type":"entrez-protein","attrs":{"text":"P01215","term_id":"121312","term_text":"P01215"}}P01215, TSHB_HUMAN {"type":"entrez-protein","attrs":{"text":"P01222","term_id":"311033515","term_text":"P01222"}}P01222), bTSH (GLHA_BOVIN {"type":"entrez-protein","attrs":{"text":"P01217","term_id":"121310","term_text":"P01217"}}P01217, TSHB_BOVIN {"type":"entrez-protein","attrs":{"text":"P01223","term_id":"136442","term_text":"P01223"}}P01223), and the superagonistic hTSH analog TR1401. The additional positively charged residues at TR1401 and at bTSH compared with wt hTSH are boxed in blue. Sequence numbering for human TSH and human analog TR1401 without signal peptide is shown in blue. B, three-dimensional structural TSH models illustrating the spatial localization of the charge related sequence differences between the TSH variants. The TSH α-subunit is shown in gray, and the β-subunit is in orange. Positively charged residues are highlighted in blue, and the C-α atoms of additional positively charged residues compared with hTSH are highlighted by blue globes. Panel i, bovine TSH, characterized by four additional positively charged residues in the α-L1 (T11K, Q13K, P16K, and Q20K) and one positively charged residue in the β-L3 (L69R); panel ii, human TSH without positively charged residues in the α-L1 and β-L3; and panel iii, the human TSH analog TR1401 is characterized by four additional positively charged residues in the α-L1 (Q13K, E14K, P16K, and Q20K) but shows a lack of the additional positively charged residue in the β-L3.TSH binds to the large extracellular region of its receptor. The extracellular region of the TSHR consists of the leucine-rich repeat domain (LRRD), which is linked with the membrane-spanning serpentine domain by the hinge region. Recently, the binding arrangements between the homologous FSH and a part of the FSH receptor ectodomain including the LRRD (FSH receptor amino acids Cys¹⁸–Ala²⁴⁶) have been identified (7). However, the hinge region is not contained in this x-ray structure (7).In vitro data provide convincing evidence for the functional importance of the hinge region for receptor activation and TSH binding (8–22). Recently, we specified positions Glu²⁹⁷ and Glu³⁰³ in the N-terminal portion and Asp³⁸² in the C-terminal portion of the hTSHR hinge region as important for bTSH binding, suggesting that in the process of bTSH binding an extended hormone-binding site is obviously essential (18). The negative charge of positions Glu²⁹⁷ and Asp³⁸² likely interact with positively charged residues of bTSH by complementary charge-charge interaction (18).To elucidate whether these hinge residues of the hTSHR are specific for interaction with bTSH, we investigated the functional characteristics of the hTSH analog TR1401 and the native ligand hTSH. For the comparison of these two TSH variants with bTSH, we used several mutations and alanine combinations at the signaling and bTSH binding-sensitive hTSHR hinge positions Glu²⁹⁷, Glu³⁰³, and Asp³⁸². Our data indicate that the higher bioactivity of the TSH variants TR1401 and bTSH are mediated by specific charged residues of the hormone and the hinge region of the hTSHR. Our findings also support the concept that the hinge region of the TSHR is an modulator of TSH potency and efficacy.     

Diversity of frankiae in soils from five continents

Clone libraries of nifH gene fragments specific for the nitrogen-fixing actinomycete Frankia were generated from six soils obtained from five continents using a nested PCR. Comparative sequence analyses of all libraries (n=247 clones) using 96 to 97% similarity thresholds revealed the presence of three and four clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively. Diversity of frankiae was represented by fewer clusters (i.e., up to four in total) within individual libraries, with one cluster generally harboring the vast majority of sequences. Meta-analysis including sequences previously published for cultures (n=48) and for uncultured frankiae in root nodules of Morella pensylvanica formed in bioassays with the respective soils (n=121) revealed a higher overall diversity with four and six clusters of frankiae representing the Elaeagnus and the Alnus host infection groups, respectively, and displayed large differences in cluster assignments between sequences retrieved from clone libraries and those obtained from nodules, with assignments to the same cluster only rarely encountered for individual soils. These results demonstrate large differences between detectable Frankia populations in soil and those in root nodules indicating the inadequacy of bioassays for the analysis of frankiae in soil and the role of plants in the selection of frankiae from soil for root nodule formation.     

Can antimicrobial peptides scavenge around a cell in less than a second?

Antimicrobial peptides, which play multiple host-defense roles, have garnered increased experimental focus because of their potential applications in the pharmaceutical and food production industries. While their mechanisms of action are richly debated, models that have been advanced share modes of peptide-lipid interactions that require peptide dynamics. Before the highly cooperative and specific events suggested in these models take place, peptides must undergo an important process of migration along the membrane surface and delivery from their site of binding on the membrane to the actual site of functional performance. This phenomenon, which contributes significantly to antimicrobial function, is poorly understood, largely due to a lack of experimental and computational tools needed to assess it. Here, we use ¹⁵N solid-state nuclear magnetic resonance to obtain molecular level data on the motions of piscidin's amphipathic helices on the surface of phospholipid bilayers. The studies presented here may help contribute to a better understanding of the speed at which the events that lead to antimicrobial response take place. Specifically, from the perspective of the kinetics of cellular processes, we discuss the possibility that piscidins and perhaps many other amphipathic antimicrobial peptides active on the membrane surface may represent a class of fast scavengers rather than static polypeptides attached to the water-lipid interface.     

Synthesis, characterization and in vitro cytotoxicity studies of platinum(IV) complexes with thiouracil ligands

Reactions of [PtMe₃(bpy)(Me₂CO)][BF₄] (2) with the thionucleobases 2-thiouracil (s²Ura), 4-thiouracil (s⁴Ura) and 2,4-dithiouracil (s²s⁴Ura) resulted in the formation of complexes of the type [PtMe₃(bpy)(L-κS)][BF₄] (L = s²Ura, 3; s⁴Ura, 4; s²s⁴Ura, 5). The complexes were characterized by NMR spectroscopy (¹H, ¹³C, ¹⁹⁵Pt), IR spectroscopy as well as microanalyses. The coordination through the C4S groups (4, 5) was additionally confirmed by DFT calculations, where it was shown that these complexes [PtMe₃(bpy)(L-κS⁴)]⁺ (L = s⁴Ura, s²s⁴Ura) are about 5.8 (4b) and 3.3 kcal/mol (5b), respectively, more stable than the respective complexes, having thiouracil ligands bound through the C2X groups (X = O, 4a; S, 5a). For [PtMe₃(bpy)(s²Ura-κS²)][BF₄] (3) no preferred coordination mode could be assigned solely based on DFT calculations. Analysis of NMR spectra showed the κS² coordination. In vitro cytotoxic studies of complexes 3−5 on nine different cell lines (8505C, A253, FaDu, A431, A549, A2780, DLD-1, HCT-8, HT-29) revealed in most cases moderate activities. However, 3 and 5 showed significant activity towards A549 and A2780, respectively, possessing IC₅₀ values comparable to those of cisplatin. Cell cycle perturbations and trypan blue exclusion test on cancer cell line A431 using [PtMe₃(bpy)(s²s⁴Ura-κS⁴)][BF₄] (5) showed induction of apoptotic cell death. Furthermore, the reaction of [PtMe₃(OAc-κ²O,O′)(Me₂CO)] (6) with 4-thiouracil yielded the dinuclear complex [(PtMe₃)₂(μ-s⁴Ura_-H)₂] (7), which has been characterized by microanalysis, NMR (¹H, ¹³C, ¹⁹⁵Pt) and IR spectroscopy as well as ESI mass spectrometry. X-ray diffraction analysis of crystals yielded in an isolated case exhibited the presence of a hexanuclear thiouracilato platinum(IV) complex, possessing each three different kinds of methyl platinum(IV) moieties and 4-thiouracilato ligands. This exhibited the ability of 4-thiouracil platinum(IV) complexes to form multinuclear complexes.     

Population structures of genebank accessions of Salvia officinalis L. (Lamiaceae) revealed by high resolution melting analysis

The genetic structure of 19 accessions of Salvia officinalis from the genebank Gatersleben (Germany) was analyzed with SNP and SSR markers developed from EST sequences of Salvia fruticosa. Analyzes were performed exclusively with HRM (high resolution melt analysis), the alleles were identified applying a beforehand developed strategy for codominant markers. Nine polymorphic markers (PIC between 0.12 and 0.61) were used for a complete allele scan in the sample set of 190 individuals from 19 accessions. AMOVA stated 51% of variance between the populations and 49% within. Principal component analysis showed a main bulk of closely related accessions, which have substantial heterogeneity within themselves. However, some populations, especially two from Romania, were clearly distant. This is in accordance to phytochemical results of the same sample set, where the Romanian populations were distant due to an exotic chemotype of the essential oil. Cluster analysis revealed connections between groups of accessions in detail.