蛋白质侧链的预测

根据Ｄｅｓｍｅｔ的死端排除定则制作了一种蛋白质侧链观测的快速软件系统,用一批实例结构数据和不同的侧链转子来检验上定则的可行性;并讨论预测的ＲＭＳ与溶剂可及性和结构元素的关系。     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

New lead discovery of insect growth regulators based on the scaffold hopping strategy

Insect growth regulators (IGRs), which can interrupt or inhibit pest life cycles, are low-toxicity pesticides widely used in integrated pest management (IPM). Ecdysone analogues and chitinase inhibitors are familiar IGRs that have attracted considerable attention because of their unique modes of action and low toxicity to non-target organisms. To find new and highly effective candidate IGRs with novel mechanisms, D-08 (N-(4-(tert-butyl)phenyl)-2-phenyl-2,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) was chosen as a lead compound, and a series of novel heptacyclic pyrazolamide derivatives were designed and synthesized using the scaffold hopping strategy. The bioassay showed that III-27 (N-(2-methylphenethyl)-1-phenyl-1,4,5,6,7,8-hexahydrocyclohepta[c]pyrazole-5-carboxamide) had excellent activity against Plutella xylostella. Protein verification and molecular docking indicated that III-27 could act on both the ecdysone receptor (EcR) and Ostrinia furnacalis chitinase (Of ChtI) and is a promising new lead IGRs. The interaction mechanism of III-27 with EcR and Of ChtI was then studied by molecular docking. These results provide important guidance for the study of new dual-target IGRs.     

Molecular Dynamics with Nonadiabatic Transitions: A Comparison of Methods

Abstract

Surface hopping (SH) and density matrix evolution (DME) methods which simulate the dynamics of quantum systems embedded in a classical environments are compared with exact quantum-dynamical calculations. These methods are applied to study the inelastic collisions of a classical particle with a five-level quantum harmonic oscillator. One-dimensional, two-state models representing electronic transitions are also treated. In addition, the methods are applied to the dynamics of a proton in a bistable potential bilinearly coupled to the bath of classical harmonic oscillators. Vibrational spectra calculated by both methods compare well with each other. The SH results are, in general, closer to the results of a full quantum treatment than the corresponding DME values. The DME method breaks down in the case of extended coupling with reflection at low energies.     

Imidazo[4,5-b]pyridine inhibitors of B-Raf kinase

This Letter details the synthesis and evaluation of imidazo[4,5-b]pyridines as inhibitors of B-Raf kinase. These compounds bind in a DFG-in, αC-helix out conformation of B-Raf, which is a binding mode associated with significant kinase selectivity. Structure–activity relationship studies involved optimization of the ATP-cleft binding region of these molecules, and led to compound 23, an inhibitor with excellent enzyme/cell potency, and kinase selectivity.     

NCK is critical for the development of deleted in colorectal cancer (DCC) sensitive spinal circuits

As our understanding of motor circuit function increases, our need to understand how circuits form to ensure proper function becomes increasingly important. Recently, deleted in colorectal cancer (DCC) has been shown to be important in the development of spinal circuits necessary for gait. Importantly, humans with mutation in DCC show mirror movement disorders pointing to the significance of DCC in the development of spinal circuits for coordinated movement. Although DCC binds a number of ligands, the intracellular signaling cascade leading to the aberrant spinal circuits remains unknown. Here, we show that the non‐catalytic region of tyrosine kinase adaptor (NCK) proteins 1 and 2 are distributed in the developing spinal cord. Using dissociated dorsal spinal neuron cultures we show that NCK proteins are necessary for the outgrowth and growth cone architecture of DCC^+ve dorsal spinal neurons. Consistent with a role for NCK in DCC signaling, we show that loss of NCK proteins leads to a reduction in the thickness of TAG1^+ve commissural bundles in the floor plate and loss of DCC mRNA in vivo. We suggest that DCC signaling functions through NCK1 and NCK2 and that both proteins are necessary for the establishment of normal spinal circuits necessary for gait.

     

Local hopping mobile DNA implicated in pseudogene formation and reductive evolution in an obligate cyanobacteria-plant symbiosis

Background

Insertion sequences (ISs) are approximately 1 kbp long “jumping” genes found in prokaryotes. ISs encode the protein Transposase, which facilitates the excision and reinsertion of ISs in genomes, making these sequences a type of class I (“cut-and-paste”) Mobile Genetic Elements. ISs are proposed to be involved in the reductive evolution of symbiotic prokaryotes. Our previous sequencing of the genome of the cyanobacterium ‘Nostoc azollae’ 0708, living in a tight perpetual symbiotic association with a plant (the water fern Azolla), revealed the presence of an eroding genome, with a high number of insertion sequences (ISs) together with an unprecedented large proportion of pseudogenes. To investigate the role of ISs in the reductive evolution of ‘Nostoc azollae’ 0708, and potentially in the formation of pseudogenes, a bioinformatic investigation of the IS identities and positions in 47 cyanobacterial genomes was conducted. To widen the scope, the IS contents were analysed qualitatively and quantitatively in 20 other genomes representing both free-living and symbiotic bacteria.

Results

Insertion Sequences were not randomly distributed in the bacterial genomes and were found to transpose short distances from their original location (“local hopping”) and pseudogenes were enriched in the vicinity of IS elements. In general, symbiotic organisms showed higher densities of IS elements and pseudogenes than non-symbiotic bacteria. A total of 1108 distinct repeated sequences over 500 bp were identified in the 67 genomes investigated. In the genome of ‘Nostoc azollae’ 0708, IS elements were apparent at 970 locations (14.3%), with 428 being full-length. Morphologically complex cyanobacteria with large genomes showed higher frequencies of IS elements, irrespective of life style.

Conclusions

The apparent co-location of IS elements and pseudogenes found in prokaryotic genomes implies earlier IS transpositions into genes. As transpositions tend to be local rather than genome wide this likely explains the proximity between IS elements and pseudogenes. These findings suggest that ISs facilitate the reductive evolution in for instance in the symbiotic cyanobacterium ‘Nostoc azollae’ 0708 and in other obligate prokaryotic symbionts.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1386-7) contains supplementary material, which is available to authorized users.     

Structure based evolution of a novel series of positive modulators of the AMPA receptor

Starting from compound 1, we utilized biostructural data to successfully evolve an existing series into a new chemotype with a promising overall profile, exemplified by 19.     

ParaFrag—an approach for surface-based similarity comparison of molecular fragments

A frequent task in computer-aided drug design is to identify novel chemotypes similar in activity but structurally different to a given reference structure. Here we report the development of a novel method for atom-independent similarity comparison of molecular fragments (substructures of drug-like molecules). The fragments are characterized by their local surface properties coded in the form of 3D pharmacophores. As surface properties, we used the electrostatic potential (MEP), the local ionization energy (IE_L), local electron affinity (EA_L) and local polarizability (POL) calculated on isodensity surfaces. A molecular fragment can then be represented by a minimal set of extremes for each surface property. We defined a tolerance sphere for each of these extremes, thus allowing us to assess the similarity of fragments in an analogous manner to classical pharmacophore comparison. As a first application of this method we focused on comparing rigid fragments suitable for scaffold hopping. A retrospective analysis of successful scaffold hopping reported for Factor Xa inhibitors [Wood MR et al (2006) J Med Chem 49:1231] showed that our method performs well where atom-based similarity metrics fail. Figure Encoding surface hotspots as a ParaFrag pharmacophore     

Discovery of EBI-1051: A novel and orally efficacious MEK inhibitor with benzofuran scaffold

A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.