Since the completion of the human genome, there has been much interest in the ‘‘druggability’’ of new potential drug targets, and what fraction of the proteome is druggable. In this paper we are concerned with protein druggability in the sense defined by Hopkins and Groom , i.e., the ability of a protein to bind small, drug-like molecules with high affinity. For many classes of protein binding sites, such as the ATP binding sites in kinases, there is little ambiguity about whether the site is druggable; the challenge in developing inhibitors in such cases is achieving selectivity and other desired properties. However, not all biological targets are druggable since only certain binding sites are complementary to drug-like compounds in terms of physicochemical properties (i.e. size, shape, polar interactions and hydrophobicity) [1,2]. An accurate method for predicting druggability would be particularly valuable for assessing emerging classes of binding sites such as protein-protein interactions (PPI)  and allosteric sites , which are generally considered more challenging but are attracting increasing interest in both academia and industry as drug targets. For example, while some PPI sites have led to potent small molecule inhibitors, others have not despite substantial effort [5,6]. A first step in evaluating target druggability is to detect the presence of binding pockets of suitable size, shape, and composition to accommodate drug-like molecules. Many such methods have been developed and tested using training sets of ligand binding sites extracted from the Protein Data Bank (PDB). Several in-depth reviews are available that summarize computa- tional methods for protein binding pocket detection [7,8,9], many
Highly active antiretroviral therapies (HAART) have been an effective way to slow the spread of HIV, but the virus continues to develop resistance to all drugs that have been used in treatment to date . HIV integrase (IN) is one of the viral targets for which small molecule therapeutics have now been approved and marketed to treat AIDS . IN is a critical enzyme in the HIV life cycle as it is required for the integration of viral DNA into the host chromatin, and therefore persistence of infection. IN performs both the 3 9 viral processing step as well as the strand transfer step to insert viral DNA into the host DNA. The structure of HIV IN has been thoroughly investigated [3–7], and consists of three domains (N-terminal DNA binding, catalytic core (CCD) and C- terminal DNA binding). The CCD has several pockets to which small molecules have been shown to bind and inhibit the enzymatic activity [6,8–12]. There are several inhibitors currently used in therapy or in late stage trials that target the catalytic site of HIV IN, making it a validated and attractive target for new therapies. IN forms a complex with viral DNA and several host cellular factors that has been termed the pre-integration complex (PIC) . One component of this complex is lens epithelium derived growth factor (LEDGF/p75), which has a conserved IN
Tuberculosis is found among the main causes of mortality in the World, although is a neglected disease since it is endemic in developing countries. The main route of therapy of tuberculosis is the inhibition of InhA, enzyme that catalyses the production of mycolic acids, which is a component of bacillus cellular wall. This reaction also is the main point of resistance against TB drugs. In this work proposed the study of InhA enzyme, working specifically in silico modeling of enzyme-ligant interactions. These ligands distinguish themselves between two distinct libraries, one of them containing organic compounds selected by its structural similarity with the enzyme substrate, NADH. Due in vitro and orally activity in murine model against tuberculosis exhibited by the compound pentacianoisoniazideferrate (II), another library, containing the pentacianoferrate II moiety bind to an auxiliary ligand studied against que InhA target. The essays realized using ligand rigid and flexible docking both, although the protein always considered rigid. Both essays had acceptable correlation within its results, regardless the scoring function used. The leading inhibitors structures had in common a high stabilization of ligand-enzyme complex due hydrophobic interactions, something expected due polarity of the enzyme bindingsite
Microtubules are the main constituents of mitotic spindles. They are nucleated in large amounts during spindle assembly, from multiprotein complexes containing c-tubulin and associated c-tubulin complex proteins (GCPs). With the aim of developing anti-cancer drugs targeting these nucleating complexes, we analyzed the interface between GCP4 and c-tubulin proteins usually located in a multiprotein complex named c-TuRC (c-Tubulin Ring Complex). 10 ns molecular dynamics simulations were performed on the heterodimers to obtain a stable complex in silico and to analyze the residues involved in persistent protein-protein contacts, responsible for the stability of the complex. We demonstrated in silico the existence of a binding pocket at the interface between the two proteins upon complex formation. By combining virtualscreening using a fragment-based approach and biophysical screening, we found several small molecules that bind specifically to this pocket. Sub-millimolar fragments have been experimentally characterized on recombinant proteins using differential scanning fluorimetry (DSF) for validation of these compounds as inhibitors. These results open a new avenue for drug development against microtubule-nucleating c-tubulin complexes.
[17–22]. The use of computational drug-design approaches would be useful here to improve the discovery of putative hits and to help obtain new leads [23–31]. However, previous virtualscreening campaigns have fallen short in the identification of new inhibitors, since none was able to find small organic compounds in the submicromolar inhibitory range. It is now widely accepted that protein flexibility is an important factor to be taken into account to ensure the success of virtualscreening campaigns . The flexibility of DENV NS3 protease is evidenced in current crystallographic structures by the lack of atomic coordinates for many residues, the difficulty in resolving an inhibitor-enzyme structure, the variable positioning of the cofactor in respect to the protease, and those of several loops [33–36]. This scenario could explain the relatively low success of previous virtualscreening attempts against this target. In fact, the flexibility of DENV NS2B/NS3 protease has already been proposed, but not proved, to explain the poor results of current drug design campaigns .
According to the environmental assessment given by the application of the RAP in three tributaries of Conquistinha River, the A site was severely impacted (18 pts), the B site was moderately changed (28 pts) and C and D sites were in better conservation condition (32 pts). The four sam‑ pling stretches at Conquistinha River (E‑H) ranged from regular to good environmental conditions (22 to 32 pts). In general, almost 70% of the evaluated stretches had margins with high degree of deforestation, soil erosion process and siltation of the stream bed, with the use of land surrounding mainly for sugarcane cultivation (44%) and pasture (33%). Domestic sewage release was observed in sampling site A, and industrial sewage, probably in the last sampling site (H). Bad sewage smell was also evident in A, E and H sites.
The mycobacterial cell wall of M. tuberculosis is rich with many unique key structural components that are necessary for the mycobacteria to survive and grow within the human host, and has long been a target for anti-TB drug development. Essential to the cell wall are the mycolic acids, which are high molecular weight 2- alkyl, 3-hydroxy fatty acids that exist in several forms of differing chemical functionality. Indeed, the first line anti-tubercular drug isoniazid (INH) works by inhibiting their biosynthesis. The complete sequencing of the TB genome  has revealed significant biochemical and genetic insight into mycolic acid biosynthesis that will aid the search for new druggable targets. These unique lipids are biosynthesised by both fatty acid synthase enzyme systems I and II (FAS I and FAS II) to produce C 56–64
The members of the group of constitutive mutants L33S, F37S, S53P, and Q61R, isolated after random mutagenesis, are discussed together because the substitutions mapped at the N terminus of AraR (Fig. 2). This region comprises a DNA- binding motif representative of the GntR family of bacterial regulatory proteins (10). The exchanges L33S and F37S re- sulted in complete loss of regulatory activity, while a twofold reduction was observed with S53P (Fig. 4A). These results do not correlate with the intracellular accumulation of the pro- teins. In fact, whereas a drastic reduction was observed with mutants L33S and S53P, only a light decrease in the amount of F37S was detected (Fig. 5). The regulatory activity of Q61R, which accumulated at levels similar to those seen with the wild type, decreased less than twofold. In addition, a mutant lacking 53 residues comprising the helix-turn-helix motif was gener- ated and characterized in vivo. Although this variant showed only a small decrease in accumulation in vivo, the regulatory activity was completely abolished (Fig. 4A and Fig. 5). The N-terminal region of AraR was modeled using the structure of the E. coli transcription factor FadR, the only member of the GntR family with the structure determined (54, 58). The DNA- binding domain contains an HTH motif of the “winged” type (4, 8). Based on the model only Q61 is predicted to contact the DNA in the minor groove (Fig. 3C), and exchanges of the corre- sponding residue in FadR (H75) and GntR (R75), the gluco- nate repressor from B. subtilis, were shown to have a trans- dominant negative phenotype (35, 59). In FadR this residue is at the tip of the wing and buried deep in the minor groove (58). The exchanges L33S and F37S, located in the second helix, may influence binding of other amino acids to the DNA, and S53P makes the third helix, the recognition helix, shorter. The specific role of residue R45 in AraR, predicted to be in contact with the DNA, was tested by exchange to an alanine (Fig. 3C). This mutation led to complete loss of regulatory activity, al- though it accumulated at wild-type levels (Fig. 4A and Fig. 5). The involvement of this particular residue in AraR-DNA bind- ing is further supported by the transdominant negative pheno- type of the araR R45A allele in B. subtilis (Table 4) and vali- dates the model presented in this work. The corresponding residue in FadR (R49) (Fig. 2) was shown to be in contact with the DNA by crystal data and mutational analysis (35, 58).
Demogines et al.  applied sophisticated evolutionary, structural, and virological analysis to infer the arms race coevolution of another common mammalian cell surface protein and viruses that use it as receptor. The protein in question is TfR1, the receptor for iron-bound transferrin, which mediates iron uptake into cells. TfR1 is known to serve as receptor for viruses of three unrelated families: mouse mammary tumor virus (MMTV), a retrovirus; several rodent and human arenaviruses, such as Machupo; and parvoviruses, including canine parvovirus. In the latter two cases, evolution of the virus to use TfR1 in a different species has been a critical factor allowing recent spread of the viruses to humans and dogs, respectively. In the Machupo virus, the crystal structure of TfR1 bound to the virus GP1 entry protein reveals that the key bindingsite is a ridge in the apical portion of the butterfly-shaped receptor dimer  (Figure 1). Other lines of evidence have identified the bindingsite for MMTV as lying on an external ridge about halfway along the outside edge of the protein. To examine the details of the molecular coevolution of these two virus groups and their receptor, Demogines et al. used phyloge- netic analyses to assess the dN/dS ratios of all extracellular TfR1 amino acids among a number of related rodents, including house mice (Mus musculus). Remarkably, only six residues exhibited ratios significantly greater than 1, and these mapped exactly to the MMTV and Machupo binding sites previously determined (Figure 1). Furthermore, when the same type of analysis was performed on the GP1 genes of Machupo virus and relatives, amino acids with dN/dS.1 were found to lie on the outward- facing side of the protein—at or near the sites of receptor binding. These results provide strong evidence for a back and forth coevolution involving the same housekeeping protein and between two different viruses and their rodent hosts. Interestingly, although arenaviruses are widespread, infectious MMTV is currently found only in M. musculus, and has not been described in any other
concomitantemente. O ponto de entrada no site traz opções que podem ser seguidas de forma aleatória, oferecendo a sensação de instantaneidade na passagem de um link para o outro, levando à exploração do caráter não-linear do texto, no sentido atribuído por Aarseth, na tentativa de construir um trajeto de leitura único. Aarseth (1993, p. 51) define o “texto não-linear como um objeto de comunicação verbal que não é apenas uma sequência fixa de letras e palavras, mas no qual a ordem de leitura pode diferir de um leitor para outro”. O link é um recurso que pode instituir uma estratégia de descentramento, podendo ser um exercício de fruição descontínua. Ao perseguir alguns links internos, de forma sucessiva, os sujeitos pesquisados demonstraram um certo desconforto pelas digressões ou avanços no tempo.
Enterotoxigenic Escherichia coli (ETEC) strains are leading causes of childhood diarrhea in developing countries. Adhesion is the first step in pathogenesis of ETEC infections and ETEC pili designated colonization factor antigens (CFAs) are believed to be important in the biofim formation, colonization and host cell adhesions. As a first step, we have determined the biofilm capability of ETEC expressing various types of pili (CFA/I, CfaE-R181A mutant/ CfaE tip mutant, CFA/II and CS2). Further, enzyme-linked immunosorbent assay (ELISA) assay were developed to compare the binding specificity of CFA/I, CFA/II (CS1 - CS3) and CS2 of ETEC, using extracted pili and piliated bacteria. CFA/II strain (E24377a) as well as extracted pili exhibited significantly higher binding both in biofilm and ELISA assays compared to non piliated wild type E24377a, CFA/I and CS2 strains. This indicates that co-expression of two or more CS2 in same strain is more efficient in increasing adherence. Significant decrease in binding specificity of DH5αF’lacI q /∆cotD (CS2) strain and MC4100/pEU2124 (CfaE-R181A) mutant strain indicated the
The leucine transporter (LeuT) has recently commanded exceptional attention due mainly to two distinctions; it provides the only crystal structures available for a protein homologous to the pharmacologically relevant neurotransmitter: sodium symporters (NSS), and, it exhibits a hallmark 5-TM inverted repeat (‘‘LeuT-fold’’), a fold recently discovered to also exist in several secondary transporter families, underscoring its general role in transporter function. Constructing the transport cycle of ‘‘LeuT-fold’’ transporters requires detailed structural and dynamic descriptions of the outward-facing (OF) and inward- facing (IF) states, as well as the intermediate states. To this end, we have modeled the structurally unknown IF state of LeuT, based on the known crystal structures of the OF state of LeuT and the IF state of vSGLT, a ‘‘LeuT-fold’’ transporter. The detailed methodology developed for the study combines structure-based alignment, threading, targeted MD and equilibrium MD, and can be applied to other proteins. The resulting IF-state models maintain the secondary structural features of LeuT. Water penetration and solvent accessibility calculations show that TM1, TM3, TM6 and TM8 line the substrate binding/unbinding pathway with TM10 and its pseudosymmetric partner, TM5, participating in the extracellular and intracellular halves of the lumen, respectively. We report conformational hotspots where notable changes in interactions occur between the IF and OF states. We observe Na2 exiting the LeuT-substrate- Na z complex in the IF state,
Melatonin, the pineal hormone produced during the dark phase of the light-dark cycle, modulates neuronal acetylcholine receptors located presynaptically on nerve terminals of the rat vas deferens. Recently we showed the presence of high affinity nicotine-binding sites during the light phase, and low and high affinity binding sites during the dark phase. The appearance of the low affinity binding sites was due to the nocturnal melatonin surge and could be mimicked by exposure to melatonin in vitro. The aim of the present research was to identify the receptor subtypes responsible for the functional response during the light and the dark phase. The rank order of potency of agonists was dimethylphenylpiperazinium (DMPP) = cytisine > nicotine > carba- chol and DMPP = nicotine = cytisine > carbachol, during the light and dark phase, respectively, due to an increase in apparent affinity for nicotine. Mecamylamine similarly blocked the DMPP response dur- ing the light and the dark phase, while the response to nicotine was more efficiently blocked during the light phase. In contrast, methyllyc- aconitine inhibited the nicotine-induced response only at 21:00 h. Since a7 nicotinic acetylcholine receptors (nAChRs) have low af- finity for nicotine in binding assays, we suggest that a mixed popula- tion composed of a3ß4 - plus a7-bearing nAChR subtypes is present at night. This plasticity in receptor subtypes is probably driven by melatonin since nicotine-induced contraction in organs from animals sacrificed at 15:00 h and incubated with melatonin (100 pg/ml, 4 h) is not totally blocked by mecamylamine. Thus melatonin, by acting directly on the short adrenergic neurons that innervate the rat vas deferens, induces the appearance of the low affinity bindingsite, probably an a7 nAChR subtype.
All organisms utilize ferrochelatase (protoheme fer- rolyase, EC 220.127.116.11) to catalyze the terminal step of the heme biosynthetic pathway, which involves the inser- tion of ferrous ion into protoporphyrin IX. Kinetic meth- ods and Mo ¨ ssbauer spectroscopy have been used in an effort to characterize the ferrous ion-binding active site of recombinant murine ferrochelatase. The kinetic stud- ies indicate that dithiothreitol, a reducing agent com- monly used in ferrochelatase activity assays, interferes with the enzymatic production of heme. Ferrochelatase specific activity values determined under strictly anaer- obic conditions are much greater than those obtained for the same enzyme under aerobic conditions and in the presence of dithiothreitol. Mo¨ssbauer spectroscopy conclusively demonstrates that, under the commonly used assay conditions, dithiothreitol chelates ferrous ion and hence competes with the enzyme for binding the ferrous substrate. Mo ¨ ssbauer spectroscopy of ferrous ion incubated with ferrochelatase in the absence of di- thiothreitol shows a somewhat broad quadrupole dou- blet. Spectral analysis indicates that when 0.1 m M Fe(II) is added to 1.75 m M ferrochelatase, the overwhelming majority of the added ferrous ion is bound to the pro- tein. The spectroscopic parameters for this bound spe- cies are d 5 1.36 6 0.03 mm/s and DE Q 5 3.04 6 0.06 mm/s, distinct from the larger DE Q of a control sample of Fe(II) in buffer only. The parameters for the bound species are consistent with an active site composed of nitrogenous/ oxygenous ligands and inconsistent with the presence of sulfur ligands. This finding is in accord with the absence of conserved cysteines among the known ferrochelatase sequences. The implications these results have with re- gard to the mechanism of ferrochelatase activity are discussed.
A structure-based drug designing method was used, and the AutoDock 4.2 tool was employed for the molecular docking study (Morris et al., 2009). This tool calculates energy values by classifying energies as internal energy and torsional free energy. Internal energy is the sum of desolvation energy, hydrogen bonding energy, van der Walls energy, and electrostatic energy. Lamarckian genetic algorithm (GA) default parameters were used to calculate ∆G for each shortlisted compound. A grid box (40×40×40 A°) was built around the IL-6R extracellular domains. The energy values generated and the binding mode with IL-6R were used to limit the list to two compounds.
p97/Valosin-containing protein (VCP) is a member of the AAA-ATPase family involved in many cellular processes including cell division, intracellular trafficking and extraction of misfolded proteins in endoplasmic reticulum-associated degradation (ERAD). It is a homohexamer with each subunit containing two tandem D1 and D2 ATPase domains and N- and C-terminal regions that function as adaptor protein binding domains. p97/VCP is directed to its many different functional pathways by associating with various adaptor proteins. The regulation of the recruitment of the adaptor proteins remains unclear. Two adaptor proteins, Ufd1/Npl4 and p47, which bind exclusively to the p97/VCP N-domain and direct p97/VCP to either ERAD- related processes or homotypic fusion of Golgi fragments, were studied here. Surface plasmon resonance biosensor-based assays allowed the study of binding kinetics in real time. In competition experiments, it was observed that in the presence of ATP, Ufd1/Npl4 was able to compete more effectively with p47 for binding to p97/VCP. By using non-hydrolysable ATP analogues and the hexameric truncated p97/N-D1 fragment, it was shown that binding rather than hydrolysis of ATP to the proximal D1 domain strengthened the Ufd1/Npl4 association with the N-domain, thus regulating the recruitment of either Ufd1/Npl4 or p47. This novel role of ATP and an assigned function to the D1 AAA-ATPase domain link the multiple functions of p97/VCP to the metabolic status of the cell.
Splice-switching oligonucleotides (SSOs) are antisense re- agents that modulate intron splicing bybinding splice site recognition or regulatory sequences and competing with cis- elements or trans-acting factors for their targets [19–21]. They have been shown to restore aberrant RNA processing, modify the relative abundance of existing mRNA isoforms, or produce novel splice variants that are not normally expressed by the cell . Most SSOs employed in preclinical and clinical devel- opment have targeted exonic sequences [19–21]. Whereas most exonic SSOs designed to induce exon skipping usually have a desired effect, functional intronic SSOs are more dif- ficult to identify, unless they block access to intronic cryptic splice sites activated by a disease-causing mutation. First, a large fraction of intronic sequences may not affect RNA pro- cessing at all, despite the wealth of intronic auxiliary splicing motifs in the human genome . In addition, a search for functional intronic SSOs that produce desirable RNA proces- sing outcomes is usually inefficient and costly and may fail completely. For example, most SSOs systematically covering exon 7 of the SMN2 (survival of motor neuron 2) gene stim- ulated exon skipping, a prerequisite for antisense therapy of spinal muscular atrophy; however, *20% SSOs increased exon inclusion . By contrast, stimulation of intron splicing was found only for *10% of SSOs targeting INS intron 1, while the majority failed to show this effect . Third, introns are enriched for many repetitive elements that preclude the SSO use with endogenous targets. Identification of effective intronic SSOs may be facilitated by global pre-mRNA folding and ultraviolet cross-linking and immunoprecipitation studies that identify binding sites for components of the spliceosome [18,25] or the exon junction complex . However, these binding sites may not reflect optimal antisense targets and their resolution may be insufficient. Thus, identification of func- tional intronic SSOs remains challenging.
The saccharide specificity of lectin bind- ing to erythrocytes was determined by inhib- iting agglutination with 100 mM sugar solu- tions in 0.15 M NaCl. The lectin dilution used for the end point was the highest dilu- tion able to cause 50% HAG. The sugars used are given in Table 2 and/or mentioned in the Results section. Fetuin and asialofetuin were also tested at 8 mg/ml concentration. To determine the minimum concentrations required for HAG inhibition by these differ- ent carbohydrates, a two-fold serial dilution of the saccharide solutions was performed. The contents of the wells were mixed by gentle shaking and covered with plastic wrap, and the extent of HAG was detected visually after 30 min of incubation. These inhibition studies were performed with the extracts that were able to cause visible HAG under the conditions described.
Co-expression has been widely used to infer regulatory interactions between TFs and their targets [16,31,32,33], but co- expression alone is not sufficient for determining direct interac- tions. Gene sets that are co-expressed with a TF are generally enriched in its targets but also contain a large proportion of non- target and indirect targets, which substantially dilute enrichment. Regulatory networks reverse engineering algorithms like ARA- CNe, on the other hand, attempt to use additional properties of the data to identify genes that are more likely to be direct transcriptional targets of the TFs. Specifically, ARACNe uses the Data Processing Inequality theorem of mutual information, as well as direct knowledge of TF identity, to remove candidate regulatory interactions that are likely to be of an indirect nature [24,25]. We used ARACNe with 100 rounds of bootstrapping to construct a regulatory network from 254 human B-cell gene-expression profiles (see ARACNe Network Inference in Materials and methods). Since activation and repression can be mediated by distinct co-factors and binding sites , we concentrated strictly on targets predicted to be activated by the TF; these constitute the majority of the interactions in the reverse-engineered regulatory network and extension to repressed subsets is straightforward. As a representative TF set for performance analysis, we selected the 70 TFs with known DNA binding motifs in TRANSFAC  that were predicted by ARACNe to be positive regulators of at least thirty targets, thus allowing appropriate statistical power for enrichment analysis. Thirty targets is also the suggested minimum for motif discovery using DME . We assembled promoter sets for each of the 70 TFs using targets predicted by ARACNe, co- expression, and co-expression*, and identified enriched TRANS- FAC motifs in each of the (7063) sets. We refer to the ARACNe- inferred promoter set as the conservation-free set because it is assembled without regard to cross-species conservation. The co- expression* set was identified by taking the top n most-co- expressed genes, where n was the total number of targets identified by ARACNe rather than based on a predefined p-value threshold (see Co-expression in Materials and methods). Note that there is no statistical threshold that could be used to reproduce the same selection a priori. Hence the co-expression* set can only be defined once ARACNe has been run and it was used only to determine if ARACNe further improves over co-expression even if only the most co-expressed targets are considered.
MicroRNAs (miRNAs) are a class of 20–24 nt non-coding RNAs that regulate gene expression primarily through post- transcriptional repression or mRNA degradation in a sequence-specific manner. The roles of miRNAs are just beginning to be understood, but the study of miRNA function has been limited by poor understanding of the general principles of gene regulation by miRNAs. Here we used CNE cells from a human nasopharyngeal carcinoma cell line as a cellular system to investigate miRNA-directed regulation of VEGF and other angiogenic factors under hypoxia, and to explore the principles of gene regulation by miRNAs. Through computational analysis, 96 miRNAs were predicted as putative regulators of VEGF. But when we analyzed the miRNA expression profile of CNE and four other VEGF-expressing cell lines, we found that only some of these miRNAs could be involved in VEGF regulation, and that VEGF may be regulated by different miRNAs that were differentially chosen from 96 putative regulatory miRNAs of VEGF in different cells. Some of these miRNAs also co-regulate other angiogenic factors (differential regulation and co-regulation principle). We also found that VEGF was regulated by multiple miRNAs using different combinations, including both coordinate and competitive interactions. The coordinate principle states that miRNAs with independent binding sites in a gene can produce coordinate action to increase the repressive effect of miRNAs on this gene. By contrast, the competitive principle states when multiple miRNAs compete with each other for a common bindingsite, or when a functional miRNA competes with a false positive miRNA for the same bindingsite, the repressive effects of miRNAs may be decreased. Through the competitive principle, false positive miRNAs, which cannot directly repress gene expression, can sometimes play a role in miRNA-mediated gene regulation. The competitive principle, differential regulation, multi-miRNA binding sites, and false positive miRNAs might be useful strategies in the avoidance of unwanted cross-action among genes targeted by miRNAs with multiple targets.