High-throughput screen for small molecules that modulate the ATPase activity of the molecular chaperone DnaK

DnaK is a molecular chaperone of Escherichia coli that belongs to a family of conserved 70-kDa heat shock proteins. The Hsp70 chaperones are well known for their crucial roles in regulating protein homeostasis, preventing protein aggregation, and directing subcellular traffic. Given the complexity of functions, a chemical method for controlling the activities of these chaperones might provide a useful experimental tool. However, there are only a handful of Hsp70-binding molecules known. To build this area, we developed a robust, colorimetric, high-throughput screening (HTS) method in 96-well plates that reports on the ATPase activity of DnaK. Using this approach, we screened a 204-member focused library of molecules that share a dihydropyrimidine core common to known Hsp70-binding leads and uncovered seven new inhibitors. Intriguingly, the candidates do not appear to bind the hydrophobic groove that normally interacts with peptide substrates. In sum, we have developed a reliable HTS method that will likely accelerate discovery of small molecules that modulate DnaK/Hsp70 function. Moreover, because this family of chaperones has been linked to numerous diseases, this platform might be used to generate new therapeutic leads.     

Design of diblock co-oligomers as low bandgap small molecules for organic solar cells

The properties of a particular kind of small molecule that is built from two oligomers of different monomers, i.e. a diblock co-oligomer, as the electron donor in the active layer of organic solar cells are investigated theoretically. For these molecules, this work shows that it is possible to predict the energies of the frontier molecular orbitals by knowing the same energies for the oligomers that constitute the diblock, opening the possibility of designing new materials with optimal energy levels and optical properties. Furthermore, it was observed that the optical absorption bands of these diblock co-oligomers were broader than that of the constituent oligomers and also of the homopolymers, allowing greater absorption of photons and possibly an improved electric current in the device. It was also shown that these phenomena are size-dependent.     

Wrapping mimicking in drug-like small molecules disruptive of protein-protein interfaces

The discovery of small-molecule drugs aimed at disrupting protein-protein associations is expected to lead to promising therapeutic strategies. The small molecule binds to the target protein thus replacing its natural protein partner. Noteworthy, structural analysis of complexes between successful disruptive small molecules and their target proteins has suggested the possibility that such ligands might somehow mimic the binding behavior of the protein they replace. In these cases, the molecules show a spatial and "chemical" (i.e., hydrophobicity) similarity with the residues of the partner protein involved in the protein-protein complex interface. However, other disruptive small molecules do not seem to show such spatial and chemical correspondence with the replaced protein. In turn, recent progress in the understanding of protein-protein interactions and binding hot spots has revealed the main role of intermolecular wrapping interactions: three-body cooperative correlations in which nonpolar groups in the partner protein promote dehydration of a two-body electrostatic interaction of the other protein. Hence, in the present work, we study some successful complexes between already discovered small disruptive drug-like molecules and their target proteins already reported in the literature and we compare them with the complexes between such proteins and their natural protein partners. Our results show that the small molecules do in fact mimic to a great extent the wrapping behavior of the protein they replace. Thus, by revealing the replacement the small molecule performs of relevant wrapping interactions, we convey precise physical meaning to the mimicking concept, a knowledge that might be exploited in future drug-design endeavors.     

Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH‐affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC‐dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non‐preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high‐carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host‐dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.     

Rational design,synthesis and biological profiling of new KDM4C inhibitors

The human histone demethylases of the KDM4 family have been related to diseases such as prostate and breast cancer. Majority of currently known inhibitors suffer from the low permeability and low selectivity between the enzyme isoforms. In this study, toxoflavin motif was used to design and synthesize new KDM4C inhibitors with improved biological activity and in vitro ADME properties. Inhibitors displayed good passive cellular permeability and metabolic stability. However, diminishing of redox liability and consequently non-specific influence on cell viability still remains a challenge.     

miRNAs modulate the drug response of tumor cells

Chemotherapy is one of the major treatments of malignant carcinomas. However,its efficiency is affected by both intrinsic and acquired resistance to anticancer drugs. The cellular mechanisms of drug resistance include the overexpression of energy-dependent transporters that eject anticancer drugs from cells such as p-glycoprotein and multidrug resistance related protein (MRP),the mutation of drug targets,the activation of DNA repair pathways,the defects in cellular death pathways and so on. The genetic and ...     

Rational design of antithrombotic peptides to target the von Willebrand Factor (vWf) - GPIb integrin interaction

Conventional antithrombotic drug discovery requires testing of large numbers of drug candidates. We used computer-aided macromolecular interaction assessment (MIAX) to select antithrombotic molecules that mimic and therefore block platelet GPIb’s binding to von Willebrand factor (vWf), an early step in thrombus formation. We screened a random array of 15-mer D-amino acid peptides for binding vWf. Structures of 4 candidate peptides were inferred by comparison to sequences in protein databases, conversion from the L to D conformations and molecular dynamics (MD) determinations of those most energetically stable. By MIAX, we deduced the amino acids and intermolecular hydrogen bonds contributing to the GPIb-vWf interaction interface. We docked the peptides onto vWf in silico to localize their binding sites and consequent potential for preventing GPIb-vWf binding. In vitro inhibition of ristocetin-initiated platelet agglutination confirmed peptide function and suitability for antithrombotic development, thereby validating this novel approach to drug discovery.     

Structure-based design of anti-mycobacterial drug leads that target the mycolic acid transporter MmpL3

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A novel binding pocket in the D2 domain of protein tyrosine phosphatase mu (PTPmu) guides AI screen to identify small molecules that modulate tumour cell adhesion,growth and migration

Approximately 40% of people will get cancer in their lifetime in the US, and 20% are predicted to die from the condition when it is invasive and metastatic. Targeted screening for drugs that interact with proteins that drive cancer cell growth and migration can lead to new therapies. We screened molecular libraries with the AtomNet® AI-based drug design tool to identify compounds predicted to interact with the cytoplasmic domain of protein tyrosine phosphatase mu. Protein tyrosine phosphatase mu (PTPmu) is proteolytically downregulated in cancers such as glioblastoma generating fragments that stimulate cell survival and migration. Aberrant nuclear localization of PTPmu intracellular fragments drives cancer progression, so we targeted a predicted drug-binding site between the two cytoplasmic phosphatase domains we termed a D2 binding pocket. The function of the D2 domain is controversial with various proposed regulatory functions, making the D2 domain an attractive target for the development of allosteric drugs. Seventy-five of the best-scoring and chemically diverse computational hits predicted to interact with the D2 binding pocket were screened for effects on tumour cell motility and growth in 3D culture as well as in a direct assay for PTPmu-dependent adhesion. We identified two high-priority hits that inhibited the migration and glioma cell sphere formation of multiple glioma tumour cell lines as well as aggregation. We also identified one activator of PTPmu-dependent aggregation, which was able to stimulate cell migration. We propose that the PTPmu D2 binding pocket represents a novel regulatory site and that inhibitors targeting this region may have therapeutic potential for treating cancer.     

Identification of small molecules that inhibit the histone chaperone Asf1 and its chromatin function

The eukaryotic genome is packed into chromatin, which is important for the genomic integrity and gene regulation. Chromatin structures are maintained through assembly and disassembly of nucleosomes catalyzed by histone chaperones. Asf1 (anti-silencing function 1) is a highly conserved histone chaperone that mediates histone transfer on/off DNA and promotes histone H3 lysine 56 acetylation at globular core domain of histone H3. To elucidate the role of Asf1 in the modulation of chromatin structure, we screened and identified small molecules that inhibit Asf1 and H3K56 acetylation without affecting other histone modifications. These pyrimidine-2,4,6-trione derivative molecules inhibited the nucleosome assembly mediated by Asf1 in vitro, and reduced the H3K56 acetylation in HeLa cells. Furthermore, production of HSV viral particles was reduced by these compounds. As Asf1 is implicated in genome integrity, cell proliferation, and cancer, current Asf1 inhibitor molecules may offer an opportunity for the therapeutic development for treatment of diseases. [BMB Reports 2015; 48(12): 685-690]     

Rational design of bis-indolylmethane-oxadiazole hybrids as inhibitors of thymidine phosphorylase

Inhibition of Thymidine phosphorylase (TP) is continuously studied for the design and development of new drugs for the treatment of neoplastic diseases. As a part of our effort to identify TP inhibitors, we performed a structure-based virtual screening (SBVS) of our compound collection. Based on the insights gained from structures of virtual screening hits, a scaffold was designed using 1,3,4-oxadiazole as the basic structural feature and SAR studies were carried out for the optimization of this scaffold. Twenty-five novel bis-indole linked 1,3,4-oxadiazoles (7–31) were designed, synthesized and tested in vitro against E. coli TP (EcTP). Compound 7 emerged as potent TP inhibitor with an IC₅₀ value of 3.50?±?0.01?μM. Docking studies were carried out using GOLD software on thymidine phosphorylase from human (hTP) and E. coli (EcTP). Various hydrogen bonding, hydrophobic interactions, and π-π stacking were observed between designed molecules and the active site amino acid residues of the studied enzymes.     

Rational design of urea-based glutamate carboxypeptidase II (GCPII) inhibitors as versatile tools for specific drug targeting and delivery

Glutamate carboxypeptidase II (GCPII), also known as prostate specific membrane antigen (PSMA), is an established prostate cancer marker and is considered a promising target for specific anticancer drug delivery. Low-molecular-weight inhibitors of GCPII are advantageous specific ligands for this purpose. However, they must be modified with a linker to enable connection of the ligand with an imaging molecule, anticancer drug, and/or nanocarrier. Here, we describe a structure–activity relationship (SAR) study of GCPII inhibitors with linkers suitable for imaging and drug delivery. Structure-assisted inhibitor design and targeting of a specific GCPII exosite resulted in a 7-fold improvement in K_i value compared to the parent structure. X-ray structural analysis of the inhibitor series led to the identification of several inhibitor binding modes. We also optimized the length of the inhibitor linker for effective attachment to a biotin-binding molecule and showed that the optimized inhibitor could be used to target nanoparticles to cells expressing GCPII.     

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.     

Rational design of selective ligands for trypanothione reductase from trypanosoma cruz Structural effects on the inhibition by dibenzazepines based on imipramine

Abstract

Trypanothione reductase, the enzyme which in trypanosomal and leishmanial parasites catalyses the reduction of trypanothione disulphide to the redox-protective dithiol and has been identified as a potential target for rational antiparasite drug design, has been found to be strongly inhibited by tricyclic compounds containing the saturated dibenzazepine (imipramine) nucleus, with K_i values in the low micromolar range. This drug lead structure was designed by molecular graphics analysis of a three-dimensional homology model, focussing on the active-site. Inhibition studies were carried out to determine the effect of inhibitor structure on the inhibitory strength towards recombinant trypanothione reductase from Trypanosoma cruzi Hansch analysis showed that inhibitory strength depended on terms in ² and s`_m indicating dependence on both lipophilicity and inductive effect for ring-substituted analogues of imipramine. The side-chain ω-aminoalkyl chain had to be longer than 2-carbon units for inhibition. The effect on inhibition strength of the substituent at the ω-amino position on the side-chain of the central ring nitrogen atom depended markedly on the detailed substitution pattern of the rest of the molecule. This provides kinetic evidence studies of multiple binding modes within a single, blanket binding site for the inhibitor with the tricyclic ring system in the general region of the hydrophobic pocket lined by Trp21, Tyr110, Met113 and Phe114. This aspect of the structural sensitivity of the precise active-site triangulation adopted by the inhibitor is probably a function of the use of hydrophobic interactions of low directional specificity in this pocket combined with an electrostatianchoring by the ω-N+HMe₂ function of the inhibitor, presumably with a glutamate sidethain, such as Glu-18, Glu-466prime; and/or Glu-467prime;.