2,530 research outputs found
Integration and mining of malaria molecular, functional and pharmacological data: how far are we from a chemogenomic knowledge space?
The organization and mining of malaria genomic and post-genomic data is
highly motivated by the necessity to predict and characterize new biological
targets and new drugs. Biological targets are sought in a biological space
designed from the genomic data from Plasmodium falciparum, but using also the
millions of genomic data from other species. Drug candidates are sought in a
chemical space containing the millions of small molecules stored in public and
private chemolibraries. Data management should therefore be as reliable and
versatile as possible. In this context, we examined five aspects of the
organization and mining of malaria genomic and post-genomic data: 1) the
comparison of protein sequences including compositionally atypical malaria
sequences, 2) the high throughput reconstruction of molecular phylogenies, 3)
the representation of biological processes particularly metabolic pathways, 4)
the versatile methods to integrate genomic data, biological representations and
functional profiling obtained from X-omic experiments after drug treatments and
5) the determination and prediction of protein structures and their molecular
docking with drug candidate structures. Progresses toward a grid-enabled
chemogenomic knowledge space are discussed.Comment: 43 pages, 4 figures, to appear in Malaria Journa
Optimization of Clustering and Database Screening Procedures for Cavbase and Virtual Screening for Novel Antimalarial and Antibacterial Molecules
Im Zyklus der rationellen Arzneimittelentwicklung werden AffinitĂ€t und SelektivitĂ€t von potentiellen Wirkstoffen intensiv erforscht. Da diese beiden Eigenschaften keine lineare AbhĂ€ngigkeit zueinander aufweisen, fĂŒhrt höhere AffinitĂ€t nicht gezwungenermaĂen auch zu einer höheren SelektivitĂ€t. Computer-basierte Verfahren spielen eine immer gröĂere Rolle fĂŒr die Analyse und Vorhersage von SelektivitĂ€tsprofilen. Da die meisten erfolgreich eingesetzten niedermolekularen Arzneistoffe in Vertiefungen auf ProteinoberflĂ€chen binden, spielen physiko-chemische Eigenschaften von Bindetaschen eine zentrale Rolle in der Erkennung und damit auch der Bindung von Liganden. Cavbase ist eine Methode, die es ermöglicht Bindetaschen anhand der physiko-chemischen Eigenschaften dort exponierter AminosĂ€uren zu beschreiben und unabhĂ€ngig von ihrer Proteinsequenz und Faltungsgeometrie zu vergleichen. Die Bindetaschen-basierte Klassifizierung von Proteinen ist ein effektiver Ansatz, um relevante Informationen fĂŒr SelektivitĂ€tsanalysen zu extrahieren, die durch Anwendung von Clustermethoden erreicht werden kann. In der vorliegenden Arbeit wurde ein neuartiger Arbeitsablauf zur Untersuchung von wichtigen Parametern einer Clusterung entwickelt. FĂŒr einen Datensatz von Proteinen wird eine Ăhnlichkeitsmatrix berechnet und anschlieĂend dem entwickelten Arbeitsablauf ĂŒbergeben. Dieser Ansatz wurde erfolgreich an zwei unterschiedlichen DatensĂ€tzen getestet. Die vorhergesagte Anzahl der Cluster, die am besten geeignete Clustermethode und die anschlieĂende Clusterstruktur waren in Ăbereinstimmung mit den Referenzklassifikation der Proteine. Im Falle der Protease-Proteinfamilie fĂŒhrte die Bindetaschen-basierte Klassifizierung zur einer signifikanten Gruppierung von ProteineintrĂ€gen, die unabhĂ€ngig von Sequenzinformation entstanden. Damit konnte auf struktureller Ebene die KreuzreaktivitĂ€t zwischen dem Protein Calpain-1 und Cysteincathepsinen detektiert werden, die bis jetzt nur auf Basis von Liganddaten beschrieben wurde. Im weiteren Verlauf wurden elf unterschiedliche Serinproteasen untersucht, indem die Topologie der Liganden, Bindetaschen- und Sequenzinformationen miteinander verglichen wurden. Die entstandenen Cluster zeigen einen Korrelationstrend zwischen der Ăhnlichkeit im Liganden- und Bindetaschenraum.
Eine steigende Anzahl von Resistenzen auf derzeitig angewandte antiparasitĂ€re und antibakterielle Arzneistoffe erfordert die Entwicklung neuartiger Antiinfektiva. FĂŒr den Parasiten Plasmodium falciparum, den Erreger der Malaria, wurde das SchlĂŒsselenzym der FettsĂ€uresynthese Typ-2, Enoyl ACP Reduktase (ENR), als potentielle Zielstruktur vorgeschlagen. In einem virtuellen Screening einer virtuellen Datenbank von fragmentartigen KleinmolekĂŒlen konnten acht vielversprechende Strukturen ausfindig gemacht werden. Ein SalicylsĂ€ureamidderivat zeigte in einem zellulĂ€ren Assay inhibitorische Wirkung im erythrozytĂ€ren Stadium. Diese Verbindung wurde in weiteren Schritten optimiert, in dem Struktur-AktivitĂ€ts-Beziehungen und kombinatorisches Docking fĂŒr Salicylamide analysiert wurden. Aus dieser Studie konnten zwei potente Verbindungen hervorgehen, die eine niedrige ZytotoxizitĂ€t aufweisen und in einstellig mikromolarer Konzentration sowohl im erythrozytĂ€ren als auch im prĂ€-erythrozytĂ€ren Stadium ihre hemmende Wirkung entfalten. Die Wirkung im prĂ€-erythrozytĂ€ren Stadium zeigte sich der Wirkung von Primaquin ĂŒberlegen.
Die Biosynthese der TetrahydrofolsĂ€ure ist ein essenzieller Stoffwechselweg fĂŒr fast alle Organismen. Das Enzym Pyruvoyltetrahydropterin Synthase im Plasmodium falciparum (PfPTPS) ĂŒbernimmt in diesem Stoffwechselweg die Katalyse einer Reaktion, die gewöhnlich von Dihydroneopterin Aldolase katalysiert wird, das jedoch im Plasmodium Genom fehlt. Die Einbettung des Enzyms PfPTPS in den Folatstoffwechsel qualifiziert es als eine potentielle Zielstruktur zur Entwicklung neuartiger Antifolate. Eine spezielle auf dieses Zielprotein hin aufgearbeitete Bibliothek weist KleinmolekĂŒle mit zink-bindenden funktionellen Gruppen auf. Die DurchfĂŒhrung eines virtuellen Screenings fĂŒhrte zur Auswahl von neun MolekĂŒlen fĂŒr die Synthese, die anschlieĂend auf ihre biologische Wirkung evaluiert werden sollen.
Eine Vielzahl pathogener Mikroorganismen sind auf die Synthese der Isoprenoide aus dem Methylerithritolphosphatweg (MEP-Weg) angewiesen, daher eignet sich die Inhibition dieses Stoffwechselweges als eine sinnvolle Strategie fĂŒr die Wirkstoffentwicklung. IspD ist eines der Enzyme des MEP-Weges und wurde als Modellprotein zur Untersuchung der bestimmenden Faktoren fĂŒr eine strukturbasierte Wirkstoffentwickung ausgewĂ€hlt. Ein Datensatz von leitstrukturartigen KleinmolekĂŒlen aus der ZINC Datenbank wurde fĂŒr ein virtuelles Screening benutzt, das zur Auswahl von sieben Kandidaten fĂŒhrte. Sechs Verbindungen konnten kommerziell erworben und getestet werden. FĂŒr drei Verbindungen konnte eine Proteinbindung gemessen werden
Using bioinformatics tools to screen for trypanosomal cathepsin B cysteine protease inhibitors from the SANCDB as a novel therapeutic modality against Human African Trypanosomiasis (HAT)
Human African Trypanosomiasis (HAT), also known as sleeping sickness, is a fatal chronic disease that is caused by flagellated protozoans, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense. HAT is spread by a bite from an infected tsetse fly of the Glosina genus. Up to 60 million people in 36 countries in sub-Saharan Africa are at a risk of infection from HAT with up to 30 000 deaths reported every year. Current chemotherapy for HAT is insufficient since the available drugs exhibit unacceptable side effects (toxicity) and parasite resistance. Novel treatments and approaches for development of specific and more potent drugs for HAT are therefore required. One approach is to target vital proteins that are essential to the life cycle of the parasite. The main interest of this study is to explore Trypanosoma brucei cathepsin B-like protease (TbCatB) structural and functional properties with the primary goal of discovering non peptide small molecule inhibitors of TbCatB using bioinformatics approaches. TbCatB is a papain family C1 cysteine protease which belongs to clan CA group and it has emerged as a potential HAT drug target. Papain family cysteine proteases of Clan CA group of Trypanosoma brucei (rhodesain and TbCatB) have demonstrated potential as chemotherapeutic targets using synthetic protease inhibitors like Z-Phe-Ala-CHN2 to kill the parasite in vitro and in vivo. TbCatB has been identified as the essential cysteine protease of T. brucei since mRNA silencing of TbCatB killed the parasite and resulted in a cure in mice infected with T. brucei while mRNA silencing of rhodesain only extended mice life. TbCatB is therefore a promising drug target against HAT and the discovery and development of compounds that can selectively inhibit TbCatB without posing any danger to the human host represent a great therapeutic solution for treatment of HAT. To understand protein-inhibitor interactions, useful information can be obtained from high resolution protease-inhibitor crystal structure complexes. This study aims to use bioinformatics approaches to carry out comparative sequence, structural and functional analysis of TbCatB protease and its homologs from T. congolense, T, cruzi, T. vivax and H. sapien as well as to identify non-peptide small molecule inhibitors of TbCatB cysteine proteases from natural compounds of South African origin. Sequences of TbCatB (PDB ID: 3HHI) homologs were retrieved by a BLAST search. Human cathepsin B (PDB ID: 3CBJ) was selected from a list of templates for homology modelling found by HHpred. MODELLER version 9.10 program was used to generate a hundred models for T. congolense, T, cruzi and T. vivax cathepsin B like proteases using 3HHI and 3CBJ as templates. The best models were chosen based on their low DOPE Z scores before validation using MetaMQAPII, ANOLEA, PROCHECK and QMEAN6. The DOPE Z scores and the RMSD (RMS) values of the calculated models indicate that the models are of acceptable energy (stability) and fold (conformation). Results from the different MQAPs indicate the models are of acceptable quality and they can be used for docking studies. High throughput screening of SANCDB using AutoDock Vina revealed nine compounds, SANC00 478, 479, 480, 481, 482, 488, 489, 490 and 491, having a strong affinity for Trypanosoma spp. cathepsin B proteases than HsCatB. SANC00488 has the strongest binding to Trypanosoma spp. cathepsin B proteases and the weakest binding to HsCatB protease. Molecular dynamics (MD) simulations show that the complexes between SANC00488 and TbCatB, TcCatB, TcrCatB and TvCatB are stable and do not come apart during simulation. The complex between this compound and HsCatB however is unstable and comes apart during simulation. Residues that are important for the stability of SANC00488-TbCatB complex are Gly328 of the S2 subsite, Phe208, and Ala256. In conclusion SANC00488 is a good candidate for development of a drug against HAT
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The design and synthesis of Novel AOX and cytochrome bc1 complex inhibitors which act as phytopathogenic fungicides
The metabolic pathway for oxidative phosphorylation has remained a reliable target for the development of novel fungicides. The reliance on single target site fungicides within this pathway has increased the selection pressure for point mutations within a number of key complexes, including the cytochrome bc1 complex. The alternative oxidase (AOX) provides an alternative route for respiration, introducing a mechanism by which selection pressure and pathogenicity can be increased. In particular, the fungal pathogen Septoria tritici has developed a highly fungicide resistant strain. The design and synthesis of inhibitors targeting the fungal AOX, or the AOX and the cytochrome bc1 complex, represents a new class of fungicides improving crop yield outcomes.
Investigation into the AOX found in S. tritici (StAOX), led to new techniques to fully characterise and overexpress the protein in a haem deficient Escherichia coli strain, which has then been compared to that of the well-studied protozoan AOX, Trypanosoma brucei brucei (TAO). The enzymatic activity of StAOX was found to be significantly lower in comparison to AOXs from other species, but responded dramatically to the nucleotide regulators, GMP and IMP. The purified protein has also been shown to be sensitive to its lipid environment with full enzymatic recovery following re-introduction into a lipid membrane.
The natural quinol analogue, ascofuranone, displays selectivity towards the AOX and was selected for further design and lead modification. Several ascofuranone derivatives were synthesised according to a new synthetic route. A variety of new methods were utilised to analyse the inhibitors providing IC50, KD, thermodynamic and cytotoxicity data. Two of the newly synthesised compounds provided selectivity for TAO over the cytochrome bc1 complex, but failed to show selectivity to the StAOX. In vitro data suggests a single phenylalanine residue restricts inhibitorâs tail length to within an 8-carbon chain length, supported by an in-silico docking screen
Supervised prediction of drugâtarget interactions using bipartite local models
Motivation: In silico prediction of drugâtarget interactions from heterogeneous biological data is critical in the search for drugs for known diseases. This problem is currently being attacked from many different points of view, a strong indication of its current importance. Precisely, being able to predict new drugâtarget interactions with both high precision and accuracy is the holy grail, a fundamental requirement for in silico methods to be useful in a biological setting. This, however, remains extremely challenging due to, amongst other things, the rarity of known drugâtarget interactions
Interfering with mRNA methylation by the 2âČO-Methyltransferase (NSP16) from SARS-CoV-2 to tackle the COVID-19 disease
The pandemic associated to Severe Acute Respiratory Syndrome Coronavirus type 2 (SARS-CoV-2) has resulted in a huge number of deaths and infected people. Although several vaccine programmes are currently underway and have reached phase 3, and a few small size drugs repurposed to aid treatment of severe cases of COVID-19 infections, effective therapeutic options for this disease do not currently exist. NSP16 is a S-adenosyl-L-Methionine (SAM) dependent 2âČO-Methyltransferase that converts mRNA cap-0 into cap-1 structure to prevent virus detection by cell innate immunity mechanisms. NSP16 methylates the ribose 2âČO-position of the first nucleotide of the mRNA only in the presence of an interacting partner, the protein NSP10. This feature suggests that inhibition of the NSP16 may represent a therapeutic window to treat COVID-19. To test this idea, we performed comparative structural analyses of the NSP16 present in human coronaviruses and developed a sinefungin (SFG) similarity-based virtual screening campaign to assess the druggability of the SARS-CoV-2 NSP16 enzyme. Through these studies, we identified the SFG analogue 44601604 as a promising more potent inhibitor of NSP16 to limit viral replication in infected cells, favouring viral clearance
Discovery of Alternative Artemisinin Binding Sites in Plasmodium falciparum ATPase-6
Malaria is a fatal yet preventable and treatable disease. It is commonly spread through the bite of an infected Anopheles mosquito. Malaria parasites belong to the Plasmodium genus and can be caused by the falciparum, malariae, ovale, vivax, and knowlesi species. Artemisinin is an endoperoxide lactone extracted from qinghaosu (Artemisia annua L. or sweet wormwood). It and its derivatives possess uncharacteristically rapid action against Plasmodium falciparum. Artemisinin is unique in its effectiveness in deadly cerebral malaria. The endoperoxide bridge is crucial for its antimalarial activity; however, how it aids in killing the parasite is unknown. While there are multiple suggested modes of action (MOA) for artemisinin, none to date have a well-characterized protein target except for PfATP6, proposed by Krishna, et al. His work suggested that artemisinin binds to the homologous thapsigargin binding site of PfATP6. Herein, knowledge of the previously proposed mechanisms was utilized along with numerous computational techniques to determine a more detailed and plausible MOA for artemisinin against PfATP6. This led to the discovery of two new, putative PfATP6 binding sites for artemisinin. None of the previous work has explained the co-dependence of antimalarial efficacy on the concentration of Fe(II). In our work, we searched for putative sites containing a cysteine residue, Fe(II) and artemisinin in a conformation allowing for the well-accepted ring-opened C4 primary artemisinin carbon radical to form a covalent bond with a cysteine thiol rather than undergoing an intramolecular self-emolative diradical ring closure. Clearly there are geometric constraints for a transition state that side-steps the latter reaction and instead allows for interception by a cysteine thiol. We have suggested mechanistic possibilities for this capture by the artemisinin C4 radical and propose that PfATP6 is deactivated by blocking the Ca(II) channel by the modification of a cysteine at either C1031 or C92. It is alternatively possible that these modifications lead to alterations in the function of the protein, rendering it dysfunctional. Structure-based virtual screening was then used to screen a commercial database of compounds to find novel inhibitors of PfATP6. Biological testing will be done to determine if targeting these new sites can produce potent antimalarials with less structural complexity than artemisinin itself
Optimiertes Design kombinatorischer Verbindungsbibliotheken durch Genetische Algorithmen und deren Bewertung anhand wissensbasierter Protein-Ligand Bindungsprofile
In dieser Arbeit sind die zwei neuen Computer-Methoden DrugScore Fingerprint (DrugScoreFP) und GARLig in ihrer Theorie und Funktionsweise vorgestellt und validiert worden.
DrugScoreFP ist ein neuartiger Ansatz zur Bewertung von computergenerierten Bindemodi potentieller Liganden fĂŒr eine bestimmte Zielstruktur. Das Programm basiert auf der etablierten Bewertungsfunktion DrugScoreCSD und unterscheidet sich darin, dass anhand bereits bekannter Kristallstrukturen fĂŒr den zu untersuchenden Rezeptor ein Referenzvektor generiert wird, der zu jedem Bindetaschenatom Potentialwerte fĂŒr alle möglichen Interaktionen enthĂ€lt. FĂŒr jeden neuen, computergenerierten Bindungsmodus eines Liganden lĂ€sst sich ein entsprechender Vektor generieren. Dessen Distanz zum Referenzvektor ist ein MaĂ dafĂŒr, wie Ă€hnlich generierte Bindungsmodi zu bereits bekannten sind. Eine experimentelle Validierung der durch DrugScoreFP als Ă€hnlich vorhergesagten Liganden ergab fĂŒr die in unserem Arbeitskreis untersuchten Proteinstrukturen Trypsin, Thermolysin und tRNA-Guanin Transglykosylase (TGT) sechs Inhibitoren fragmentĂ€rer GröĂe und eine Thermolysin Kristallstruktur in Komplex mit einem der gefundenen Fragmente.
Das in dieser Arbeit entwickelte Programm GARLig ist eine auf einem Genetischen Algorithmus basierende Methode, um chemische Seitenkettenmodifikationen niedermolekularer Verbindungen hinsichtlich eines untersuchten Rezeptors effizient durchzufĂŒhren. Zielsetzung ist hier die Zusammenstellung einer Verbindungsbibliothek, welche eine benutzerdefiniert groĂe Untermenge aller möglichen chemischen Modifikationen Ligand-Ă€hnlicher GrundgerĂŒste darstellt. Als zentrales QualitĂ€tskriterium einzelner Vertreter der Verbindungsbibliothek dienen durch Docking erzeugte Ligand-Geometrien und deren Bewertungen durch Protein-Ligand-Bewertungsfunktionen. In mehreren Validierungsszenarien an den Proteinen Trypsin, Thrombin, Faktor Xa, Plasmin und Cathepsin D konnte gezeigt werden, dass eine effiziente Zusammenstellung Rezeptor-spezifischer Substrat- oder Ligand-Bibliotheken lediglich eine Durchsuchung von weniger als 8% der vorgegebenen SuchrĂ€ume erfordert und GARLig dennoch im Stande ist, bekannte Inhibitoren in der Zielbibliothek anzureichern
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