Fragment-based interrogation of a bacterial replisome

Chromosomes are replicated by the replisome, a molecular machine comprised of a set of interacting proteins with interdependent functions. In previous work, a fragment library was screened in vitro against a bacterial replisome, reconstituted from thirteen different subunits. In this thesis, the results of this phenotypic assay were validated, analysed and extended with functional and biophysical experiments to explore different mechanisms of inhibition. �e work shows that phenotypic fragment screening can be used as a starting point for target identification. Assays for the individual protein complexes in the replisome revealed that most fragment inhibitors acted through specific proteins. For one fragment inhibitor, the results were consistent with inhibition of primase, an enzyme that synthesises short primers to initiate elongation. �e mode of action of this fragment and analogues were studied in detail. �e bacterial primase consists of three domains: a helicase-binding, a zinc-binding, and a polymerase domain. �e fragment was not observed to bind to the polymerase domain in X-ray crystallography and nuclear magnetic resonance experiments. Molecular dynamics simulations showed a clear preference for binding to a conserved protein-protein interaction site on the helicase-binding domain, rather than to the zincbinding domain. �is prediction was validated by protein-observed nuclear magnetic resonance experiments and structure-activity and -affinity relationships. Some of the replisome fragment inhibitors acted only on the full machine and do not show inhibition of the available assays for individual components. Methods were developed to generate and test photoreactive fragment hit analogues and to screen covalent cysteine-targeting fragments. �is work did not identify any new site of interaction

Current perspectives in fragment based lead discovery (FBLD)

It is over 20 years since the first fragment-based discovery projects were disclosed. The methods are now mature for most ‘conventional’ targets in drug discovery such as enzymes (kinases and proteases) but there has also been growing success on more challenging targets, such as disruption of protein–protein interactions. The main application is to identify tractable chemical startpoints that non-covalently modulate the activity of a biological molecule. In this essay, we overview current practice in the methods and discuss how they have had an impact in lead discovery – generating a large number of fragment-derived compounds that are in clinical trials and two medicines treating patients. In addition, we discuss some of the more recent applications of the methods in chemical biology – providing chemical tools to investigate biological molecules, mechanisms and systems

La Prospección de placeres del oro y otros minerales densos

[eng] Alluvial prospecting is the most widely used method in heavy mineral exploration. Heavy minerals are resistant to meteoric agents. Economically, the most important heavy minerals are: precious metals (gold, silver and platinum group minerals), gems (diamonds, rubies, sapphires, emeralds, etc), the tin base metal group (cassiterite), technologic metals such as titanium (ilmenite and rutile), tungsten (wolframite and scheelite), zirconium (zircon) and the rare earths. The most usual techniques used in alluvial prospecting and the different criteria employed in exploration, mineral benefit and evaluation of alluvial ore deposits known as placers are presented.[cat] La prospecció al.luvionar es un mètode excepcional per la recerca de minerals de interès econòmic, com los minerals densos d'elevada resistència física i química. Entre ells son d' interès els metalls preciosos (or, plata i platí), gemmes (diamant, rubí, zèfirs, maragdes, aigua-marina, etc.), metalls de base com l'estany (cassiterites), minerals tecnològics com el titani (ilmenites i rutils), wolfram (volframita i scheelita), zirconi (zircó), de terres rares (monacites), etc.. En aquest text, és presenten las tècniques mas usuals en Prospecció al.luvionar com la batea, així com els distints criteris a seguir per dur a termini una prospecció, un benefici i una avaluació dels seus jaciments que es coneixen com ¿placeres¿.[spa] La prospección aluvionar es un método excepcional para la búsqueda de minerales de interés económico, como los minerales densos de elevada resistencia físico-química. Entre ellos son de interés los metales preciosos (oro, plata y platinoides), gemas (diamante, rubíes, zafiros, esmeraldas, aguamarinas, etc.), metales de base como el estaño (casiteritas), minerales tecnológicos como el titanio (ilmenitas y rutilos), wólfram (volframita y scheelita), circonio (circón), de tierras raras (monacitas), etc.. En este texto, se presentan las técnicas mas usuales en prospección aluvionar como la batea, así como los distintos criterios a seguir para llevar a cabo una prospección, un beneficio y una evaluación de sus yacimientos que se conocen como ¿placeres¿