Martin R. Saunders

Bio: Martin R. Saunders is an academic researcher from GlaxoSmithKline. The author has contributed to research in topics: RNase P & Topoisomerase. The author has an hindex of 4, co-authored 5 publications receiving 862 citations.

Type IIA topoisomerase inhibition by a new class of antibacterial agents.

Abstract: Despite the success of genomics in identifying new essential bacterial genes, there is a lack of sustainable leads in antibacterial drug discovery to address increasing multidrug resistance. Type IIA topoisomerases cleave and religate DNA to regulate DNA topology and are a major class of antibacterial and anticancer drug targets, yet there is no well developed structural basis for understanding drug action. Here we report the 2.1 A crystal structure of a potent, new class, broad-spectrum antibacterial agent in complex with Staphylococcus aureus DNA gyrase and DNA, showing a new mode of inhibition that circumvents fluoroquinolone resistance in this clinically important drug target. The inhibitor 'bridges' the DNA and a transient non-catalytic pocket on the two-fold axis at the GyrA dimer interface, and is close to the active sites and fluoroquinolone binding sites. In the inhibitor complex the active site seems poised to cleave the DNA, with a single metal ion observed between the TOPRIM (topoisomerase/primase) domain and the scissile phosphate. This work provides new insights into the mechanism of topoisomerase action and a platform for structure-based drug design of a new class of antibacterial agents against a clinically proven, but conformationally flexible, enzyme class.

Glycoforms modify the dynamic stability and functional activity of an enzyme.

Abstract: Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide proton exchange rates for individual glycoforms of RNase B and unglycosylated RNase A showed that while the 3D structure was unaffected, glycosylation decreased dynamic fluctuations throughout the molecule. There was individual variation in the NH-ND exchange rates of the same protons in different glycoforms, demonstrating the effects of variable glycosylation on dynamic stability. Consistent with the overall decrease in flexibility, and with the possibility that all of the sugars may afford steric protection to susceptible sites, was the finding that each of the glycoforms tested showed increased resistance to Pronase compared with the unglycosylated protein. In a novel sensitive assay using double-stranded RNA substrate, the different glycoforms showed nearly a 4-fold variation in functional activity; molecular modeling suggested that steric factors may also play a role in modulating this interaction.

Orientation of the headgroup of phosphatidylinositol in a model biomembrane as determined by neutron diffraction

Abstract: Derivatives of the sodium salt of dimyristoylphosphatidylinositol (DMPI) have been synthesized specifically deuterated in the headgroup. A 50:50 (molar) mixture of DMPI with dimyristoylphosphatidylcholine (DMPC) hydrated to the level of 16 waters/lipid gives a biomembrane-like Lalpha phase at 50 degrees C. Comparison of the neutron diffraction scattering profiles for deuterated and undeuterated membranes allowed the depth of each deuterium (hydrogen) within the bilayer to be determined to +/-0.5 A. This gave the orientation of the inositol ring which lies more-or-less along the bilayer normal projecting directly out into the water. This orientation is similar to that of the sugar residue in glycolipids and confirms previous models for PI. On the assumption that the (P)O-DAG bond is more-or-less parallel to the bilayer normal, it is consistent with a roughly trans, trans, trans, gauche- conformation for the glyceryl-phosphate-inositol link. In the case of DMPI, it is the C4-hydroxy group which is most fully extended into the water layer, but when this is phosphorylated, the inositol ring turns over and tilts so that the C5-hydroxy group is now the one furthest extended into the water layer. Hence, at each stage in the pathway PI --> PI-4P --> PI-4,5-P2, it is the hydroxy position most exposed to the water which undergoes phosphorylation. Whereas the orientation of the inositol ring in DMPI can be seen simply as maximizing its hydration, the tilt of the ring in DMPI-4P cannot be explained in this way. It is suggested that it is due to an electrostatic interaction.

PIP2 and Proteins: Interactions, Organization, and Information Flow

Abstract: ▪ Abstract We review the physical properties of phosphatidylinositol 4,5-bisphosphate (PIP2) that determine both its specific interactions with protein domains of known structure and its nonspecific electrostatic sequestration by unstructured domains. Several investigators have postulated the existence of distinct pools of PIP2 within the cell to account for the myriad functions of this lipid. Recent experimental work indicates certain regions of the plasma membrane—membrane ruffles and nascent phagosomes—do indeed concentrate PIP2. We consider two mechanisms that could account for this phenomenon: local synthesis and electrostatic sequestration. We conclude by considering the hypothesis that proteins such as MARCKS bind a significant fraction of the PIP2 in a cell, helping to sequester it in lateral membrane domains, then release this lipid in response to local signals such as an increased concentration of Ca++/calmodulin or activation of protein kinase C.

Mechanism of quinolone action and resistance.

Abstract: Quinolones are one of the most commonly prescribed classes of antibacterials in the world and are used to treat a variety of bacterial infections in humans. Because of the wide use (and overuse) of these drugs, the number of quinolone-resistant bacterial strains has been growing steadily since the 1990s. As is the case with other antibacterial agents, the rise in quinolone resistance threatens the clinical utility of this important drug class. Quinolones act by converting their targets, gyrase and topoisomerase IV, into toxic enzymes that fragment the bacterial chromosome. This review describes the development of the quinolones as antibacterials, the structure and function of gyrase and topoisomerase IV, and the mechanistic basis for quinolone action against their enzyme targets. It will then discuss the following three mechanisms that decrease the sensitivity of bacterial cells to quinolones. Target-mediated resistance is the most common and clinically significant form of resistance. It is caused by specific mutations in gyrase and topoisomerase IV that weaken interactions between quinolones and these enzymes. Plasmid-mediated resistance results from extrachromosomal elements that encode proteins that disrupt quinolone–enzyme interactions, alter drug metabolism, or increase quinolone efflux. Chromosome-mediated resistance results from the underexpression of porins or the overexpression of cellular efflux pumps, both of which decrease cellular concentrations of quinolones. Finally, this review will discuss recent advancements in our understanding of how quinolones interact with gyrase and topoisomerase IV and how mutations in these enzymes cause resistance. These last findings suggest approaches to designing new drugs that display improved activity against resistant strains.

Concepts and Principles of O-Linked Glycosylation

Abstract: The biosynthesis, structures, and functions of O-glycosylation, as a complex posttranslational event, is reviewed and compared for the various types of O-glycans. Mucin-type O-glycosylation is initiated by tissue-specific addition of a GalNAc-residue to a serine or a threonine of the fully folded protein. This event is dependent on the primary, secondary, and tertiary structure of the glycoprotein. Further elongation and termination by specific transferases is highly regulated. We also describe some of the physical and biological properties that O-glycosylation confers on the protein to which the sugars are attached. These include providing the basis for rigid conformations and for protein stability. Clustering of O-glycans in Ser/Thr(/Pro)-rich domains allows glycan determinants such as sialyl Lewis X to be presented as multivalent ligands, essential for functional recognition. An additional level of regulation, imposed by exon shuffling and alternative splicing of mRNA, results in the expression of prot...

Drugging Topoisomerases: Lessons and Challenges

Abstract: Topoisomerases are ubiquitous enzymes that control DNA supercoiling and entanglements. They are essential during transcription and replication, and topoisomerase inhibitors are among the most effective and most commonly used anticancer and antibacterial drugs. This review consists of two parts. In the first part (“Lessons”), it gives background information on the catalytic mechanisms of the different enzyme families (6 different genes in humans and 4 in most bacteria), describes the “interfacial inhibition” by which topoisomerase-targeted drugs act as topoisomerase poisons, and describes clinically relevant topoisomerase inhibitors. It generalizes the interfacial inhibition principle, which was discovered from the mechanism of action of topoisomerase inhibitors, and discusses how topoisomerase inhibitors kill cells by trapping topoisomerases on DNA rather than by classical enzymatic inhibition. Trapping protein–DNA complexes extends to a novel mechanism of action of PARP inhibitors and could be applied to...