Multiplexed experimental strategies for fragment library screening against challenging drug targets using SPR biosensors.

Surface plasmon resonance (SPR) biosensor methods are ideally suited for fragment-based lead discovery.  However, generally applicable experimental procedures and detailed protocols are lacking, especially for structurally or physico-chemically challenging targets or when tool compounds are not available. Success depends on accounting for the features of both the target and the chemical library, purposely designing screening experiments for identification and validation of hits with desired specificity and mode-of-action, and availability of orthogonal methods capable of confirming fragment hits. The range of targets and libraries amenable to an SPR biosensor-based approach for identifying hits is considerably expanded by adopting multiplexed strategies, using multiple complementary surfaces or experimental conditions. Here we illustrate principles and multiplexed approaches for using flow-based SPR biosensor systems for screening fragment libraries of different sizes (90 and 1056 compounds) against a selection of challenging targets. It shows strategies for the identification of fragments interacting with 1) large and structurally dynamic targets, represented by acetyl choline binding protein (AChBP), a Cys-loop receptor ligand gated ion channel homologue, 2) targets in multi protein complexes, represented by lysine demethylase 1 and a corepressor (LSD1/CoREST), 3) structurally variable or unstable targets, represented by farnesyl pyrophosphate synthase (FPPS), 4) targets containing intrinsically disordered regions, represented by protein tyrosine phosphatase 1B  (PTP1B), and 5) aggregation-prone proteins, represented by an engineered form of human tau  (tau K18M). Practical considerations and procedures accounting for the characteristics of the proteins and libraries, and that increase robustness, sensitivity, throughput and versatility are highlighted. The study shows that the challenges for addressing these types of targets is not identification of potentially useful fragments per se, but establishing methods for their validation and evolution into leads.

A unique cell division machinery in the Archaea.

In contrast to the cell division machineries of bacteria, euryarchaea, and eukaryotes, no division components have been identified in the second main archaeal phylum, Crenarchaeota. Here, we demonstrate that a three-gene operon, cdv, in the crenarchaeon Sulfolobus acidocaldarius, forms part of a unique cell division machinery. The operon is induced at the onset of genome segregation and division, and the Cdv proteins then polymerize between segregating nucleoids and persist throughout cell division, forming a successively smaller structure during constriction. The cdv operon is dramatically down-regulated after UV irradiation, indicating division inhibition in response to DNA damage, reminiscent of eukaryotic checkpoint systems. The cdv genes exhibit a complementary phylogenetic range relative to FtsZ-based archaeal division systems such that, in most archaeal lineages, either one or the other system is present. Two of the Cdv proteins, CdvB and CdvC, display homology to components of the eukaryotic ESCRT-III sorting complex involved in budding of luminal vesicles and HIV-1 virion release, suggesting mechanistic similarities and a common evolutionary origin.

Early absorption and distribution analysis of antitumor and anti-AIDS drugs: lipid membrane and plasma protein interactions.

The interactions of a set of compounds of potential importance for anticancer and AIDS chemotherapy with lipid membranes and plasma proteins were studied with a surface plasmon resonance (SPR) based optical biosensor, giving valuable information on the absorption and distribution of the compounds. The technique allowed both effective screening of compounds and more detailed kinetic and mechanistic analysis of specific interactions. The interaction with two different types of lipid membranes could be reliably measured at a drug concentration as low as 20 microM, allowing analysis of poorly soluble compounds. Distribution was evaluated by investigation of the interactions with two human plasma proteins, human serum albumin (HSA) and alpha(1)-acid glycoprotein (AGP). Two apparent binding sites were clearly defined for HSA: one with rapid and one with slow association and dissociation rates. The sites appear to differ in accessibility and recognition characteristics rather than in their capacities to form strong complexes with drugs.