Key Points• We present an antidote for dabigatran that effectively reverses its anticoagulative effect in human plasma in vitro and in rats in vivo.• The antidote shares structural features with thrombin in the mode of binding but has no activity in coagulation tests.Dabigatran etexilate is a direct thrombin inhibitor and used widely as an anticoagulant for the prevention of stroke in patients with atrial fibrillation. However, anticoagulation therapy can be associated with an increased risk of bleeding. Here, we present data on the identification, humanization, and in vitro pharmacology of an antidote for dabigatran (aDabi-Fab). The X-ray crystal structure of dabigatran in complex with the antidote reveals many structural similarities of dabigatran recognition compared with thrombin. By a tighter network of interactions, the antidote achieves an affinity for dabigatran that is ∼350 times stronger than its affinity for thrombin. Despite the structural similarities in the mode of dabigatran binding, the antidote does not bind known thrombin substrates and has no activity in coagulation tests or platelet aggregation. In addition we demonstrate that the antidote rapidly reversed the anticoagulant activity of dabigatran in vivo in a rat model of anticoagulation. This is the first report of a specific antidote for a next-generation anticoagulant that may become a valuable tool in patients who require emergency procedures. (Blood. 2013;121(18):3554-3562)
The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the
coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable
efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this
enzyme. On the basis of the X-ray crystal structure of the peptidelike thrombin inhibitor NAPAP
complexed with bovine thrombin, we have designed a new structural class of nonpeptidic
inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported
by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin
inhibition in the lower nanomolar range could be achieved although the binding energy mainly
results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the
overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar
compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic
molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral
activity in different animal species. On the basis of these results, 31 was chosen for clinical
development.
Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound-target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.
Genetically encoded fusion constructs derived from fluorescent proteins (FPs) can be designed to report on a multitude of events and signals in cells, tissues, and entire organs without interfering with the complex machinery of life. EosFP is a novel FP from the scleractinian coral Lobophyllia hemprichii that switches its fluorescence emission from green (516 nm) to red (581 nm) upon irradiation with Ϸ400-nm light. This property enables localized tagging of proteins and thus provides a valuable tool for tracking protein movements within live cells. Here, we present the x-ray structures of the green and red forms of WT EosFP. They reveal that formation of the red chromophore is associated with cleavage of the peptide backbone, with surprisingly little change elsewhere in the structure, and provide insights into the mechanism that generates this interesting posttranslational polypeptide modification.photochemistry ͉ photoconversion ͉ x-ray structure ͉ Anthozoa
Components of the chromatin remodelling
switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated
in tumors, suggesting that altering the activity of the complex plays
a role in oncogenesis. However, the role that the individual subunits
play in this process is not clear. We set out to develop an inhibitor
compound targeting the bromodomain of BRD9 in order to evaluate its
function within the SWI/SNF complex. Here, we present the discovery
and development of a potent and selective BRD9 bromodomain inhibitor
series based on a new pyridinone-like scaffold. Crystallographic information
on the inhibitors bound to BRD9 guided their development with respect
to potency for BRD9 and selectivity against BRD4. These compounds
modulate BRD9 bromodomain cellular function and display antitumor
activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were
identified that should prove to be useful in further exploring BRD9
bromodomain biology in both in vitro and in vivo settings.
Azurin*, a by-product of heterologous expression of the gene encoding the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli, was characterized by chemical analysis and electrospray ionization mass spectrometry, and its structure determined by X-ray crystallography. It was shown that azurin* is native azurin with its copper atom replaced by zinc in the metal binding site. Zinc is probably incorporated in the apo-protein after its expression and transport into the periplasm. Holo-azurin can be reconstituted from azurin* by prolonged exposure of the protein to high copper ion concentrations or unfolding of the protein and refolding in the presence of copperions.An X-ray crystallographic analysis of azurin* at 0.21-nm resolution revealed that the overall structure of azurin is not perturbed by the metal exchange. However, the geometry of the co-ordination sphere changes from trigonal bipyramidal in the case of copper azurin to distorted tetrahedral for the zinc protein. The copper ligand Met121 is no longer co-ordinated to zinc which adopts a position close to the carbonyl oxygen atom from residue Gly45.The polypeptide structure surrounding the metal site undergoes moderate reorganization upon zinc binding. The largest displacement observed is for the carbonyl oxygen from residue Gly45, whch is involved in copper and zinc binding. It moves by 0.03 nm towards the zinc, thereby reducing its distance to the metal from 0.29 nm in the copper protein to 0.23 nm in the derivative.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.