Deacetoxycephalosporin-C synthase (DAOCS) is a mononuclear ferrous enzyme that transforms penicillins into cephalosporins by inserting a carbon atom into the penicillin nucleus. In the first half-reaction, dioxygen and 2-oxoglutarate produce a reactive iron-oxygen species, succinate and CO2. The oxidizing iron species subsequently reacts with penicillin to give cephalosporin and water. Here we describe high-resolution structures for ferrous DAOCS in complex with penicillins, the cephalosporin product, the cosubstrate and the coproduct. Steady-state kinetic data, quantum-chemical calculations and the new structures indicate a reaction sequence in which a 'booby-trapped' oxidizing species is formed. This species is stabilized by the negative charge of succinate on the iron. The binding sites of succinate and penicillin overlap, and when penicillin replaces succinate, it removes the stabilizing charge, eliciting oxidative attack on itself. Requisite groups of penicillin are within 1 A of the expected position of a ferryl oxygen in the enzyme-penicillin complex.
Bioassay-guided fractionation of a CH2Cl2/MeOH extract of the sponge Suberea clavata using the serine protease factor XIa to detect antithrombotic activity led to the isolation of the new marine natural products, clavatadines A and B. Clavatadines A and B inhibited factor XIa with IC50's of 1.3 and 27 microM, respectively. A crystal structure of protein-inhibitor (clavatadine A) complex was obtained and revealed interesting selective binding and irreversible inhibition of factor XIa. The cocrystal structure provides guidance for the design and synthesis of future factor XIa inhibitors as antithrombotic agents.
• In the clinic, all oral antiplatelet medicines have a risk of bleeding complications.• We present an antidote for ticagrelor that reverses its antiplatelet effect in human platelet-rich plasma and its bleeding effect in mice.Ticagrelor is a direct-acting reversibly binding P2Y 12 antagonist and is widely used as an antiplatelet therapy for the prevention of cardiovascular events in acute coronary syndrome patients. However, antiplatelet therapy can be associated with an increased risk of bleeding. Here, we present data on the identification and the in vitro and in vivo pharmacology of an antigen-binding fragment (Fab) antidote for ticagrelor. The Fab has a 20 pM affinity for ticagrelor, which is 100 times stronger than ticagrelor's affinity for its target, P2Y 12 . Despite ticagrelor's structural similarities to adenosine, the Fab is highly specific and does not bind to adenosine, adenosine triphosphate, adenosine 59-diphosphate, or structurally related drugs. The antidote concentration-dependently neutralized the free fraction of ticagrelor and reversed its antiplatelet activity both in vitro in human platelet-rich plasma and in vivo in mice. Lastly, the antidote proved effective in normalizing ticagrelor-dependent bleeding in a mouse model of acute surgery. This specific antidote for ticagrelor may prove valuable as an agent for patients who require emergency procedures. (Blood. 2015;125(22):3484-3490)
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