National Research Council of Canada

Dmitri Tolkatchev

Feng Ni

Maureen D O'Connor-McCourt

Anne E G Lenferink

Zhengding Su

Anna Natapova

Ping Xu

Anatol Koutychenko

Sazzard Hossain

Kenji Tonan

Rana Filfil

Anne Eg Lenferink

Sazzad Hossain

A locally-activatable bivalent thrombin binding agent is provided having two thrombin binding moieties for non-overlapping sites on a surface of thrombin linked together by a linker. The linker is a polypeptide having 5 to 30 amino acid residues existing in a folded state under an environmental condition where the binding agent is inactive. The linker changes conformation from the folded state to an unfolded state in response to a change in bulk temperature and/or to the presence of hyper-mobile water thereby activating the binding agent. Such locally-activatable thrombin binding agents can be administered systemically while only targeting specific sites of coagulation or inflammation since the thrombin binding agent will only activate at the site where the existence of atherosclerotic plaques has changed the local bulk temperature and/or created hyper-mobile water sufficiently to unfold the linker and activate the binding agent. Such binding agents are useful as site-specific anti-coagulant, anti-thrombotic and/or antiinflammatory agents.

Locally-active anticoagulants and anti-inflammatory agents

Peptides are disclosed that are useful for molecular imaging or diagnosis of a disease state, such as cancer, in which clusterin is upregulated.

Peptide ligands for clusterin and uses thereof

There is provided herein a multivalent binding molecule and uses thereof. The molecule is useful in binding a target under certain conditions and releasing it under other conditions. The molecule has the general formula (1) of BM1-L-(BM2)(1) wherein, BM1 is a binding moiety 1 having an affinity for site 1 on the target, BM2 is a binding moiety 2 having an affinity for a site other than site 1 on the target, n is 1 or greater, and L is a linker joining BM1 and BM2, said linker being adapted to respond to a change in its environment with a change in conformation and/or flexibility, wherein BM1 and BM2 may be the same or different and are selected such that in use each of the BM1 and BM2 existing separately has a lower binding affinity then the complex of BM1 and BM2 does when they are linked to form the molecule. BM2 may have a single binding region or multiple binding regions with affinity for the target. The binding affinity of BM1 or BM2 to the target alone is no more than &#xbd; the binding affinity of the molecule of formula (1). The molecule of formula (1) can be constructed using an oligomeric or polymeric linker, such as a polypeptide sequence. Such molecules can be useful in the delayed release of drugs, in screening assays, for stabilizing enzymes such as proteases, and for controlling reactions such as blood clotting.

Bivalent thrombin binding molecules comprising linkers

The tetrapeptide Phe-Asn-Pro-Arg (SEQ ID NO: 3) is a structurally-optimized sequence for binding to the active site of thrombin. By conjugating this tetrapeptide or variants thereof to a C-terminal fragment of hirudin, we were able to generate a series of new multivalent inhibitors of thrombin containing only genetically encodable natural amino acids. We found that synergistic binding to both the active site and an exosite of thrombin can be enhanced through substitutions of amino acid residues at the P, Pand P' sites of the active-site directed sequence, Xaa (P)-Yaa (P)-Pro (P)-Arg (P)-Pro(P)-Gln(P&#x2032;)-Zaa(P&#x2032;). Complementary to rational design, a phage library was constructed to explore further the residue requirements at the P, Pand P&#x2032; sites for multivalent and optimized bridge-binding. Panning of the phage library has led to thrombin-inhibitory peptides possessing strong anti-cloning activities in the low nanomolar range and yet interfering only partially with the catalytic active site of thrombin. In all, the availability of potent and genetically-encodable polypepticle inhibitors of thrombin opens the door for much wider applications of this clinically-successful class of anticoagulants, e.g. through more cost-effective recombinant peptide production, in areas such as gene therapy as well as to improve clinical efficacy/safety through the incorporation of homing peptides for targeted delivery.

Peptide inhibitors of thrombin as potent anticoagulants

Growing community of inventors

La Prairie, Canada

Dmitri Tolkatchev