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SMCCCTHER Report Summary

Project ID: 626890
Funded under: FP7-PEOPLE
Country: United Kingdom

Final Report Summary - SMCCCTHER (Small molecule cytokine conjugates for cancer treatment)

Standard of care strategies for the treatment of cancer still relies heavily on non-specific radiation- and chemotherapy. They are commonly associated with severe toxicities and in many cases only offer limited benefit for the patient. The targeted delivery of radionuclides, cytotoxic drugs and proinflammatory cytokines into malignant tissue, on the other hand, can markedly improve the therapeutic index and overall efficacy of such substances. Whilst monoclonal antibodies are the most widely used delivery vehicles to date, we believe that small molecule ligands will offer improved targeting properties. Most importantly, depth of tumour penetration and tumour-to-blood distribution ratio after injection should be significantly superior. We therefore propose to systematically investigate the targeting performance of somatostatin receptor non-internalizing ligands for the targeted delivery of cytokines into somatostatin receptor-overexpressing tumours. This project aims at (1) developing a general chemical approach for building safe small molecule cytokine conjugates (SMCC) (2) demonstrate that a somatostatin antagonist, non-internalizing ligand can selectively accumulate in somatostatin receptor-overexpressing cancers and (3) when site-specifically conjugated to proinflammatory interleukin-12, these novel SMCC are highly effective for the treatment of acute myeloid leukaemia (AML). A brief summary of the work developed and the results obtained is listed below:

1. We have investigated the stabilization of disulfide bonds present in a truncated somatostatin analogue (octreotide) via isosteric replacement using a homocysteine-cysteine thioether bridge. Attempts involve the reaction of either (a) O-mesitylsulfonylhydroxylamine (MSH) with reduced (2xSH) cyclic peptide model containing both HomoCys and Cys residues or (b) hexamethyl phosphorus triamide (HMPT) with the corresponding cyclic (-SS-) peptide model to exploit the differences between the sulfhydryl groups in HomoCys and Cys towards their elimination and subsequent rebridging reaction. The in silico conformational evaluation of isosteric octreotide analogues by molecular dynamic (MD) simulations has been also studied.
2. The stabilization of disulfide bonds present in somatostatin and its truncated analogue (octreotide) was further explored using 3,3-bis(bromomethyl)oxetane as a novel stapling agent. The evaluation of the conformational behaviour of such a new motif has been also studied using MD simulations.
3. The selective oxetane bis-alkylation of cysteine residues on peptides and proteins stabilizes folded structures and enhances binding activity as demonstrated for a selection of biologically relevant cyclic peptides and proteins including a Fab arm of the antibody Herceptin®. Furthermore, oxetane stapling of genetically detoxified Diphteriae toxin CRM197 produced a significant improvement in its immunogenicity in mice, which demonstrates the utility of this method to modulate the stability and biological activity of therapeutic proteins containing disulfides. The evaluation of the conformational behaviour of such a new motif to stabilize the pharmacophore β-turn has been studied using MD simulations.
4. We have efficiently explored novel, complementary methods for chemical protein modification (and the conjugation of small molecule payloads) with a particular focus in those compatible with the presence of disulfides moieties, which occur in natural somatostatin. Some of the methods explored involve:

(a) Formation of C-N bonds via aza-Michael additions of N-nucleophiles to dehydroalanine(Dha)-tagged proteins.
(b) Post-expression Regioselective Mutagenesis (Cys-to-Dha) Reveals a Water-Bridged Cysteine-Cysteine Redox Regulation Mechanism in Mtb Protein Tyrosine Phosphatase A.
(c) Preparation of protein-drug conjugates using Bortezomib (BTZ) as a model boron-containing drug.
(d) Exploration of hydrosilylation reaction as a novel metal-mediated bioorthogonal ligation between an alkyne-tagged protein and a Si-H small molecule using a commercial Ru(II) catalyst.
(e) Preparation of Ru-metalloproteins for controlled CO delivery.
(f) Use of 2+2 cycloadditions and inverse electron demand Diels-Alder reactions (IEDDA) with small alkene-tagged proteins. In particular, the use of minimalistic, unstrained S-Allyl-tagged apoptotic protein markers for pretargeting Diels–Alder bioorthogonal labelling in live cells as well as vinyl ether-tetrazine pair for the traceless release of drugs containing OH motifs. These are two representative examples of targeted imaging or delivery of cytotoxic drugs (developed to be further applied for the targeted delivery of cytokines into somatostatin receptor-overexpressing tumours).
(g) Development of a simple methodology for the ready incorporation of oxetanes into proteins (apoptotic protein markers, proteins used in drug formulation, and a therapeutic antibody) through chemoselective alkylation of cysteine. The evaluation of the mechanistic basis underlying the display and reactivity of oxetane reagents with proteins has been studied using MD and DFT calculations.

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