Hans-Ulrich Schmoldt

Engineered Cystine Knot Miniproteins as Potent Inhibitors of Human Mast Cell Tryptase β

Here we report the design, chemical and recombinant synthesis, and functional properties of a series of novel inhibitors of human mast cell tryptase beta, a protease of considerable interest as a therapeutic target for the treatment of allergic asthma and inflammatory disorders. These inhibitors are derived from a linear variant of the cyclic cystine knot miniprotein MCoTI-II, originally isolated from the seeds of Momordica cochinchinensis. A synthetic cyclic miniprotein that bears additional positive charge in the loop connecting the N- and C-termini inhibits all monomers of the tryptase beta tetramer with an overall equilibrium dissociation constant K(i) of 1 nM and thus is one of the most potent proteinaceous inhibitors of tryptase beta described to date. These cystine knot miniproteins may therefore become valuable scaffolds for the design of a new generation of tryptase inhibitors.

Grafting of thrombopoietin‐mimetic peptides into cystine knot miniproteins yields high‐affinity thrombopoietin antagonists and agonists

Thrombopoietin is the primary regulator of platelet production. We exploited two naturally occurring miniproteins of the inhibitor cystine knot family as stable and rigid scaffolds for the incorporation of peptide sequences that have been shown to act as high‐affinity thrombopoietin antagonists. Several miniproteins that antagonistically block thrombopoietin‐mediated receptor activation were identified using a microscale reporter assay. Covalent miniprotein dimerization yielded potent bivalent c‐Mpl receptor agonists with EC50 values in the low nanomolar or picomolar range. One selected miniprotein‐derived thrombopoietin agonist was almost as active as natural thrombopoietin with regard to stimulation of megakaryocyte colony formation from human bone marrow mononuclear cells, and elicited doubling of platelet counts in mice. Our data suggest that dimeric cystine knot miniproteins have considerable potential for the future development of small and stable receptor agonists. This approach may provide a promising strategy for pharmaceutical interference with other receptors activated by ligand‐induced dimerization.

Trypsin inhibition by macrocyclic and open-chain variants of the squash inhibitor MCoTI-II

Abstract MCoTI-I and MCoTI-II from the seeds of Momordica cochinchinensis are inhibitors of trypsin-like proteases and the only known members of the large family of squash inhibitors that are cyclic and contain an additional loop connecting the amino- and the carboxy-terminus. To investigate the contribution of macrocycle formation to biological activity, we synthesized a set of open-chain variants of MCoTI-II that lack the cyclization loop and contain various natural and non-natural amino acid substitutions in the reactive-site loop. Upon replacement of P1 lysine residue #10 within the open-chain variant of MCoTI-II by the non-natural isosteric nucleo amino acid AlaG [β-(guanin-9-yl)-L-alanine], a conformationally restricted arginine mimetic, residual inhibitory activity was detected, albeit reduced by four orders of magnitude. While the cyclic inhibitors MCoTI-I and MCoTI-II were found to be very potent trypsin inhibitors, with picomolar inhibition constants, the open-chain variants displayed an approximately 10-fold lower affinity. These data suggest that the formation of a circular backbone in the MCoTI squash inhibitors results in enhanced affinity and therefore is a determinant of biological activity.

Head-to-Tail Cyclized Cystine-Knot Peptides by a Combined Recombinant and Chemical Route of Synthesis

Cyclic peptides form an important class of naturally occurring or synthetic compounds with a large variety of biological activities as, for example, hormones, ion carriers, cancerostatics, antibiotics, antimycotics, or toxins. Biological studies with cyclopeptides have often indicate increased metabolic stability, improved receptor selectivity, and improved activity profiles in comparison with their linear counterparts. Among the group of natural circular peptides and proteins isolated in the last few years from microorganisms, plants, and even from humans, cyclotides provide an especially interesting topology. This family of circular plant proteins displays a head-totail cyclized peptide backbone together with a cystine knot (CK) motif based on disulfide bonds formed by six conserved Cys residues (Figure 1). Two disulfide bonds and their connecting backbone segments form a ring that is penetrated by the third disulfide bond to give a pseudo-knot structure that is ACHTUNGTRENNUNGinvariably associated with the nearby b sheet structure. The cystine knot in combination with the cyclic backbone appears to be a highly efficient motif for structure stabilization, resulting in exceptional conformational rigidity, together with stability against denaturing conditions, as well as against proteolytic degradation. CK-containing peptides are found in almost 20 different protein families with activities such as ion channel blocking (conotoxins and spider toxins), protease inhibition (squash inhibitors), and antiinsecticidal activity (plant cyclotides). Head-to-tail macrocyclic cystine knot peptides have been isolated from plants in the Rubiaceae, Violaceae, and Cucurbitaceae families. Several members of these family have been introduced as versatile scaffolds in drug design and biomolecular engineering. Because of their sizes, in the range of 30–40 amino acids, ACHTUNGTRENNUNGcyclotides are amenable both to recombinant production through bacterial expression and to chemical synthesis. In both routes, two steps of post-synthetic processing—oxidation of six cysteines to form three disulfide bonds and head-to-tail cyclization—are required to obtain the final cyclic product. Although the processes by which cyclotide backbone cyclization occurs naturally are largely unknown, two major strategies have been applied to generate synthetic macrocyclic CK peptides. The first approach relies on recombinant synthesis and makes use of modified protein splicing elements known as inteins to form a C-terminal thioester that reacts with the N terminus to result in macrocyclization. The second strategy is based on a solid-phase synthesis of the target peptide, followed by oxidation and cyclization. Fully deprotected peptides have successfully been “zipped” into macrocycles, followed by oxidation and cystine knot formation. Here we present a strategy for the backbone cyclization of already folded miniproteins based on the formation of a stable hydrazone. This method takes advantage of the combination of cheap and high-yielding recombinant production of linear peptide precursors that are already folded and oxidized. Chemical synthesis efficiently provides the artificial linkage of the termini, not interfering with the fold of the knotted motif stabilized by disulfide bonds. In a comparison of linear and cyclized derivatives, an increased efficiency in tryptase inhibition is reported for a representative iminocyclotide; this also indicates Figure 1. Structure of the cystine knot peptide McoEeTI. b Strand secondary structure elements are indicated as arrows. The disulfide bonds forming the cystine knot architecture are indicated as sticks; cysteine residues are numbered from I to VI beginning from the N terminus. The macrocycleforming loop was tentatively added and is shown as a dashed line.

Cystine-knot peptides targeting cancer-relevant human cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)

Cystine‐knot peptides sharing a common fold but displaying a notably large diversity within the primary structure of flanking loops have shown great potential as scaffolds for the development of therapeutic and diagnostic agents. In this study, we demonstrated that the cystine‐knot peptide MCoTI‐II, a trypsin inhibitor from Momordica cochinchinensis, can be engineered to bind to cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4), an inhibitory receptor expressed by T lymphocytes, that has emerged as a target for the treatment of metastatic melanoma. Directed evolution was used to convert a cystine‐knot trypsin inhibitor into a CTLA‐4 binder by screening a library of variants using yeast surface display. A set of cystine‐knot peptides possessing dissociation constants in the micromolar range was obtained; the most potent variant was synthesized chemically. Successive conjugation with neutravidin, fusion to antibody Fc domain or the oligomerization domain of C4b binding protein resulted in oligovalent variants that possessed enhanced (up to 400‐fold) dissociation constants in the nanomolar range. Our data indicate that display of multiple knottin peptides on an oligomeric scaffold protein is a valid strategy to improve their functional affinity with ramifications for applications in diagnostics and therapy. Copyright