Design, synthesis and in vitro evaluation of novel bivalent S-adenosylmethionine analogues

In optimal cases, bivalent ligands can bind with exceptionally high affinity to their protein targets. However, designing optimised linkers, that orient the two binding groups perfectly, is challenging, and yet crucial in both fragment-based ligand design and in the discovery of bisubstrate enzyme inhibitors. To further our understanding of linker design, a series of novel bivalent S-adenosylmethionine (SAM) analogues were designed with the aim of interacting with the MetJ dimer in a bivalent sense (1:1 ligand/MetJ dimer). A range of ligands was synthesised and analyzed for ability to promote binding of the Escherichia coli methionine repressor, MetJ, to its operator DNA. Binding of bivalent SAM analogues to the MetJ homodimer in the presence of operator DNA was evaluated by fluorescence anisotropy and the effect of linker length and structure was investigated. The most effective bivalent ligand identified had a flexible linker, and promoted the DNA-protein interaction at 21-times lower concentration than the corresponding monovalent control compound.     

Quantitative relation between intermolecular and intramolecular binding of pro-rich peptides to SH3 domains

Flexible linkers are often found to tether binding sequence motifs or connect protein domains. Here we analyze three usages of flexible linkers: 1), intramolecular binding of proline-rich peptides (PRPs) to SH3 domains for kinase regulation; 2), intramolecular binding of PRP for increasing the folding stability of SH3 domains; and 3), covalent linking of PRPs and other ligands for high-affinity bivalent binding. The basis of these analyses is a quantitative relation between intermolecular and intramolecular binding constants. This relation has the form K(i) = K(e0)p for intramolecular binding and K(e) = K(e01)K(e02)p for bivalent binding. The effective concentration p depends on the length of the linker and the distance between the linker attachment points in the bound state. Several applications illustrate the usefulness of the quantitative relation. These include intramolecular binding to the Itk SH3 domain by an internal PRP and to a circular permutant of the alpha-spectrin SH3 domain by a designed PRP, and bivalent binding to the two SH3 domains of Grb2 by two linked PRPs. These and other examples suggest that flexible linkers and sequence motifs tethered to them, like folded protein domains, are also subject to tight control during evolution.     

Synthesis and evaluation of bivalent ligands for binding to the human melanocortin-4 receptor

Membrane proteins, especially G-protein coupled receptors (GPCRs), are interesting and important theragnostic targets since many of them serve in intracellular signaling critical for all aspects of health and disease. The potential utility of designed bivalent ligands as targeting agents for cancer diagnosis and/or therapy can be evaluated by determining their binding to the corresponding receptors. As proof of concept, GPCR cell surface proteins are shown to be targeted specifically using multivalent ligands. We designed, synthesized, and tested a series of bivalent ligands targeting the over-expressed human melanocortin 4 receptor (hMC4R) in human embryonic kidney (HEK) 293 cells. Based on our data suggesting an optimal linker length of 25 ± 10 Å inferred from the bivalent melanocyte stimulating hormone (MSH) agonist, the truncated heptapeptide, referred to as MSH(7): Ac-Ser-Nle-Glu-His-D-Phe-Arg-Trp-NH₂ was used to construct a set of bivalent ligands incorporating a hMC4R antagonist, SHU9119: Ac-Nle-_c[Asp-His-2′-D-Nal-Arg-Trp-Lys]-NH₂ and another set of bivalent ligands containing the SHU9119 antagonist pharmacophore on both side of the optimized linkers. These two binding motifs within the bivalent constructs were conjoined by semi-rigid (Pro-Gly)₃ units with or without the flexible poly(ethylene glycol) (PEGO) moieties. Lanthanide-based competitive binding assays showed bivalent ligands binds to the hMC4R with up to 240-fold higher affinity than the corresponding linked monovalent ligands.     

A study of the structure-activity relationship of GABA(A)-benzodiazepine receptor bivalent ligands by conformational analysis with low temperature NMR and X-ray analysis

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The stable conformations in solution correlated well with those in the solid state. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity and was illustrated for alpha5 subtype selective ligands. Bivalent ligands with an oxygen-containing linker folded back upon themselves both in solution and the solid state. Dimers which are folded do not bind to Bz receptors.     

Bivalent inhibitors of the tyrosine kinases ABL and SRC: determinants of potency and selectivity

We recently reported a chemical genetic method for generating bivalent inhibitors of protein kinases. This method relies on the use of the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (AGT) to display an ATP-competitive inhibitor and a ligand that targets a secondary binding domain. With this method potent and selective inhibitors of the tyrosine kinases SRC and ABL were identified. Here, we dissect the molecular determinants of the potency and selectivity of these bivalent ligands. Systematic analysis of ATP-competitive inhibitors with varying linker lengths revealed that SRC and ABL have differential sensitivities to ligand presentation. Generation of bivalent constructs that contain ligands with differential affinities for the ATP-binding sites and SH3 domains of SRC and ABL demonstrated the modular nature of inhibitors based on the AGT scaffold. Furthermore, these studies revealed that the interaction between the SH3 domain ligand and the kinase SH3 domain is the major selectivity determinant amongst closely-related tyrosine kinases. Finally, the potency of bivalent inhibitors against distinct phospho-isoforms of SRC was determined. Overall, these results provide insight into how individual ligands can be modified to provide more potent and selective bivalent inhibitors of protein kinases.     

On the benefit of bivalency in peptide ligand/pin1 interactions

The human peptidyl prolyl cis/trans isomerase (PPIase) Pin1 has a key role in developmental processes and cell proliferation. Pin1 consists of an N-terminal WW domain and a C-terminal catalytic PPIase domain both targeted specifically to Ser(PO₃H₂)/Thr(PO₃H₂)-Pro sequences. Here, we report the enhanced affinity originating from bivalent binding of ligands toward Pin1 compared to monovalent binding. We developed composite peptides where an N-terminal segment represents a catalytic site-directed motif and a C-terminal segment exhibits a predominant affinity to the WW domain of Pin1 tethered by polyproline linkers of different chain length. We used NMR shift perturbation experiments to obtain information on the specific interaction of a bivalent ligand to both targeted sites of Pin1. The bivalent ligands allowed a considerable range of thermodynamic investigations using isothermal titration calorimetry and PPIase activity assays. They expressed up to 350-fold improved affinity toward Pin1 in the nanomolar range in comparison to the monovalent peptides. The distance between the two binding motifs was highly relevant for affinity. The optimum in affinity manifested by a linker length of five prolyl residues between active site- and WW domain-directed peptide fragments suggests that the corresponding domains in Pin1 are allowed to adopt preferred spatial arrangement upon ligand binding.     

Cell-specific targeting by heterobivalent ligands

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 ? is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.     

Ligand association rates to the inner-variable-domain of a dual-variable-domain immunoglobulin are significantly impacted by linker design

The DVD-Ig^TM protein is a dual-specific immunoglobulin. Each of the two arms of the molecule contains two variable domains, an inner variable domain and an outer variable domain linked in tandem, each with binding specificity for different targets or epitopes. One area of on-going research involves determining how the proximity of the outer variable domain affects the binding of ligands to the inner variable domain. To explore this area, we prepared a series of DVD-Ig proteins with binding specificities toward TNFα and an alternate therapeutic target. Kinetic measurements of TNFα binding to this series of DVD-Ig proteins were used to probe the effects of variable domain position and linker design on ligand on- and off-rates. We found that affinities for TNFα are generally lower when binding to the inner domain than to the outer domain and that this loss of affinity is primarily due to reduced association rate. This effect could be mitigated, to some degree, by linker design. We show several linker sequences that mitigate inner domain affinity losses in this series of DVD-Ig proteins. Moreover, we show that single chain proteolytic cleavage between the inner and outer domains, or complete outer domain removal, can largely restore inner domain TNFα affinity to that approaching the reference antibody. Taken together, these results suggest that a loss of affinity for inner variable domains in this set of DVD-Ig proteins may be largely driven by simple steric hindrance effects and can be reduced by careful linker design.     

ER subtype selectivity of m-carborane-containing phenols: C-alkyl groups on the m-carborane cage enhance ERα selectivity

Estrogen receptor (ER) exhibits two subtypes, ERα and ERβ, whose biological functions are quite different despite expression in the same tissues. We developed diiodo-m-carborane derivative 3a, which showed 14-fold selectivity for ERβ with high binding affinity toward ERβ. Interestingly, introduction of an alkyl group into the carbon atom of the m-carborane cage of 3a markedly enhanced the binding affinity toward ERα and decreased affinity toward ERβ. C-n-propyl derivative 3d showed 28-fold selectivity for ERα in an ER binding assay and promoted proliferation of MCF-7 breast cancer cells. Docking simulation studies suggest that the directions of the n-propyl group and the diiodo substituent introduced on the m-carborane cage play important roles for the control of ER subtype selectivity. As 3a and 3d showed ERβ and ERα selectivity with high binding affinity, respectively, these ligands may be useful as biological tools to aid in understanding the different roles of ER subtypes.     

Determination of the stable conformation of GABA(A)-benzodiazepine receptor bivalent ligands by low temperature NMR and X-ray analysis

The stable conformations of GABA(A)-benzodiazepine receptor bivalent ligands 2 and 3 were determined by low temperature NMR spectroscopy and confirmed by single crystal X-ray analysis. The linear conformation was important for these dimers to access the binding site and exhibit potent in vitro affinity as illustrated for alpha5 subtype selective ligand 2 (15 nM). Bivalent ligand 3 with the 5 atom linker folded back upon itself both in solution and in the solid state, moreover, it did not bind to Bz receptors.     

Use of hydrophilic diamines for bridging of two opioid peptide pharmacophores. Synthesis and receptor binding of two new analogues.

The bivalent ligand approach, which assumes that two pharmacophores are connected by a spacer, was used to design receptor type-selective ligands for opioid receptors. The first two opioid peptide bivalent ligands with different spacer lengths containing different numbers of hydroxyl groups, (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-)2 (Tyr-D-Ala-Gly-Phe-NH-CH2-CHOH-CHOH-)2, were synthesized and their binding to mu, delta, and kappa opioid receptors was characterized. Both analogues were found to possess high opioid in vitro activities. The length of the hydrophilic spacer does not affect the affinity for delta receptors, whereas shorter spacer length increases affinity for mu and even more so for kappa receptors. Thus receptor type-selective peptides for opioid receptors can be designed using the bivalent approach.     

scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies.

Single-chain Fv antibody fragments (scFvs) incorporate a polypeptide linker to tether the VH and VL domains together. An scFv molecule with a linker 5-12 residues long cannot fold into a functional Fv domain and instead associates with a second scFv molecule to form a bivalent dimer (diabody). Direct ligation of VH and VL domains further restricts association and forces three scFv molecules to associate into a trivalent trimer (triabody). We have defined the effect of linker length on scFv association by constructing a series of scFvs from anti-neuraminidase antibody NC10 in which the linker varied from one to four glycine residues. NC10 scFv molecules containing linkers of three and four residues showed a strong preference for dimer formation (diabodies), whereas a linker length of one or two glycine residues prevented the formation of diabodies and directed scFv association into trimers (triabodies). The data suggest a relatively strict transition from dimer (diabody) to trimer (triabody) upon reduction of the linker length from three to two glycine residues. Modelling studies are consistent with three residues as the minimum linker length compatible with diabody formation. Electron microscope images of complexes formed between the NC10 scFv multimers and an anti-idiotype Fab' showed that the dimer was bivalent for antigen binding and the trimer was trivalent.     

Design and evaluation of novel bivalent thrombin inhibitors based on amidinophenylalanines.

Two bivalent thrombin inhibitors were synthesized, which consist of a benzamidine-based active-site-blocking segment, a fibrinogen recognition exosite inhibitor and a peptidic linker connecting these fragments. BZA-1 hirulog contains an Nalpha-(2-naphthylsulfonyl)-S-3-amidinophenylalanyl-is onipecotic acid residue connected via the carboxyl group to the linker segment. The active-site-directed moiety of BZA-2 hirulog [Nalpha-(2-naphthylsulfonyl-glutamyl)-R-4-amidinophenylal anyl-piperid ide] was coupled to the linker via the side chain of the glutamic acid. Both BZA-hirulogs contain almost identical linker-exo site inhibitor parts, except for the substitution of a glycine as the first linker residue in BZA-1 hirulog by a gamma-amino butyric acid in BZA-2 hirulog, thus increasing flexibility and linker length by two additional atoms. BZA-1 hirulog showed moderate potency (Ki = 0. 50 +/- 0.14 nM), while BZA-2 hirulog was characterized as a slow, tight binding inhibitor of thrombin (Ki = 0.29 +/- 0.08 pM). The stability in human plasma of both analogs was strongly improved compared with hirulog-1. For BZA-2 hirulog a significantly reduced plasma clearance was observed after intravenous injection in rats compared with BZA-1 hirulog and hirulog-1. The X-ray structure of the BZA-2 hirulog in complex with human alpha-thrombin was solved and confirmed the expected bivalent binding mode.     

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.     

Linker Dependence of Avidity in Multivalent Interactions Between Disordered Proteins

Multidomain proteins often interact through several independent binding sites connected by disordered linkers. The architecture of such linkers affects avidity by modulating the effective concentration of intramolecular binding. The linker dependence of avidity has been estimated theoretically using simple physical models, but such models have not been tested experimentally because the effective concentrations could not be measured directly. We have developed a model system for bivalent protein interactions connected by disordered linkers, where the effective concentration can be measured using a competition experiment. We characterized the bivalent protein interactions kinetically and thermodynamically for a variety of linker lengths and interaction strengths. In total, this allowed us to critically assess the existing theoretical models of avidity in disordered, multivalent interactions. As expected, the onset of avidity occurs when the effective concentration reached the dissociation constant of the weakest interaction. Avidity decreased monotonously with linker length, but only by a third of what is predicted by theoretical models. We suggest that the length dependence of avidity is attenuated by compensating mechanisms such as linker interactions or entanglement. The direct role of linkers in avidity suggests they provide a generic mechanism for allosteric regulation of disordered, multivalent proteins.     

Bivalent ligands with rigid double-stranded DNA spacers reveal structural constraints on signaling by Fc epsilon RI

Degranulation of mast cells and basophils during the allergic response is initiated by Ag-induced cross-linking of cell surface IgE-Fc epsilon RI receptor complexes. To investigate how separation distances between cross-linked receptors affect the competency of signal transduction, we synthesized and characterized bivalent dinitrophenyl (DNP)-modified dsDNA oligomers with rigid spacing lengths of approximately 40-100 A. All of these bivalent ligands effectively bind and cross-link anti-DNP IgE with similar affinities in the nanomolar range. The 13-mer (dsDNA length of 44 A), 15-mer (51 A), and flexible 30-mer ligands stimulate similar amounts of cellular degranulation, about one-third of that with multivalent Ag, whereas the 20-mer (68 A) ligand is less effective and the rigid 30-mer (102 A) ligand is ineffective. Surprisingly, all stimulate tyrosine phosphorylation of Fc epsilon RI beta, Syk, and linker for activation of T cells to similar extents as multivalent Ag at optimal ligand concentrations. The magnitudes of Ca(2+) responses stimulated by these bivalent DNP-dsDNA ligands are small, implicating activation of Ca(2+) mobilization by stimulated tyrosine phosphorylation as a limiting process. The results indicate that structural constraints on cross-linked IgE-Fc epsilon RI complexes imposed by these rigid DNP-dsDNA ligands prevent robust activation of signaling immediately downstream of early tyrosine phosphorylation events. To account for these results, we propose that activation of a key downstream target is limited by the spacing between cross-linked, phosphorylated receptors and their associated components.     

Synthesis and pharmacological characterization of bivalent ligands of epibatidine at neuronal nicotinic acetylcholine receptors

A series of bivalent ligands 6a-d of epibatidine were synthesized. All four ligands showed nanomolar binding affinities at six neuronal nicotinic acetylcholine receptor (nAChR) subtypes in competition binding assays. In contrast to epibatidine, these bivalent ligands are weak partial agonists at the alpha3beta4 nAChR as shown by functional assays.     

Synthesis and evaluation of aryl-substituted diarylpropionitriles,selective ligands for estrogen receptor β, as positron-emission tomographic imaging agents

We have investigated halogen-substituted non-steroidal estrogens with selective binding affinity for the estrogen receptor β (ERβ that might be used for imaging the levels of this ER-subtype in breast tumors by positron emission tomography (PET). Based on diarylpropionitrile (DPN, 1a), a compound previously reported that has a 72-fold binding selectivity for ERβ, we developed a series of DPN analogs having methyl-, hydroxyl-, and halogen substituents, including fluoroethyl and fluoropropyl groups. In competitive radiometric binding assays with [³H]estradiol, all of these DPN analogs showed high ERβ/ERα selectivity; while the selectivity varied, in some cases it reached nearly 300-fold (RBA: ERα, 0.023%; ERβ, 6.25%). The absolute ERβ binding affinities, however, were not sufficient to merit further consideration for developing these ligands as PET imaging agents.