Oliver Demmer

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop.

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90‐binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α‐helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A–TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide‐binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C‐terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1–DP1 module may serve as an Hsp70–client delivery system for the TPR2A–TPR2B–DP2 segment, which is required for client activation in vivo.

PET of CXCR4 expression by a (68)Ga-labeled highly specific targeted contrast agent.

The overexpression of the chemokine receptor CXCR4 plays an important role in oncology, since together with its endogenous ligand, the stromal cell–derived factor (SDF1-α), CXCR4 is involved in tumor development, growth, and organ-specific metastasis. As part of our ongoing efforts to develop highly specific CXCR4-targeted imaging probes and with the aim to assess the suitability of this ligand for first proof-of-concept studies in humans, we further evaluated the new 68Ga-labeled high-affinity cyclic CXCR4 ligand, 68Ga-CPCR4-2 (cyclo(D-Tyr1-[NMe]-D-Orn2-[4-(aminomethyl) benzoic acid,68Ga-DOTA]-Arg3-2-Nal4-Gly5)). Methods: Additional biodistribution and competitions studies in vivo, dynamic PET studies, and investigations on the metabolic stability and plasma protein binding were performed in nude mice bearing metastasizing OH1 human small cell lung cancer xenografts. CXCR4 expression on OH1 tumor sections was determined by immunohistochemical staining. Results: natGa-CPCR4-2 exhibits high CXCR4 affinity with a half maximum inhibitory concentration of 4.99 ± 0.72 nM. 68Ga-CPCR4-2 showed high in vivo stability and high and specific tumor accumulation, which was reduced by approximately 80% in competition studies with AMD3100. High CXCR4 expression in tumors was confirmed by immunohistochemical staining. 68Ga-CPCR4-2 showed low uptake in nontumor tissue and particularly low kidney accumulation despite predominant renal excretion, leading to high-contrast delineation of tumors in small-animal PET studies. Conclusion: The small and optimized cyclic peptide CPCR4-2 labeled with 68Ga is a suitable tracer for targeting and imaging of human CXCR4 receptor expression in vivo. The high affinity for CXCR4, its in vivo stability, and the excellent pharmacokinetics recommend the further evaluation of 68Ga-CPCR4-2 in a proof-of-concept study in humans.

PET imaging of CXCR4 receptors in cancer by a new optimized ligand.

CXCR4 Imaging: Based on a high-affinity CXCR4 ligand, an imaging agent for CXCR4-positive tumors was developed through structure–activity relationship studies. The best compound was evaluated in vivo and shown to have excellent properties as a positron emission tomography (PET) tracer.

Design, Synthesis, and Functionalization of Dimeric Peptides Targeting Chemokine Receptor CXCR4

The chemokine receptor CXCR4 is a critical regulator of inflammation and immune surveillance, and it is specifically implicated in cancer metastasis and HIV-1 infection. On the basis of the observation that several of the known antagonists remarkably share a C(2) symmetry element, we constructed symmetric dimers with excellent antagonistic activity using a derivative of a cyclic pentapeptide as monomer. To optimize the binding affinity, we investigated the influence of the distance between the monomers and the pharmacophoric sites in the synthesized constructs. The affinity studies in combination with docking computations support a two-site binding model. In a final step, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was introduced as chelator for (radio-)metals, thus allowing to exploit these compounds as a new group of CXCR4-binding peptidic probes for molecular imaging and endoradiotherapeutic purposes. Both the DOTA conjugates and some of their corresponding metal complexes retain good CXCR4 affinity, and one (68)Ga labeled compound was studied as PET tracer.

BclxL Changes Conformation upon Binding to Wild-type but Not Mutant p53 DNA Binding Domain *□

p53 can induce apoptosis through mitochondrial membrane permeabilization by interaction of its DNA binding region with the anti-apoptotic proteins BclxL and Bcl2. However, little is known about the action of p53 at the mitochondria in molecular detail. By using NMR spectroscopy and fluorescence polarization we characterized the binding of wild-type and mutant p53 DNA binding domains to BclxL and show that the wild-type p53 DNA binding domain leads to structural changes in the BH3 binding region of BclxL, whereas mutants fail to induce such effects due to reduced affinity. This was probed by induced chemical shift and residual dipolar coupling data. These data imply that p53 partly achieves its pro-apoptotic function at the mitochondria by facilitating interaction between BclxL and BH3-only proteins in an allosteric mode of action. Furthermore, we characterize for the first time the binding behavior of Pifithrin-μ, a specific small molecule inhibitor of the p53-BclxL interaction, and present a structural model of the protein-ligand complex. A rather unusual behavior is revealed whereby Pifithrin-μ binds to both sides of the protein-protein complex. These data should facilitate the rational design of more potent specific BclxL-p53 inhibitors.

Introduction of functional groups into peptides via N-alkylation.

An optimized protocol for the mild and selective Fukuyama-Mitsunobu reaction was used for mono- and di- N-alkylation on solid support. Thereby, nonfunctionalized aliphatic and aromatic residues are quickly introduced into transiently protected, primary amines of a linear peptide. N-Alkylation can also be used to implement alkyl chains carrying (protected) functionalities suited for subsequent modification. Applicability of this method is demonstrated by various N-alkylated analogues of a cyclic CXCR4 receptor antagonist originally developed by Fujii et. al.

A Conformationally Frozen Peptoid Boosts CXCR4 Affinity and Anti‐HIV Activity

The chemokine receptor subtype CXCR4 belongs to the G-protein coupled receptors (GPCRs) and is, together with itsnatural ligand CXCL12 (or SDF-1), a central part of thesignaling system in the human body. Its functions range fromstem-cell trafficking during embryogenesis, through cardio-vascular, hematopoietic, and brain development, to signalingin the nervous and immune system.