Selective and potent proteomimetic inhibitors of intracellular protein–protein interactions

Inhibition of protein–protein interactions (PPIs) represents a major challenge in chemical biology and drug discovery. α-Helix mediated PPIs may be amenable to modulation using generic chemotypes, termed “proteomimetics”, which can be assembled in a modular manner to reproduce the vectoral presentation of key side chains found on a helical motif from one partner within the PPI. In this work, it is demonstrated that by using a library of N-alkylated aromatic oligoamide helix mimetics, potent helix mimetics which reproduce their biophysical binding selectivity in a cellular context can be identified

An α-Helix-Mimicking 12,13-Helix: Designed α/β/γ-Foldamers as Selective Inhibitors of Protein-Protein Interactions

A major current challenge in bioorganic chemistry is the identification of effective mimics of protein secondary structures that act as inhibitors of protein–protein interactions (PPIs). In this work, trans-2-aminocyclobutanecarboxylic acid (tACBC) was used as the key β-amino acid component in the design of α/β/γ-peptides to structurally mimic a native α-helix. Suitably functionalized α/β/γ-peptides assume an α-helix-mimicking 12,13-helix conformation in solution, exhibit enhanced proteolytic stability in comparison to the wild-type α-peptide parent sequence from which they are derived, and act as selective inhibitors of the p53/hDM2 interaction

Cyclic and macrocyclic peptides as chemical tools to recognise protein surfaces and probe protein-protein interactions

Targeting protein surfaces and protein–protein interactions (PPIs) with small molecules is a frontier goal of chemical biology and provides attractive therapeutic opportunities in drug discovery. The molecular properties of protein surfaces, including their shallow features and lack of deep binding pockets, pose significant challenges, and as a result have proved difficult to target. Peptides are ideal candidates for this mission due to their ability to closely mimic many structural features of protein interfaces. However, their inherently low intracellular stability and permeability and high in vivo clearance have thus far limited their biological applications. One way to improve these properties is to constrain the secondary structure of linear peptides by cyclisation. Herein we review various classes of cyclic and macrocyclic peptides as chemical probes of protein surfaces and modulators of PPIs. The growing interest in this area and recent advances provide evidence of the potential of developing peptide‐like molecules that specifically target these interactions

Stereocontrolled protein surface recognition using chiral oligoamide proteomimetic foldamers

The development of foldamers capable of selective molecular recognition of solvent exposed protein surfaces represents an outstanding challenge in supramolecular chemical biology. Here we introduce an oligoamide foldamer with well-defined conformation that bears all the hallmarks of an information rich oligomer. Specifically, the foldamer recognizes its target protein hDM2 leading to inhibition of its protein–protein interaction with p53 in a manner that depends upon the composition, spatial projection and stereochemistry of functional groups appended to the scaffold. Most significantly, selective inhibition of p53/hDM2 can be achieved against four other targets and the selectivity for p53/hDM2 inhibition versus Mcl-1/NOXA-B inhibition is critically dependent upon the stereochemistry of the helix mimetic

Site-specific labeling uncovers differences in levels and distribution of B-cell receptors of different isotypes on primary B cells

Expression of the BCR is essential for survival, development, and effector functions of B cells. Naive B cells express surface IgM and IgD, while surface IgG1 is expressed by class-switched (memory) B cells. Despite similar overall structures, the different BCR isotypes show differences in distribution and expression levels. The dynamics of BCR behavior have been difficult to explore owing to a lack of appropriate tools that can track the BCR without causing concomitant activation. Using CRISPR-Cas9, we inserted a sortase recognition motif (LPETG [LeuProGluThrGly]) at the C-terminus of the OB1 transnuclear ovalbumin-specific Cκ chain (Igκ-LPETG mice). The surface BCR from Igκ-LPETG mice is fully functional and can be labeled site-specifically with biotin or fluorophores. Igκ-LPETG mice show near-normal B-cell development, with an increase in Igλ-producing cells, presumably due to massive contraction of the κ locus V-region cluster upon V-J recombination to generate the OB1 light chain. Using the Igκ-LPETG mice, we compared organization and density of BCRs on the surface of IgM/IgD+ B cells bearing a wild-type (WT) heavy chain locus and IgG1 B cells in the OB1 model. The density of IgG1 BCRs is much reduced compared to IgM/IgD BCRs on primary B cells. Upon activation, IgM/IgD BCRs are found in detergent-insoluble domains, whereas IgG1 BCRs are not. The isotype of the Ig heavy chain thus contributes to surface expression and nanoscale organization of the BCR

Backbone modification of a polypeptide drug alters duration of action in vivo

because of protease-catalyzed degradation. We used PTHR1 signaling to evaluate a strategy for creating active and biostable backbone-modified analogs of the well-known agonist PTH(1-34). PTH is an 84-residue protein that controls key physiological processes, including the maintenance of extracellular levels of calcium and phosphate and bone homeostasis 1 . PTH(1-34) matches full-length PTH in potency and efficacy at PTHR1 and is the active ingredient in the osteoporosis drug teriparatide (Forteo). As with many other peptide-based therapeutics, PTH(1-34) has a short half-life in the bloodstream (<30 min) 2 . Therapeutic effects for osteoporosis treatment appear to be maximized by pulsatile rather than continuous exposure to PTH(1-34), but the optimal exposure cycle is unclear 3 . We generated new analogs of PTH(1-34) by replacing selected α-amino acid residues with homologous β-amino acid residues, an unconventional approach that alters the backbone but can maintain the natural side chain complement. The results show that this technically straightforward strategy can provide hormone analogs that display native-like receptor activation potencies and prolonged residency in the bloodstream. The C-terminal portion of PTH(1-34) forms an α-helix upon binding to the receptor, but the bioactive conformation of the N-terminal segment is unknown. The backbone-modification strategy described here is based on previous studies showing that α-helical segments Systematic modification of the backbone of bioactive polypeptides through b-amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein-coupled receptor in the B-family, we present an approach for a→b residue replacement that enables both high activity and improved pharmacokinetic properties in vivo. involved in protein-recognition processes can be mimicked by oligomers containing mixtures of α and β residues 4,5 . Other types of unnatural oligomers, such as peptides composed of D-α-amino acid residues 6 , peptoids 7 and β-peptides 8 , have been explored for functional mimicry of bioactive α-helices; however, none of these alternative molecular scaffolds allows faithful mimicry of a long α-helix 5 , as required for potent analogs of PTH. In previous studies, PTH analogs containing one to three β-residue replacements were used to probe local conformational requirements, and many of these replacements caused profound declines in agonist activity We prepared all four PTH(1-34) analogs containing five α→β 3 replacements in an αααβ pattern 13 within the C-terminal region (A5-D5 in PTHR1 has two distinct functional states: RG, which forms when the intracellular portion contacts G αS (a heterotrimeric G-protein responsible for activating adenylate cyclase upon receptor activation); and R 0 , which forms independent of G αS 15,16 . An agonist's affinity for the RG state correlates with PTHR1 activation potency, whereas R 0 affinity correlates with the duration of some in vivo response

Altered signaling and desensitization responses in PTH1R mutants associated with Eiken syndrome

The parathyroid hormone receptor type 1 (PTH1R) is a G protein-coupled receptor that plays key roles in regulating calcium homeostasis and skeletal development via binding the ligands, PTH and PTH-related protein (PTHrP), respectively. Eiken syndrome is a rare disease of delayed bone mineralization caused by homozygous PTH1R mutations. Of the three mutations identified so far, R485X, truncates the PTH1R C-terminal tail, while E35K and Y134S alter residues in the receptor\u27s amino-terminal extracellular domain. Here, using a variety of cell-based assays, we show that R485X increases the receptor\u27s basal rate of cAMP signaling and decreases its capacity to recruit β-arrestin2 upon ligand stimulation. The E35K and Y134S mutations each weaken the binding of PTHrP leading to impaired β-arrestin2 recruitment and desensitization of cAMP signaling response to PTHrP but not PTH. Our findings support a critical role for interaction with β-arrestin in the mechanism by which the PTH1R regulates bone formation

Generation of Site‐Specifically Labeled Affinity Reagents via Use of a Self‐Labeling Single Domain Antibody

Several chemical and enzymatic methods have been used to link antibodies to moieties that facilitate visualization of cognate targets. Emerging evidence suggests that the extent of labeling, dictated by the type of chemistry used, has a substantial impact on performance, especially in the context of antibodies used for the visualization of tumors in vivo. These effects are particularly pronounced in studies using small antibody fragments, such as single‐domain antibodies, or nanobodies. Here, we leverage a new variety of conjugation chemistry, based on a nanobody that forms a crosslink with a specialized high‐affinity epitope analogue, to label target‐specific nanobody constructs with functionalities of choice, including fluorophores, chelators, and click chemistry handles. Using heterodimeric nanobody conjugates, comprised of an antigen recognition module and a self‐labeling module, enables us to attach the desired functional group at a location distal to the site of antigen recognition. Constructs generated using this approach bound to antigens expressed on xenograft murine models of liver cancer and allowed for non‐invasive diagnostic imaging. The modularity of our approach using a self‐labeling nanobody offers a novel method for site‐specific functionalization of biomolecules and can be extended to other applications for which covalent labeling is required

Matrix Organization Theory

Matrix organization management is a rather new concept of organization and management. Variations of traditional and project organizations have been utilized for centuries. However, the organization structure denoted by the term "matrix” has been recognized and documented only over the past two decades. Its development was greatly accelerated by the needs of the aerospace industry. Rapidly changing conditions in the business world in general have led to creation and use of new relationships to augment established organizational and management concepts. | Like many new concepts, the concept of matrix organization management has not been consistently understood. Consequently, it has been used verv appropriately in some situations and grossly misused in others. Some managers have hailed the matrix concept as a boon to mankind and the heir apparent to traditional organization structure. Managers at the other extreme feel that the matrix concept has been greatly overrated and is little more than a passing fad that creates more problems than it cures. | Educators and businessmen have written numerous articles on various aspects of matrix organization and management wherein conflicting and opposing statements and varying viewpoints have been expressed. Many of these efforts have tended to thoroughly muddy the water.ProQuest Traditional Publishing Optio

Rapid covalent labeling of a GPCR on living cells using a nanobody-epitope tag pair to interrogate receptor pharmacology

AbstractSynthetic molecules that form a covalent bond upon binding to a targeted biomolecule (proximity-induced reactivity) are the subject of intense biomedical interest for the unique pharmacological properties imparted by irreversible binding. However, off-target covalent labeling and the lack of molecules with sufficient specificity limit more widespread applications. We describe the first example of a crosslinking platform that uses a synthetic peptide epitope and a single domain antibody (or nanobody) pair to form a covalent linkage rapidly and specifically. The rate of the crosslinking reaction between peptide and nanobody is faster than most other biocompatible crosslinking reactions, and it can be used to label live cells expressing receptor-nanobody fusions. The rapid kinetics of this system allowed us to probe the consequences on signaling for ligand crosslinking to the A2A-adenosine receptor. Our method may be generally useful to site-specifically link synthetic molecules to receptors on mammalian cell surfaces.</jats:p