N-tert-Butoxycarbonyl-N-(2-(tritylthio)ethoxy)glycine as a Building Block for Peptide Ubiquitination.

N-Boc-N-(2-(tritylthio)ethoxy)glycine has been developed as a building block for peptide ubiquitination, which is fully compatible with solid-phase Fmoc chemistry and common peptide modifications including phosphorylation, methylation, acetylation, biotinylation, and fluorescence labeling. The optimal conditions for peptide cleavage and auxiliary removal were obtained. The utility of this building block in peptide ubiquitination was demonstrated by the synthesis of seven ubiquitinated histone and Tau peptides bearing various modifications. Cys residues were well tolerated and did not require orthogonal protection. The structural integrity and folding of the synthesized ubiquitinated peptides were confirmed by enzymatic deubiquitination of a fluorescently labeled ubiquitin conjugate. The synthetic strategy using this building block provides a practical approach for the preparation of ubiquitinated peptides with diverse modifications.

Evaluating Translational Efficiency of Noncanonical Amino Acids to Inform the Design of Druglike Peptide Libraries.

Advances in genetic code reprogramming have allowed the site-specific incorporation of noncanonical functionalities into polypeptides and proteins, providing access to wide swaths of chemical space via in vitro translation techniques like mRNA display. Prior efforts have established that the translation machinery can tolerate amino acids with modifications to both the peptide backbone and side chains, greatly broadening the chemical space that can be interrogated in ligand discovery efforts. However, existing methods for confirming the translation yield of new amino acid building blocks for these technologies necessitate multistep workups and, more importantly, are not relevant for measuring translation within the context of a combinatorial library consisting of multiple noncanonical amino acids. In this study, we developed a luminescence-based assay to rapidly assess the relative translation yield of any noncanonical amino acid in real time. Among the 59 amino acids tested here, we found that many translate with high efficiency, but translational yield is not necessarily correlated to whether the amino acid is proteinogenic or has high tRNA acylation efficiency. Interestingly, we found that single-template translation data can inform the library-scale translation yield and that shorter peptide libraries are more tolerant of lower-efficiency amino acid monomers. Together our data show that the luminescence-based assay described herein is an essential tool in evaluating new building blocks and codon table designs within mRNA display toward the goal of developing druglike peptide-based libraries for drug discovery campaigns.

Incorporating NOE-Derived Distances in Conformer Generation of Cyclic Peptides with Distance Geometry.

Nuclear magnetic resonance (NMR) data from NOESY (nuclear Overhauser enhancement spectroscopy) and ROESY (rotating frame Overhauser enhancement spectroscopy) experiments can easily be combined with distance geometry (DG) based conformer generators by modifying the molecular distance bounds matrix. In this work, we extend the modern DG based conformer generator ETKDG, which has been shown to reproduce experimental crystal structures from small molecules to large macrocycles well, to include NOE-derived interproton distances. In noeETKDG, the experimentally derived interproton distances are incorporated into the distance bounds matrix as loose upper (or lower) bounds to generate large conformer sets. Various subselection techniques can subsequently be applied to yield a conformer bundle that best reproduces the NOE data. The approach is benchmarked using a set of 24 (mostly) cyclic peptides for which NOE-derived distances as well as reference solution structures obtained by other software are available. With respect to other packages currently available, the advantages of noeETKDG are its speed and that no prior force-field parametrization is required, which is especially useful for peptides with unnatural amino acids. The resulting conformer bundles can be further processed with the use of structural refinement techniques to improve the modeling of the intramolecular nonbonded interactions. The noeETKDG code is released as a fully open-source software package available at www.github.com/rinikerlab/customETKDG.

A novel method to produce synthetic murine CXCL10 for efficient screening of functional variants.

Antitumor immune responses depend on the infiltration of solid tumors by effector T cells, a process guided by chemokines. In particular, the chemokine CXCL10 has been shown to play a critical role in mediating recruitment of CXCR3 + cytolytic T and NK cells in tumors, though its use as a therapeutic agent has not been widely explored. One of the limitations is due to the rapid inactivation of CXCL10 by dipeptidyl peptidase 4 (DPP4), a broadly expressed enzyme that is active in plasma and other bodily fluids. In the present study, we describe a novel method to produce synthetic CXCL10 that is resistant to DPP4 N-terminal truncation. Using a Fmoc solid-phase peptide synthesis approach, synthetic murine WT CXCL10 was produced, showing similar biochemical and biological properties to the recombinant protein. This synthesis method supported production of natural (amino acid substitution, insertion or deletion) and non-natural (chemical modifications) variants of CXCL10. In association with a functional screening cascade that assessed DPP4-mediated cleavage, CXCR3 signaling potency and chemotactic activity, we successfully generated 20 murine CXCL10 variants. Among those, two non-natural variants with N-methylated Leu3 (MeLeu3) and a reduced amide bond between Pro2 and Leu3 (rLeu3), respectively, showed resistance to DPP4 truncation but decreased CXCR3 signaling and chemotactic activity. Interestingly, MeLeu3 and rLeu3 CXCL10 behaved as DPP4 inhibitors, preventing the truncation of WT CXCL10. This study highlights the potential of using Fmoc solid-phase chemistry in association with biochemical and biological characterization to rapidly identify CXCL10 variants with desired properties. These novel methods unlock the opportunity to develop DPP4 resistant CXCL10 variants, as well as other chemokine substrates, while maintaining chemotactic properties.

Small Molecule Dysregulation of TEAD Lipidation Induces a Dominant-Negative Inhibition of Hippo Pathway Signaling.

The transcriptional enhanced associate domain (TEAD) family of transcription factors serves as the receptors for the downstream effectors of the Hippo pathway, YAP and TAZ, to upregulate the expression of multiple genes involved in cellular proliferation and survival. Recent work identified TEAD S-palmitoylation as critical for protein stability and activity as the lipid tail extends into a hydrophobic core of the protein. Here, we report the identification and characterization of a potent small molecule that binds the TEAD lipid pocket (LP) and disrupts TEAD S-palmitoylation. Using a variety of biochemical, structural, and cellular methods, we uncover that TEAD S-palmitoylation functions as a TEAD homeostatic protein level checkpoint and that dysregulation of this lipidation affects TEAD transcriptional activity in a dominant-negative manner. Furthermore, we demonstrate that targeting the TEAD LP is a promising therapeutic strategy for modulating the Hippo pathway, showing tumor stasis in a mouse xenograft model.

Heterobifunctional Molecules Induce Dephosphorylation of Kinases-A Proof of Concept Study.

Heterobifunctional molecules have proven powerful tools to induce ligase-dependent ubiquitination of target proteins. We describe here a chemical strategy for controlling a different post-translational modification (PTM): phosphorylation. Heterobifunctional molecules were designed to promote the proximity of a protein phosphatase (PP1) to protein targets. The synthesized molecules induced the PP1-dependent dephosphorylation of AKT and EGFR. To our knowledge, this work represents the first examples of small molecules recruiting non-native partners to induce removal of a PTM.

Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

Temporal trends in lipid screening and therapy among youth from 2002 to 2012.

BACKGROUND: Pediatric lipid management recommendations have evolved from selective screening to universal screening to identify and target therapy for genetic dyslipidemias. Data on the success of the selective screening guidelines for lipid testing, dyslipidemia detection, and lipid management are conflicting. OBJECTIVE: To determine temporal trends in lipid testing, dyslipidemia categories and pharmacotherapy in a cohort of 653,642 individual youth aged 2 to 20 years from 2002 to 2012. METHODS: Summary data on lipid test results, lipid-lowering medicine (LLM) dispensings, and International Classification of Diseases, Ninth Revision diagnoses were compiled from the virtual data warehouses of 5 sites in the Cardiovascular Research Network. Temporal trends were determined using linear regression. RESULTS: Among the average 255,160 ± 25,506 children enrolled each year, lipid testing declined from 16% in 2002 to 11% in 2012 (P < .001 for trend). Among the entire population, the proportion newly detected each year with low-density lipoprotein cholesterol >190 mg/dL, a value commonly used to define familial hypercholesterolemia, increased over time from 0.03% to 0.06% (P = .03 for trend). There was no significant change over time in the proportion of the yearly population initiated on LLM or statins specifically (0.045 ± 0.009%, P = .59 [LLM] and 0.028 ± 0.006%, P = .25 [statin]). CONCLUSIONS: Although lipid testing declined during 2002 to 2012, the detection of familial hypercholesterolemia-level low-density lipoprotein cholesterol increased. Despite this increased detection, pharmacotherapy did not increase over time. These findings highlight the need to enhance lipid screening and management strategies in high-risk youth.

Development of orthogonally protected hypusine for solid-phase peptide synthesis.

An orthogonally protected hypusine reagent was developed for solid-phase synthesis of hypusinated peptides using the Fmoc/t-Bu protection strategy. The reagent was synthesized in an overall yield of 27% after seven steps from Cbz-Lys-OBzl and (R)-3-hydroxypyrrolidin-2-one. The side-chain protecting groups (Boc and t-Bu) are fully compatible with standard Fmoc chemistry and can be readily removed during the peptide cleavage step. The utility of the reagent was demonstrated by solid-phase synthesis of hypusinated peptides.

An allosteric switch for pro-HGF/Met signaling using zymogen activator peptides.

Stimulation of hepatocyte growth factor (HGF) signaling through the Met receptor is an attractive approach for promoting tissue repair and preventing fibrosis. Using structure-guided peptide phage display combined with an activity-based sorting strategy, we engineered allosteric activators of zymogen-like pro-HGF to bypass proteolytic activation and reversibly stimulate pro-HGF signaling through Met. Biochemical, structural and biological data showed that zymogen activator peptides (ZAPtides) potently and selectively bind the activation pocket within the serine protease-like ß-chain of pro-HGF and display titratable activation of pro-HGF-dependent Met signaling, leading to cell survival and migration. To further demonstrate the versatility of our ZAPtide platform, we identified allosteric activators for pro-macrophage stimulating protein and a zymogen serine protease, Protein C, which also provides evidence for target selectivity. These studies reveal that ZAPtides use molecular mimicry of the trypsin-like N-terminal insertion mechanism and establish a new paradigm for selective pharmacological activation of plasminogen-related growth factors and zymogen serine proteases.

Identification of a small peptide that inhibits PCSK9 protein binding to the low density lipoprotein receptor.

PCSK9 (proprotein convertase subtilisin/kexin type 9) is a negative regulator of the hepatic LDL receptor, and clinical studies with PCSK9-inhibiting antibodies have demonstrated strong LDL-c-lowering effects. Here we screened phage-displayed peptide libraries and identified the 13-amino acid linear peptide Pep2-8 as the smallest PCSK9 inhibitor with a clearly defined mechanism of inhibition that has been described. Pep2-8 bound to PCSK9 with a KD of 0.7 µm but did not bind to other proprotein convertases. It fully restored LDL receptor surface levels and LDL particle uptake in PCSK9-treated HepG2 cells. The crystal structure of Pep2-8 bound to C-terminally truncated PCSK9 at 1.85 Å resolution showed that the peptide adopted a strand-turn-helix conformation, which is remarkably similar to its solution structure determined by NMR. Consistent with the functional binding site identified by an Ala scan of PCSK9, the structural Pep2-8 contact region of about 400 Å(2) largely overlapped with that contacted by the EGF(A) domain of the LDL receptor, suggesting a competitive inhibition mechanism. Consistent with this, Pep2-8 inhibited LDL receptor and EGF(A) domain binding to PCSK9 with IC50 values of 0.8 and 0.4 µm, respectively. Remarkably, Pep2-8 mimicked secondary structural elements of the EGF(A) domain that interact with PCSK9, notably the ß-strand and a discontinuous short α-helix, and it engaged in the same ß-sheet hydrogen bonds as EGF(A) does. Although Pep2-8 itself may not be amenable to therapeutic applications, this study demonstrates the feasibility of developing peptidic inhibitors to functionally relevant sites on PCSK9.

Geographic variation in cardiovascular procedure use among Medicare fee-for-service vs Medicare Advantage beneficiaries.

IMPORTANCE: Little is known about how different financial incentives between Medicare Advantage and Medicare fee-for-service (FFS) reimbursement structures influence use of cardiovascular procedures. OBJECTIVE: To compare regional cardiovascular procedure rates between Medicare Advantage and Medicare FFS beneficiaries. DESIGN, SETTING, AND PARTICIPANTS: Cross-sectional study of Medicare beneficiaries older than 65 years between 2003-2007 comparing rates of coronary angiography, percutaneous coronary intervention (PCI), and coronary artery bypass graft (CABG) surgery across 32 hospital referral regions in 12 states. MAIN OUTCOMES AND MEASURES: Rates of coronary angiography, PCI, and CABG surgery. RESULTS: We evaluated a total of 878,339 Medicare Advantage patients and 5,013,650 Medicare FFS patients. Compared with Medicare FFS patients, Medicare Advantage patients had lower age-, sex-, race-, and income-adjusted procedure rates per 1000 person-years for angiography (16.5 [95% CI, 14.8-18.2] vs 25.9 [95% CI, 24.0-27.9]; P < .001) and PCI (6.8 [95% CI, 6.0-7.6] vs 9.8 [95% CI, 9.0-10.6]; P < .001) but similar rates for CABG surgery (3.1 [95% CI, 2.8-3.5] vs 3.4 [95% CI, 3.1-3.7]; P = .33). There were no significant differences between Medicare Advantage and Medicare FFS patients in the rates per 1000 person-years of urgent angiography (3.9 [95% CI, 3.6-4.2] vs 4.3 [95% CI, 4.0-4.6]; P = .24) or PCI (2.4 [95% CI, 2.2-2.7] vs 2.7 [95% CI, 2.5-2.9]; P = .16). Procedure rates varied widely across hospital referral regions among Medicare Advantage and Medicare FFS patients. For angiography, the rates per 1000 person-years ranged from 9.8 to 40.6 for Medicare Advantage beneficiaries and from 15.7 to 44.3 for Medicare FFS beneficiaries. For PCI, the rates ranged from 3.5 to 16.8 for Medicare Advantage and from 4.7 to 16.1 for Medicare FFS. The rates for CABG surgery ranged from 1.5 to 6.1 for Medicare Advantage and from 2.5 to 6.0 for Medicare FFS. Across regions, we found no statistically significant correlation between Medicare Advantage and Medicare FFS beneficiary utilization for angiography (Spearman r = 0.19, P = .29) and modest correlations for PCI (Spearman r = 0.33, P = .06) and CABG surgery (Spearman r = 0.35, P = .05). Among Medicare Advantage beneficiaries, adjustment for additional cardiac risk factors had little influence on procedure rates. CONCLUSIONS AND RELEVANCE: Although Medicare beneficiaries enrolled in capitated Medicare Advantage programs had lower angiography and PCI procedure rates than those enrolled in Medicare FFS, the degree of geographic variation in procedure rates was substantial among Medicare Advantage beneficiaries and was similar in magnitude to that observed among Medicare FFS beneficiaries.

Discovery of a potent small-molecule antagonist of inhibitor of apoptosis (IAP) proteins and clinical candidate for the treatment of cancer (GDC-0152).

A series of compounds were designed and synthesized as antagonists of cIAP1/2, ML-IAP, and XIAP based on the N-terminus, AVPI, of mature Smac. Compound 1 (GDC-0152) has the best profile of these compounds; it binds to the XIAP BIR3 domain, the BIR domain of ML-IAP, and the BIR3 domains of cIAP1 and cIAP2 with K(i) values of 28, 14, 17, and 43 nM, respectively. These compounds promote degradation of cIAP1, induce activation of caspase-3/7, and lead to decreased viability of breast cancer cells without affecting normal mammary epithelial cells. Compound 1 inhibits tumor growth when dosed orally in the MDA-MB-231 breast cancer xenograft model. Compound 1 was advanced to human clinical trials, and it exhibited linear pharmacokinetics over the dose range (0.049 to 1.48 mg/kg) tested. Mean plasma clearance in humans was 9 ± 3 mL/min/kg, and the volume of distribution was 0.6 ± 0.2 L/kg.

Multiple protein interactions involving proposed extracellular loop domains of the tight junction protein occludin.

Occludin is a tetraspan integral membrane protein in epithelial and endothelial tight junction (TJ) structures that is projected to have two extracellular loops. We have used peptides emulating central regions of human occludin's first and second loops, termed O-A:101-121 and O-B:210-228, respectively, to examine potential molecular interactions between these two regions of occludin and other TJ proteins. A superficial biophysical assessment of A:101-121 and O-B:210-228 showed them to have dissimilar solution conformation characteristics. Although O-A:101-121 failed to strongly interact with protein components of the human epithelial intestinal cell line T84, O-B:210-228 selectively associated with occludin, claudin-one and the junctional adhesion molecule (JAM)-A. Further, the presence of O-B:210-228, but not O-A:101-121, impeded the recovery of functional TJ structures. A scrambled peptide sequences of O-B:210-228 failed to influence TJ assembly. These studies demonstrate distinct properties for these two extracellular segments of the occludin protein and provide an improved understanding of how specific domains of occludin may interact with proteins present at TJ structures.

A membrane-permeant peptide that inhibits MLC kinase restores barrier function in in vitro models of intestinal disease.

BACKGROUND & AIMS: Maintenance of the mucosal barrier is a critical function of intestinal epithelia. Myosin regulatory light chain (MLC) phosphorylation is a common intermediate in the pathophysiologic regulation of this barrier. The aim of this study was to determine whether a membrane permeant inhibitor of MLC kinase (PIK) could inhibit intracellular MLC kinase and regulate paracellular permeability. METHODS: Recombinant MLC and Caco-2 MLC kinase were used for kinase assays. T84 and Caco-2 monolayers were treated with enteropathogenic Escherichia coli (EPEC) or tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma to induce barrier dysfunction. RESULTS: PIK inhibited MLC kinase in vitro and was able to cross cell membranes and concentrate at the perijunctional actomyosin ring. Consistent with these properties, apical addition of PIK reduced intracellular MLC phosphorylation by 22% +/- 2%, increased transepithelial resistance (TER) by 50% +/- 1%, and decreased paracellular mannitol flux rates by 5.2 +/- 0.2-fold. EPEC infection induced TER decreases of 37% +/- 6% that were limited to 16% +/- 5% by PIK. TNF-alpha and IFN-gamma induced TER decreases of 22% +/- 3% that were associated with a 172% +/- 1% increase in MLC phosphorylation. Subsequent PIK addition caused MLC phosphorylation to decrease by 25% +/- 4% while TER increased to 97% +/- 6% of control. CONCLUSIONS: PIK can prevent TER defects induced by EPEC and reverse MLC phosphorylation increases and TER decreases induced by TNF-alpha and IFN-gamma. The data also suggest that TNF-alpha and IFN-gamma regulate TER, at least in part, via the perijunctional cytoskeleton. Thus, PIK may be the prototype for a new class of targeted therapeutic agents that can restore barrier function in intestinal disease states.

Solution structure of a phage-derived peptide antagonist in complex with vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent endothelial cell-specific mediator of angiogenesis and vasculogenesis. VEGF is involved pathologically in cancer, proliferative retinopathy and rheumatoid arthritis, and as such represents an important therapeutic target. Three classes of disulfide-constrained peptides that antagonize binding of the VEGF dimer to its receptors, KDR and Flt-1, were identified previously using phage display methods. NMR studies of a representative peptide from the most potent class of these peptide antagonists, v107 (GGNECDAIRMWEWECFERL), were undertaken to characterize its interactions with VEGF. v107 has no defined structure free in solution, but binding to VEGF induces folding of the peptide. The solution structure of the VEGF receptor-binding domain-v107 complex was determined using 3940 (1970 per VEGF monomer) internuclear distance and 476 (238 per VEGF monomer) dihedral angle restraints derived from NMR data obtained using samples containing either (13)C/(15)N-labeled protein plus excess unlabeled peptide or (13)C/(15)N-labeled peptide plus excess unlabeled protein. Residual dipolar coupling restraints supplemented the structure determination of the complex and were found to increase significantly both the global precision of VEGF in the complex and the agreement with available crystal structures of VEGF. The calculated ensemble of structures is of high precision and is in excellent agreement with the experimental restraints. v107 has a turn-helix conformation with hydrophobic residues partitioned to one face of the peptide and polar or charged residues at the other face. Contacts between two v107 peptides and the VEGF dimer are mediated by primarily hydrophobic side-chain interactions. The v107-binding site on VEGF overlaps partially with the binding site of KDR and is similar to that for domain 2 of Flt-1. The structure of the VEGF-v107 complex provides new insight into how binding to VEGF can be achieved that may be useful for the design of small molecule antagonists.