Exploration of macrocyclic peptide binders to the extracellular CRD domain of human receptor tyrosine kinase-like orphan receptor 1 (ROR1).

Elevated levels of receptor tyrosine kinase-like orphan receptor 1 (RORl) expression are observed in multiple hematological and solid tumors, but not in most of the healthy adult tissues, identifying ROR1 as an attractive target for tumor-specific therapy. Herein we will describe the discovery of macrocyclic peptides as binders of the extracellular Cysteine-Rich Domain (CRD) of human ROR1 via mRNA in vitro selection technology using the PDPS platform, followed by exploration of sidechain SAR of parent macrocycle peptides, fluorescently labeled analogs, and a Peptide Drug Conjugate (PDC). The parent macrocyclic peptides represented by Compound 1 and Compound 14 displayed nanomolar cell-based binding to ROR1 and relatively good internalization in 786-O and MDA-MB-231 tumor cell lines. However, these peptides were not observed to induce apoptosis in Mia PaCa-2 cells, a model pancreatic tumor cell line with a relatively low level of cell surface expression of ROR1.

Biochemical and Structural Characterization of a Peptidic Inhibitor of the YAP:TEAD Interaction That Binds to the α-Helix Pocket on TEAD.

The TEAD transcription factors are the most distal elements of the Hippo pathway, and their transcriptional activity is regulated by several proteins, including YAP. In some cancers, the Hippo pathway is deregulated and inhibitors of the YAP:TEAD interaction are foreseen as new anticancer drugs. The binding of YAP to TEAD is driven by the interaction of an α-helix and an Ω-loop present in its TEAD-binding domain with two distinct pockets at the TEAD surface. Using the mRNA-based display technique to screen a library of in vitro-translated cyclic peptides, we identified a peptide that binds with a nanomolar affinity to TEAD. The X-ray structure of this peptide in complex with TEAD reveals that it interacts with the α-helix pocket. Under our experimental conditions, this peptide can form a ternary complex with TEAD and YAP. Furthermore, combining it with a peptide binding to the Ω-loop pocket gives an additive inhibitory effect on the YAP:TEAD interaction. Overall, our results show that it is possible to identify nanomolar inhibitors of the YAP:TEAD interaction that bind to the α-helix pocket, suggesting that developing such compounds might be a strategy to treat cancers where the Hippo pathway is deregulated.

Identification of a PCSK9-LDLR disruptor peptide with in vivo function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates plasma low-density lipoprotein cholesterol (LDL-C) levels by promoting hepatic LDL receptor (LDLR) degradation. Therapeutic antibodies that disrupt PCSK9-LDLR binding reduce LDL-C concentrations and cardiovascular disease risk. The epidermal growth factor precursor homology domain A (EGF-A) of the LDLR serves as a primary contact with PCSK9 via a flat interface, presenting a challenge for identifying small molecule PCSK9-LDLR disruptors. We employ an affinity-based screen of 1013in vitro-translated macrocyclic peptides to identify high-affinity PCSK9 ligands that utilize a unique, induced-fit pocket and partially disrupt the PCSK9-LDLR interaction. Structure-based design led to molecules with enhanced function and pharmacokinetic properties (e.g., 13PCSK9i). In mice, 13PCSK9i reduces plasma cholesterol levels and increases hepatic LDLR density in a dose-dependent manner. 13PCSK9i functions by a unique, allosteric mechanism and is the smallest molecule identified to date with in vivo PCSK9-LDLR disruptor function.

Discovery, X-ray structure and CPP-conjugation enabled uptake of p53/MDM2 macrocyclic peptide inhibitors.

Mouse double minute 2 homolog (MDM2, Hdm2) is an important negative regulator of the tumor suppressor p53. Using a mRNA based display technique to screen a library of >1012 in vitro-translated cyclic peptides, we have identified a macrocyclic ligand that shows picomolar potency on MDM2. X-Ray crystallography reveals a novel binding mode utilizing a unique pharmacophore to occupy the Phe/Trp/Leu pockets on MDM2. Conjugation of a cyclic cell-penetrating peptide (cCPP) to the initially non cell-permeable ligand enables cellular uptake and a pharmacodynamic response in SJSA-1 cells. The demonstrated enhanced intracellular availability of cyclic peptides that are identified by a display technology exemplifies a process for the application of intracellular tools for drug discovery projects.

State of the union: a review of lumbar fusion indications and techniques for degenerative spine disease.

Lumbar fusion is an accepted and effective technique for the treatment of lumbar degenerative disease. The practice has evolved continually since Albee and Hibbs independently reported the first cases in 1913, and advancements in both technique and patient selection continue through the present day. Clinical and radiological indications for surgery have been tested in trials, and other diagnostic modalities have developed and been studied. Fusion practices have also advanced; instrumentation, surgical approaches, biologics, and more recently, operative planning, have undergone stark changes at a seemingly increasing pace over the last decade. As the general population ages, treatment of degenerative lumbar disease will become a more prevalent-and costlier-issue for surgeons as well as the healthcare system overall. This review will cover the evolution of indications and techniques for fusion in degenerative lumbar disease, with emphasis on the evidence for current practices.

TRAP display: a high-speed selection method for the generation of functional polypeptides.

Here, we describe a novel method that enables high-speed in vitro selection of functional peptides, peptidomimetics, and proteins via a simple procedure. We first developed a new cell-free translation system, the TRAP system (transcription-translation coupled with association of puromycin linker), which automatically produces a polypeptide library through a series of sequential reactions: transcription, association of puromycin-DNA linker, translation, and conjugation between the nascent polypeptide and puromycin-DNA linker. We then applied the TRAP system for the selection of macrocyclic peptides against human serum albumin. Six rounds of selection using TRAP display were performed in approximately 14 h, yielding macrocyclic peptides with nanomolar affinity to their target protein. Because TRAP display enables high-speed selection of functional polypeptides, it will facilitate the generation of various polypeptides that are useful for biological and therapeutic applications.

In vitro selection of multiple libraries created by genetic code reprogramming to discover macrocyclic peptides that antagonize VEGFR2 activity in living cells.

We report the in vitro selection of thioether-macrocyclized peptides against vascular endothelial growth factor receptor 2 (VEGFR2) from multiple, highly diverse peptide libraries constructed utilizing genetic code reprogramming. The macrocyclic peptide libraries consisted of combinations of four types of amino acid linkers for cyclization and two types of elongator amino acid compositions, including four backbone-modified non-proteinogenic amino acids. Affinity selection from these libraries, using our recently developed TRAP (Transcription-translation coupled with Association of Puromycin-linker) display, yielded multiple anti-VEGFR2 macrocyclic peptide leads. Further antagonizing activity-based screening of the chemically synthesized lead peptides identified a potent macrocyclic peptide that inhibited VEGF-induced VEGFR2 autophosphorylation, proliferation, and angiogenesis of living vascular endothelial cells. The TRAP display-based selection from multiple, highly diverse peptide libraries followed by activity-based screening of selected peptides is a powerful strategy for discovering biologically active peptides targeted to various biomolecules.

Charging of tRNAs using ribozymes and selection of cyclic peptides containing thioethers.

In vitro selection methods represent a powerful approach toward identifying high-affinity peptide ligands from highly diverse peptide libraries against a desired target. We herein describe a method for the display and selection of cyclic thioether peptide libraries. Reprogramming the initiation event from fMet to an N-chloroacetyl-amino acid by utilizing flexizyme to rapidly and efficiently prepare the aa-tRNA can be effectively used to initiate translation, upon which the thiol group of an inserted cysteine at the C terminus of the designed library spontaneously reacts to yield a nonreducible cyclic thioether peptide readily compatible with any in vitro display methods. Thus, cyclic peptides already in a nonreducible stable form can be selected directly against the target of interest.

The Down syndrome critical region gene 1 short variant promoters direct vascular bed-specific gene expression during inflammation in mice.

Down syndrome critical region gene 1 (DSCR-1) short variant (DSCR-1s) is an inhibitor of calcineurin/NFAT signaling encoded by exons 4-7 of DSCR1. We previously reported that VEGF induces DSCR-1s expression in endothelial cells, which in turn negatively feeds back to attenuate endothelial cell activation. Here, in order to characterize the role of the promoter that drives DSCR-1s expression in mediating inducible expression in vivo and to determine the functional relevance of DSCR-1s in inflammation, we targeted a DNA construct containing 1.7 kb of the human DSCR1s promoter coupled to the lacZ reporter to the hypoxanthine guanine phosphoribosyl transferase (Hprt) locus of mice. We determined that lacZ was uniformly expressed in the endothelium of transgenic embryos but was markedly downregulated postnatally. Systemic administration of VEGF or LPS in adult mice resulted in cyclosporine A-sensitive reactivation of the DSCR1s promoter and endogenous gene expression in a subset of organs, including the heart and brain. The DSCR1s promoter was similarly induced in the endothelium of tumor xenografts. In a mouse model of endotoxemia, DSCR-1s-deficient mice demonstrated increased sepsis mortality, whereas adenovirus-mediated DSCR-1s overexpression protected against LPS-induced lethality. Collectively, these data suggest that the DSCR1s promoter directs vascular bed-specific expression in activated endothelium and that DSCR-1s serves to dampen the host response to infection.

Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.

Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.

Histone deacetylase inhibitor reduces monocyte adhesion to endothelium through the suppression of vascular cell adhesion molecule-1 expression.

OBJECTIVE: Tumor necrosis factor (TNF)-alpha initiates numerous changes in endothelial cell (EC) gene expression that contributes to the pathology of various diseases including inflammation. We hypothesized that TNF-alpha-mediated gene induction involves multiple signaling pathways, and that inhibition of one or more of these pathways may selectively target subsets of TNF-alpha-responsive genes and functions. METHODS AND RESULTS: Human umbilical vein endothelial cells (ECs) were preincubated with inhibitors of PI3 kinase (LY294002), histone deacetylases (HDAC) (trichostatin A [TSA]), de novo protein synthesis (CHX), proteasome (MG-132), and GATA factors (K-11430) before exposure to TNF-alpha at 4 hours and analyzed by microarray. TNF-alpha-mediated induction of vascular cell adhesion molecule-1 (VCAM-1) was attenuated by all of these inhibitors, whereas in contrast, stimulation of intercellular adhesion molecule-1 (ICAM-1) was blocked by MG-132 alone. Moreover TSA blocked TNF-alpha-mediated induction of monocyte adhesion both in vitro and in vivo through the suppression of VCAM-1. Further analysis demonstrated that HDAC3 plays a significant role in the regulation of TNF-alpha-mediated VCAM-1 expression. CONCLUSIONS: TNF-alpha activates ECs via multiple signaling pathways, and these pathways may be selectively targeted to modulate EC function. Moreover, TSA treatment reduced monocyte adhesion via VCAM-1 suppression in vitro and in vivo, suggesting that TSA might be useful for the attenuation of the inflammatory response in EC.

Association of active gamma-secretase complex with lipid rafts.

Cholesterol has been implicated in the pathogenesis of Alzheimer's disease (AD). Although the underlying mechanisms are not yet clear, several studies have provided evidence for the involvement of cholesterol-rich lipid rafts in the production of amyloid beta peptide (Abeta), the major component of amyloid deposits in AD. In this regard, the gamma-secretase complex is responsible for the final cleavage event in the processing of beta-amyloid precursor protein (betaAPP), resulting in Abeta generation. The gamma-secretase complex is a multiprotein complex composed of presenilin, nicastrin (NCT), APH-1, and PEN-2. Recent reports have suggested that gamma-secretase activity is predominantly localized in lipid rafts, and presenilin and NCT have been reported to be localized in lipid rafts. In this study, various biochemical methods, including coimmunoprecipitation, in vitro gamma-secretase assay, and methyl-beta-cyclodextrin (MbetaCD) treatment, are employed to demonstrate that all four components of the active endogenous gamma-secretase complex, including APH-1 and PEN-2, are associated with lipid rafts in human neuroblastoma cells (SH-SY5Y). Treatment with statins, 3-hydroxy-3-methylglutaryl-CoA-reductase inhibitors, significantly decreased the association of the gamma-secretase complex with lipid rafts without affecting the distribution of flotillin-1. This effect was partially abrogated by the addition of geranylgeraniol. These results suggest that both cholesterol and protein isoprenylation influence the active gamma-secretase complex association with lipid rafts.