Synthesis, derivatization, and conformational scanning of peptides containing N-Aminoglycine.

N-alkylated glycine residues are the main constituent of peptoids and peptoid-peptide hybrids that are employed across the biomedical and materials sciences. While the impact of backbone N-alkylation on peptide conformation has been extensively studied, less is known about the effect of N-amination on the secondary structure propensity of glycine. Here, we describe a convenient protocol for the incorporation of N-aminoglycine into host peptides on solid support. Amide-to-hydrazide substitution also affords a nucleophilic handle for further derivatization of the backbone. To demonstrate the utility of late-stage hydrazide modification, we synthesized and evaluated the stability of polyproline II helix and ß-hairpin model systems harboring N-aminoglycine derivatives. The described procedures provide facile entry into peptidomimetic libraries for conformational scanning.

Beyond N-Alkylation: Synthesis, Structure, and Function of N-Amino Peptides.

The growing list of physiologically important protein-protein interactions (PPIs) has amplified the need for compounds to target topologically complex biomolecular surfaces. In contrast to small molecules, peptide and protein mimics can exhibit three-dimensional shape complementarity across a large area and thus have the potential to significantly expand the "druggable" proteome. Strategies to stabilize canonical protein secondary structures without sacrificing side-chain content are particularly useful in the design of peptide-based chemical probes and therapeutics.Substitution of the backbone amide in peptides represents a subtle chemical modification with profound effects on conformation and stability. Studies focused on N-alkylation have already led to broad-ranging applications in peptidomimetic design. Inspired by nonribosomal peptide natural products harboring amide N-oxidations, we envisioned that main-chain hydrazide and hydroxamate bonds would impose distinct conformational preferences and offer unique opportunities for backbone diversification. This Account describes our exploration of peptide N-amination as a strategy for stabilizing canonical protein folds and for the structure-based design of soluble amyloid mimics.We developed a general synthetic protocol to access N-amino peptides (NAPs) on solid support. In an effort to stabilize ß-strand conformation, we designed stitched peptidomimetics featuring covalent tethering of the backbone N-amino substituent to the preceding residue side chain. Using a combination of NMR, X-ray crystallography, and molecular dynamics simulations, we discovered that backbone N-amination alone could significantly stabilize ß-hairpin conformation in multiple models of folding. Our studies revealed that the amide NH2 substituent in NAPs participates in cooperative noncovalent interactions that promote ß-sheet secondary structure. In contrast to Cα-substituted α-hydrazino acids, we found that N-aminoglycine and its N'-alkylated derivatives instead stabilize polyproline II (PPII) conformation. The reactivity of hydrazides also allows for late-stage peptide macrocyclization, affording novel covalent surrogates of side-chain-backbone H-bonds.The pronounced ß-sheet propensity of Cα-substituted α-hydrazino acids prompted us to target amyloidogenic proteins using NAP-based ß-strand mimics. Backbone N-amination was found to render aggregation-prone lead sequences soluble and resistant to proteolysis. Inhibitors of Aß and tau identified through N-amino scanning blocked protein aggregation and the formation of mature fibrils in vitro. We further identified NAP-based single-strand and cross-ß tau mimics capable of inhibiting the prion-like cellular seeding activity of recombinant and patient-derived tau fibrils.Our studies establish backbone N-amination as a valuable addition to the peptido- and proteomimetic tool kit. α-Hydrazino acids show particular promise as minimalist ß-strand mimics that retain side-chain information. Late-stage derivatization of hydrazides also provides facile entry into libraries of backbone-edited peptides. We anticipate that NAPs will thus find applications in the development of optimally constrained folds and modulators of PPIs.

ß-Bracelets: Macrocyclic Cross-ß Epitope Mimics Based on a Tau Conformational Strain.

The aggregation of misfolded tau into neurotoxic fibrils is linked to the progression of Alzheimer's disease (AD) and related tauopathies. Disease-associated conformations of filamentous tau are characterized by hydrophobic interactions between side chains on unique and distant ß-strand modules within each protomer. Here, we report the design and diversity-oriented synthesis of ß-arch peptide macrocycles composed of the aggregation-prone PHF6 hexapeptide of tau and the cross-ß module specific to the AD tau fold. Termed "ß-bracelets", these proteomimetics assemble in a sequence- and macrocycle-dependent fashion, resulting in amyloid-like fibrils that feature in-register parallel ß-sheet structure. Backbone N-amination of a selected ß-bracelet affords soluble inhibitors of tau aggregation. We further demonstrate that the N-aminated macrocycles block the prion-like cellular seeding activity of recombinant tau as well as mature fibrils from AD patient extracts. These studies establish ß-bracelets as a new class of cross-ß epitope mimics and demonstrate their utility in the rational design of molecules targeting amyloid propagation and seeding.

N-Aminoglycine and Its Derivatives Stabilize PPII Secondary Structure.

The identification of unnatural residues that stabilize polyproline type 2 (PPII) folds can aid in the design of peptidomimetics targeting PPII-binding domains. Here, we examine the impact of peptide backbone N-amination on PPII helix stability and find N-aminoglycine (aGly) to be an effective PPII promoter. Further derivatization of an aGly-containing peptide affords N'-alkylated analogues with increased helical propensity. Backbone N-amination of glycine represents a convenient approach to stabilize PPII conformation and allows for the diversity-oriented synthesis of optimally constrained folds.

Total Synthesis of Pargamicin A.

We report the total synthesis and configurational assignment of pargamicin A, a highly oxidized nonribosomal peptide that potently inhibits the growth of drug-resistant bacteria. Our synthetic approach relies on late-stage piperazine ring formation and careful selection of condensation reagents to assemble the densely substituted hexapeptide backbone. This work enables the synthesis of pargamicin congeners for the development of structure-activity relationships and informs strategies for accessing other sterically congested piperazic acid-containing natural products.

IRE-1-Targeting Caged Prodrug with Endoplasmic Reticulum Stress-Inducing and XBP-1S-Inhibiting Activities for Cancer Therapy.

Activation of the IRE-1/XBP-1s pathway supports tumor progression. Here, we report a novel prodrug, TC-D-F07, in which a thiol-reactive dinitrobenzenesulfonyl (Dns) cage was installed onto the C8 hydroxyl of the covalent IRE-1 inhibitor D-F07. The electron-withdrawing Dns group in TC-D-F07 stabilizes the neighboring 1,3-dioxane acetal, allowing for stimulus-mediated control of its inhibitory activity. TC-D-F07 exhibits high sensitivity to intracellular thiols. Because tumor cells exhibit higher concentrations of glutathione and cysteine, treatment with TC-D-F07 results in more sustained levels of D-F07 in transformed versus normal cells. In addition, we show that a dinitrophenyl cysteine adduct resulting from cleavage of the Dns group induces endoplasmic reticulum (ER) stress, causing tumor cells to increase the expression of XBP-1s. The accumulated levels of D-F07 and its gradual decomposition into the active IRE-1 inhibitor eventually deprive tumor cells of XBP-1s, leading to more severe apoptosis than those treated with its uncaged analogue.

Late-Stage Sidechain-to-Backbone Macrocyclization of N-Amino Peptides.

Cysteine-containing N-amino peptides undergo chemoselective reactions with haloaldehydes to afford ethylene-bridged cyclic peptides. This bis-alkylation strategy provides macrocycles harboring a novel covalent H-bond surrogate. Mimicry of a native sidechain-to-backbone (sb) H-bond is demonstrated in the context of a model loop-helix peptide. The described method is amenable to the synthesis of diverse ring sizes from crude unprotected linear substrates under aqueous conditions.

N-Amination Converts Amyloidogenic Tau Peptides into Soluble Antagonists of Cellular Seeding.

The spread of neurofibrillary tangles composed of tau protein aggregates is a hallmark of Alzheimer's and related neurodegenerative diseases. Early oligomerization of tau involves conformational reorganization into parallel ß-sheet structures and supramolecular assembly into toxic fibrils. Despite the need for selective inhibitors of tau propagation, ß-rich protein assemblies are inherently difficult to target with small molecules. Here, we describe a minimalist approach to mimic the aggregation-prone modules within tau. We carried out a backbone residue scan and show that amide N-amination completely abolishes the tendency of these peptides to self-aggregate, rendering them soluble mimics of ordered ß-strands from the tau R2 and R3 domains. Several N-amino peptides (NAPs) inhibit tau fibril formation in vitro. We further demonstrate that NAPs 12 and 13 are effective at blocking the cellular seeding of endogenous tau by interacting with monomeric or fibrillar forms of extracellular tau. Peptidomimetic 12 is serum stable, non-toxic to neuronal cells, and selectivity inhibits the fibrilization of tau over Aß42. Structural analysis of our lead NAPs shows considerable conformational constraint imposed by the N-amino groups. The described backbone N-amination approach provides a rational basis for the mimicry of other aggregation-prone peptides that drive pathogenic protein assembly.

Synthesis and conformation of backbone N-aminated peptides.

The chemical modification of peptides is a promising approach for the design of protein-protein interaction inhibitors and peptide-based drug candidates. Among several peptidomimetic strategies, substitution of the amide backbone maintains side-chain functionality that may be important for engagement of biological targets. Backbone amide substitution has been largely limited to N-alkylation, which can promote cis amide geometry and disrupt important H-bonding interactions. In contrast, N-amination of peptides induces distinct backbone geometries and maintains H-bond donor capacity. In this chapter we discuss the conformational characteristics of designed N-amino peptides and present a detailed protocol for their synthesis on solid support. The described methods allow for backbone N-amino scanning of biologically active parent sequences.

STING regulates BCR signaling in normal and malignant B cells.

STING is an endoplasmic reticulum (ER)-resident protein critical for sensing cytoplasmic DNA and promoting the production of type I interferons; however, the role of STING in B cell receptor (BCR) signaling remains unclear. We generated STING V154M knock-in mice and showed that B cells carrying constitutively activated STING specifically degraded membrane-bound IgM, Igα, and Igß via SEL1L/HRD1-mediated ER-associated degradation (ERAD). B cells with activated STING were thus less capable of responding to BCR activation by phosphorylating Igα and Syk than those without activated STING. When immunized with T-independent antigens, STING V154M mice produced significantly fewer antigen-specific plasma cells and antibodies than immunized wild-type (WT) mice. We further generated B cell-specific STINGKO mice and showed that STINGKO B cells indeed responded to activation by transducing stronger BCR signals than their STING-proficient counterparts. When B cell-specific STINGKO mice were T-independently immunized, they produced significantly more antigen-specific plasma cells and antibodies than immunized STINGWT mice. Since both human and mouse IGHV-unmutated malignant chronic lymphocytic leukemia (CLL) cells downregulated the expression of STING, we explored whether STING downregulation could contribute to the well-established robust BCR signaling phenotype in malignant CLL cells. We generated a STING-deficient CLL mouse model and showed that STING-deficient CLL cells were indeed more responsive to BCR activation than their STING-proficient counterparts. These results revealed a novel B cell-intrinsic role of STING in negatively regulating BCR signaling in both normal and malignant B cells.

Development of Tumor-Targeting IRE-1 Inhibitors for B-cell Cancer Therapy.

The IRE-1 kinase/RNase splices the mRNA of the XBP-1 gene, resulting in the spliced XBP-1 (XBP-1s) mRNA that encodes the functional XBP-1s transcription factor that is critically important for the growth and survival of B-cell leukemia, lymphoma, and multiple myeloma (MM). Several inhibitors targeting the expression of XBP-1s have been reported; however, the cytotoxicity exerted by each inhibitor against cancer cells is highly variable. To design better therapeutic strategies for B-cell cancer, we systematically compared the ability of these compounds to inhibit the RNase activity of IRE-1 in vitro and to suppress the expression of XBP-1s in mouse and human MM cell lines. Tricyclic chromenone-based inhibitors B-I09 and D-F07, prodrugs harboring an aldehyde-masking group, emerged as the most reliable inhibitors for potent suppression of XBP-1s expression in MM cells. The cytotoxicity of B-I09 and D-F07 against MM as well as chronic lymphocytic leukemia and mantle cell lymphoma could be further enhanced by combination with inhibitors of the PI3K/AKT pathway. Because chemical modifications of the salicylaldehyde hydroxy group could be used to tune 1,3-dioxane prodrug stability, we installed reactive oxygen species-sensitive structural cage groups onto these inhibitors to achieve stimuli-responsive activities and improve tumor-targeting efficiency.

N-Hydroxy peptides: solid-phase synthesis and ß-sheet propensity.

Peptide backbone amide substitution can dramatically alter the conformational and physiochemical properties of native sequences. Although uncommon relative to N-alkyl substituents, peptides harboring main-chain N-hydroxy groups exhibit unique conformational preferences and biological activities. Here, we describe a versatile method to prepare N-hydroxy peptide on solid support and evaluate the impact of backbone N-hydroxylation on secondary structure stability. Based on previous work demonstrating the ß-sheet-stabilizing effect of α-hydrazino acids, we carried out an analogous study with N-hydroxy-α-amino acids using a model ß-hairpin fold. In contrast to N-methyl substituents, backbone N-hydroxy groups are accommodated in the ß-strand region of the hairpin without energetic penalty. An enhancement in ß-hairpin stability was observed for a di-N-hydroxylated variant. Our results facilitate access to this class of peptide derivatives and inform the use of backbone N-hydroxylation as a tool in the design of constrained peptidomimetics.

Inhibition of Human Dendritic Cell ER Stress Response Reduces T Cell Alloreactivity Yet Spares Donor Anti-tumor Immunity.

Acute graft- vs. -host disease (GVHD) is an important cause of morbidity and death after allogeneic hematopoietic cell transplantation (HCT). We identify a new approach to prevent GVHD that impairs monocyte-derived dendritic cell (moDC) alloactivation of T cells, yet preserves graft- vs.-leukemia (GVL). Exceeding endoplasmic reticulum (ER) capacity results in a spliced form of X-box binding protein-1 (XBP-1s). XBP-1s mediates ER stress and inflammatory responses. We demonstrate that siRNA targeting XBP-1 in moDCs abrogates their stimulation of allogeneic T cells. B-I09, an inositol-requiring enzyme-1α (IRE1α) inhibitor that prevents XBP-1 splicing, reduces human moDC migration, allo-stimulatory potency, and curtails moDC IL-1ß, TGFß, and p40 cytokines, suppressing Th1 and Th17 cell priming. B-I09-treated moDCs reduce responder T cell activation via calcium flux without interfering with regulatory T cell (Treg) function or GVL effects by cytotoxic T lymphocytes (CTL) and NK cells. In a human T cell mediated xenogeneic GVHD model, B-I09 inhibition of XBP-1s reduced target-organ damage and pathogenic Th1 and Th17 cells without impacting donor Tregs or anti-tumor CTL. DC XBP-1s inhibition provides an innovative strategy to prevent GVHD and retain GVL.

Secretory IgM Exacerbates Tumor Progression by Inducing Accumulations of MDSCs in Mice.

Chronic lymphocytic leukemia (CLL) cells can secrete immunoglobulin M. However, it is not clear whether secretory IgM (sIgM) plays a role in disease progression. We crossed the Eµ-TCL1 mouse model of CLL, in which the expression of human TCL1 oncogene was driven by the V(H) promoter-Ig(H)-Eµ enhancer, with MD4 mice whose B cells produced B-cell receptor (membrane-bound IgM) and sIgM with specificity for hen egg lysozyme (HEL). CLL cells that developed in these MD4/Eµ-TCL1 mice reactivated a parental Ig gene allele and secreted IgM, and did not recognize HEL. The MD4/Eµ-TCL1 mice had reduced survival, increased myeloid-derived suppressor cells (MDSC), and decreased numbers of T cells. We tested whether sIgM could contribute to the accumulation of MDSCs by crossing µS-/- mice, which could not produce sIgM, with Eµ-TCL1 mice. The µS-/-/Eµ-TCL1 mice survived longer than Eµ-TCL1 mice and developed decreased numbers of MDSCs which were less able to suppress proliferation of T cells. We targeted the synthesis of sIgM by deleting the function of XBP-1s and showed that targeting XBP-1s genetically or pharmacologically could lead to decreased sIgM, accompanied by decreased numbers and reduced functions of MDSCs in MD4/Eµ-TCL1 mice. Additionally, MDSCs from µS-/- mice grafted with Lewis lung carcinoma were inefficient suppressors of T cells, resulting in slower tumor growth. These results demonstrate that sIgM produced by B cells can upregulate the functions of MDSCs in tumor-bearing mice to aggravate cancer progression. In a mouse model of CLL, production of secretory IgM led to more MDSCs, fewer T cells, and shorter survival times for the mice. Thus, secretory IgM may aggravate the progression of this cancer. Cancer Immunol Res; 6(6); 696-710. ©2018 AACR.

Inhibition of the IRE-1α/XBP-1 pathway prevents chronic GVHD and preserves the GVL effect in mice.

Hematopoietic stem cell transplantation (HCT) is a curative procedure for hematological malignancies, but chronic graft-versus-host disease (cGVHD) remains a major complication after allogeneic HCT. Because donor B cells are essential for cGVHD development and B cells are sensitive to endoplasmic reticulum (ER) stress, we hypothesized that the IRE-1α/XBP-1 pathway is required for B-cell activation and function and for the development of cGVHD. To test this hypothesis, we used conditional knock-out mice deficient of XBP-1 specifically in B cells. Recipients transplanted with donor grafts containing XBP-1-deficient B cells displayed reduced cGVHD compared with controls. Reduction of cGVHD correlated with impaired B-cell functions, including reduced production of anti-double-stranded DNA immunoglobulin G antibodies, CD86, Fas, and GL7 surface expression, and impaired T-cell responses, including reduced interferon-γ production and follicular helper T cells. In a bronchiolitis obliterans cGVHD model, recipients of transplants containing XBP-1-deficient B cells demonstrated improved pulmonary function correlated with reduced donor splenic follicular helper T cells and increased B cells compared with those of wild-type control donor grafts. We then tested if XBP-1 blockade via an IRE-1α inhibitor, B-I09, would attenuate cGVHD and preserve the graft-versus-leukemia (GVL) effect. In a cutaneous cGVHD model, we found that prophylactic administration of B-I09 reduced clinical features of cGVHD, which correlated with reductions in donor T-cell and dendritic cell skin infiltrates. Inhibition of the IRE-1α/XBP-1 pathway also preserved the GVL effect against chronic myelogenous leukemia mediated by allogeneic splenocytes. Collectively, the ER stress response mediated by the IRE-1α/XBP-1 axis is required for cGVHD development but dispensable for GVL activity.

IRE1α RNase-dependent lipid homeostasis promotes survival in Myc-transformed cancers.

Myc activation is a primary oncogenic event in many human cancers; however, these transcription factors are difficult to inhibit pharmacologically, suggesting that Myc-dependent downstream effectors may be more tractable therapeutic targets. Here, we show that Myc overexpression induces endoplasmic reticulum (ER) stress and engages the inositol-requiring enzyme 1α (IRE1α)/X-box binding protein 1 (XBP1) pathway through multiple molecular mechanisms in a variety of c-Myc- and N-Myc-dependent cancers. In particular, Myc-overexpressing cells require IRE1α/XBP1 signaling for sustained growth and survival in vitro and in vivo, dependent on elevated stearoyl-CoA-desaturase 1 (SCD1) activity. Pharmacological and genetic XBP1 inhibition induces Myc-dependent apoptosis, which is alleviated by exogenous unsaturated fatty acids. Of note, SCD1 inhibition phenocopies IRE1α RNase activity suppression in vivo. Furthermore, IRE1α inhibition enhances the cytotoxic effects of standard chemotherapy drugs used to treat c-Myc-overexpressing Burkitt's lymphoma, suggesting that inhibiting the IRE1α/XBP1 pathway is a useful general strategy for treatment of Myc-driven cancers.

Synthesis and ß-sheet propensity of constrained N-amino peptides.

The stabilization of ß-sheet secondary structure through peptide backbone modification represents an attractive approach to protein mimicry. Here, we present strategies toward stable ß-hairpin folds based on peptide strand N-amination. Novel pyrazolidinone and tetrahydropyridazinone dipeptide constraints were introduced via on-resin Mitsunobu cyclization between α-hydrazino acid residues and a serine or homoserine side chain. Acyclic and cyclic N-amino peptide building blocks were then evaluated for their effect on ß-hairpin stability in water using a GB1-derived model system. Our results demonstrate the strong ß-sheet stabilizing effect of the peptide N-amino substituent, and provide useful insights into the impact of covalent dipeptide constraint on ß-sheet folding.

Agonist-Mediated Activation of STING Induces Apoptosis in Malignant B Cells.

Endoplasmic reticulum (ER) stress responses through the IRE-1/XBP-1 pathway are required for the function of STING (TMEM173), an ER-resident transmembrane protein critical for cytoplasmic DNA sensing, IFN production, and cancer control. Here we show that the IRE-1/XBP-1 pathway functions downstream of STING and that STING agonists selectively trigger mitochondria-mediated apoptosis in normal and malignant B cells. Upon stimulation, STING was degraded less efficiently in B cells, implying that prolonged activation of STING can lead to apoptosis. Transient activation of the IRE-1/XBP-1 pathway partially protected agonist-stimulated malignant B cells from undergoing apoptosis. In Eµ-TCL1 mice with chronic lymphocytic leukemia, injection of the STING agonist 3'3'-cGAMP induced apoptosis and tumor regression. Similarly efficacious effects were elicited by 3'3'-cGAMP injection in syngeneic or immunodeficient mice grafted with multiple myeloma. Thus, in addition to their established ability to boost antitumoral immune responses, STING agonists can also directly eradicate malignant B cells. Cancer Res; 76(8); 2137-52. ©2016 AACR.

ß-Strand mimics based on tetrahydropyridazinedione (tpd) peptide stitching.

Short peptides featuring a tetrahydropyridazinedione (tpd) backbone tether exhibit reduced conformational flexibility external to the heterocyclic constraint. Analysis by NMR, molecular modeling and X-ray crystallography suggests both covalent and non-covalent stabilization of extended peptide conformations. An efficient solid-phase protocol was developed for the synthesis of a new class of ß-strand mimics based on oligomeric tpd subunits.

Solid-phase synthesis of tetrahydropyridazinedione-constrained peptides.

The design and solid-phase synthesis of tetrahydropyridazine-3,6-dione (Tpd) peptidomimetics derived from backbone-aminated peptides is reported. The described protocol features the synthesis of chiral α-hydrazino acids suitable for chemoselective incorporation into growing peptide chains. Acid-catalyzed cyclization to form the Tpd ring during cleavage affords the target peptidomimetics in good yield and purity. The scope of Tpd incorporation is demonstrated through the synthesis of constrained peptides featuring nucleophilic/electrophilic side chains and sterically encumbered α-substituted hydrazino acid residues.