Imidazo[1,2-a]pyridine-based peptidomimetics as inhibitors of Akt.

We report the design, synthesis, and biological evaluation of imidazopyridine-based peptidomimetics based on the substrate consensus sequence of Akt, an AGC family serine/threonine kinase hyperactivated in over 50% of human tumors. Our ligand-based approach led to the identification of novel substrate mimetic inhibitors of Akt1 featuring an unnatural extended dipeptide surrogate. Compound 11 inhibits Akt isoforms in the sub-micromolar range and exhibits improved proteolytic stability relative to a parent pentapeptide.

Total synthesis and structural revision of lucentamycin A.

Lucentamycin A is a marine-derived peptide natural product harboring a unique 4-ethylidene-3-methylproline (Emp) subunit. The proposed structure of lucentamycin A and the core Emp residue have recently been called into question through synthesis. Here, we report the first total synthesis of lucentamycin A, which confirms that the ethylidene substituent in Emp bears an E geometry, in contrast to the originally assigned Z configuration. Synthesis of the desired (E)-Emp subunit required the implementation of a novel strategy starting from Garner's aldehyde.

TCR-Independent Metabolic Reprogramming Precedes Lymphoma-Driven Changes in T-cell Fate.

Chronic T-cell receptor (TCR) signaling in the tumor microenvironment is known to promote T-cell dysfunction. However, we reasoned that poorly immunogenic tumors may also compromise T cells by impairing their metabolism. To address this, we assessed temporal changes in T-cell metabolism, fate, and function in models of B-cell lymphoma driven by Myc, a promoter of energetics and repressor of immunogenicity. Increases in lymphoma burden most significantly impaired CD4+ T-cell function and promoted regulatory T cell (Treg) and Th1-cell differentiation. Metabolomic analyses revealed early reprogramming of CD4+ T-cell metabolism, reduced glucose uptake, and impaired mitochondrial function, which preceded changes in T-cell fate. In contrast, B-cell lymphoma metabolism remained robust during tumor progression. Finally, mitochondrial functions were impaired in CD4+ and CD8+ T cells in lymphoma-transplanted OT-II and OT-I transgenic mice, respectively. These findings support a model, whereby early, TCR-independent, metabolic interactions with developing lymphomas limits T cell-mediated immune surveillance.

An ester enolate-Claisen rearrangement route to substituted 4-alkylideneprolines. studies toward a definitive structural revision of lucentamycin A.

Substituted 4-alkylideneprolines represent a rare class of naturally occurring amino acids with promising biological activities. Lucentamycin A is a cytotoxic, marine-derived tripeptide that harbors a 4-ethylidine-3-methylproline (Emp) residue unique among known peptide natural products. In this paper, we examine the synthesis of Emp and related 4-alkylideneprolines employing a versatile ester enolate-Claisen rearrangement. The scope and selectivity of the key rearrangement reaction are described with a number of diversely substituted glycine ester substrates. Treatment of the allyl esters with excess NaHMDS at ambient temperature gives rise to highly substituted α-allylglycine products with good to excellent diastereoselectivities. Resolution of dipeptide diastereomers and cyclization to form the pyrrolidine rings provide rapid access to stereopure prolyl dipeptides. We have applied this strategy to the synthesis of four Emp-containing isomers of lucentamycin A in pursuit of a definitive stereochemical revision of the natural product. Our studies indicate that the Emp stereogenic centers are not the source of structural misassignment. The current strategy should find broad utility in the synthesis of additional natural product analogues and related 3-alkyl-4-alkylidene prolines.

Synthesis of GSK3ß mimetic inhibitors of Akt featuring a novel extended dipeptide surrogate.

Akt is a cardinal nodal point in PI3K signaling pathway and confers resistance to apoptosis through inactivation of regulatory substrates such as GSK3ß. Efforts to inhibit the kinase activity of Akt have largely focused on targeting the ATP-binding domain of Akt. Here, we present the design and synthesis of conformationally constrained GSK3ß mimics featuring a novel extended dipeptide surrogate core. This effort resulted in the identification of a novel substrate mimetic Akt inhibitor (11) with low micromolar activity in vitro (Akt1 IC(50)=3.1 µM).

Synthesis and conformational analysis of bicyclic extended dipeptide surrogates.

Regio- and diastereoselective reactions of a homoproline enolate enable the synthesis of novel extended dipeptide surrogates. Bicyclic carbamate 9 and fused beta-lactam scaffold 11 were prepared from L-pyroglutamic acid via substrate-controlled electrophilic azidation. Synthesis of orthogonally protected hexahydropyrrolizine, hexahydropyrrolizinone, and hexahydropyrroloazepinone dipeptide surrogates relied on allylation of proline derivative 5, followed by Curtius rearrangement to introduce the N-terminal carbamate group. A total of six azabicycloalkane derivatives were evaluated for conformational mimicry of extended dipeptides by a combination of X-ray diffraction and molecular modeling. Analysis of putative backbone dihedral angles and N- to C-terminal dipeptide distances indicate that compounds (alpha'S)-14b and 21 approximate the conformation of dipeptides found in beta-sheets, while tripeptide mimic 28 is also highly extended in the solid state. Structural data suggest that ring size and relative stereochemistry have a profound effect on the ability of these scaffolds to act as beta-strand mimetics and should inform the design of related conformational probes.

Synthesis of Boc-protected bicycloproline.

The synthesis of a highly constrained quaternary carbocyclic α-amino acid, (+)-N-Boc-bicycloproline, has been achieved starting from sodium cyclopentadienylide. Key steps include a rhodium-catalyzed nitrenoid C-H insertion to install the tert-alkylamine and a ring-closing metathesis reaction to form the pyrrolidine ring.

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.

Synthesis of novel tricyclic chromenone-based inhibitors of IRE-1 RNase activity.

Inositol-requiring enzyme 1 (IRE-1) is a kinase/RNase ER stress sensor that is activated in response to excessive accumulation of unfolded proteins, hypoxic conditions, calcium imbalance, and other stress stimuli. Activation of IRE-1 RNase function exerts a cytoprotective effect and has been implicated in the progression of cancer via increased expression of the transcription factor XBP-1s. Here, we describe the synthesis and biological evaluation of novel chromenone-based covalent inhibitors of IRE-1. Preparation of a family of 8-formyltetrahydrochromeno[3,4-c]pyridines was achieved via a Duff formylation that is attended by an unusual cyclization reaction. Biological evaluation in vitro and in whole cells led to the identification of 30 as a potent inhibitor of IRE-1 RNase activity and XBP-1s expression in wild type B cells and human mantle cell lymphoma cell lines.

Inhibition of ER stress-associated IRE-1/XBP-1 pathway reduces leukemic cell survival.

Activation of the ER stress response is associated with malignant progression of B cell chronic lymphocytic leukemia (CLL). We developed a murine CLL model that lacks the ER stress-associated transcription factor XBP-1 in B cells and found that XBP-1 deficiency decelerates malignant progression of CLL-associated disease. XBP-1 deficiency resulted in acquisition of phenotypes that are disadvantageous for leukemic cell survival, including compromised BCR signaling capability and increased surface expression of sphingosine-1-phosphate receptor 1 (S1P1). Because XBP-1 expression requires the RNase activity of the ER transmembrane receptor IRE-1, we developed a potent IRE-1 RNase inhibitor through chemical synthesis and modified the structure to facilitate entry into cells to target the IRE-1/XBP-1 pathway. Treatment of CLL cells with this inhibitor (B-I09) mimicked XBP-1 deficiency, including upregulation of IRE-1 expression and compromised BCR signaling. Moreover, B-I09 treatment did not affect the transport of secretory and integral membrane-bound proteins. Administration of B-I09 to CLL tumor-bearing mice suppressed leukemic progression by inducing apoptosis and did not cause systemic toxicity. Additionally, B-I09 and ibrutinib, an FDA-approved BTK inhibitor, synergized to induce apoptosis in B cell leukemia, lymphoma, and multiple myeloma. These data indicate that targeting XBP-1 has potential as a treatment strategy, not only for multiple myeloma, but also for mature B cell leukemia and lymphoma.

Total synthesis of the putative structure of lucentamycin A.

A rapid and stereoselective enolate-Claisen rearrangement provides access to the 4-ethylidene-3-methylproline (Emp) subunit of lucentamycin A. Synthesis of the putative structure of the cytotoxic natural product suggests the need for structural revision.