Wayne David Kohn

Akt activation, but not extracellular signal-regulated kinase activation, is required for SDF-1α./CXCR4- mediated migration of epitheloid carcinoma cells

Emerging evidence shows that the stromal cell–derived factor 1 (SDF-1)/CXCR4 interaction regulates multiple cell signaling pathways and a variety of cellular functions such as cell migration, proliferation, and survival. There is little information linking the cellular functions and individual signaling pathways mediated by SDF-1 and CXCR4 in human cancer cells. In this study, we have shown that human epitheloid carcinoma HeLa cells express functional CXCR4 by reverse transcription-PCR, immunofluorescent staining, and 125I-SDF-1α ligand binding analyses. The treatment of HeLa cells with recombinant SDF-1α results in time-dependent Akt and extracellular signal–regulated kinase 1/2 (ERK1/2) activations. The SDF-1α–induced Akt and ERK1/2 activations are CXCR4 dependent as confirmed by their total inhibition by T134, a CXCR4-specific peptide antagonist. Cell signaling analysis with pathway-specific inhibitors reveals that SDF-1α–induced Akt activation is not required for ERK1/2 activation and vice versa, indicating that activations of Akt and ERK1/2 occur independently. Functional analysis shows that SDF-1α induces a CXCR4-dependent migration of HeLa cells. The migration can be totally blocked by phosphoinositide 3-kinase inhibitors, wortmannin or LY294002, whereas mitogen-activated protein/ERK kinase inhibitors, PD98059 and U0126, have no significant effect on SDF-1α–induced migration, suggesting that Akt activation, but not ERK1/2 activation, is required for SDF-1α–induced migration of epitheloid carcinoma cells.

pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity

A long-acting (basal) insulin capable of delivering flat, sustained, reproducible glycemic control with once daily administration represents an improvement in the treatment paradigm for both type 1 and type 2 diabetes. Optimization of insulin pharmacodynamics is achievable through structural modification, but often at the expense of alterations in receptor affinity and selectivity. A series of isoelectric point (pI)-shifted insulin analogs based on the human insulin sequence or the GlyA21 acid stable variant were prepared by semi-synthetic methods. The pI shift was achieved through systematic addition of one or more arginine (Arg) or lysine (Lys) residues at the N terminus of the A chain, the N terminus of the B chain, the C terminus of the B chain, or through a combination of additions at two of the three sites. The analogs were evaluated for their affinity for the insulin and IGF-1 receptors, and aqueous solubility under physiological pH conditions. Notably, the presence of positively charged amino acid residues at the N terminus of the A chain was consistently associated with an enhanced insulin to IGF-1 receptor selectivity profile. Increased IGF-1 receptor affinity that results from Arg addition to the C terminus of the B chain was attenuated by cationic extension at the N terminus of the A chain. Analogs 10, 17, and 18 displayed in vitro receptor selectivity similar to that of native insulin and solubility at physiological pH that suggested the potential for extended time action. Accordingly, the in vivo pharmacokinetic and pharmacodynamic profiles of these analogs were established in a somatostatin-induced diabetic dog model. Analog 18 (A0:Arg, A21:Gly, B31:Arg, B32:Arg human insulin) exhibited a pharmacological profile comparable to that of analog 15 (insulin glargine) but with a 4.5-fold more favorable insulin:IGF-1 receptor selectivity. These results demonstrate that the selective combination of positive charge to the N terminus of the A chain and the C terminus of the B chain generates an insulin with sustained pharmacology and a near-native receptor selectivity profile.

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for β- and β′-cleavage sites by human and murine BACE1

β‐amyloid peptides (Aβ) are produced by a sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. The lack of Aβ production in beta‐APP cleaving enzyme (BACE1)–/– mice suggests that BACE1 is the principal β‐secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild‐type and BACE1–/– mice supports cleavage of APP at the canonical β‐secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as β′, resulting in Aβ peptides starting at Glu11. The apparent inability of human BACE1 to make this β′‐cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species‐specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the β′‐cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the β‐ and β′‐cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the β‐ and β′‐sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated β′‐cleavage site. It does not appear to be a significant species specificity to this cleavage.

Rapid and recurrent neutrophil mobilization regulated by T134, a CXCR4 peptide antagonist

The CXCR4/stromal cell-derived factor-1 (SDF-1) axis plays important roles in development, leukocyte trafficking, HIV infection, and tumorigenesis. Its critical function in bone marrow stem cell and hematopoietic progenitor cell retention, homing and release has been well-characterized by genetic and pharmacological analyses. However, its role in neutrophil retention and release is still poorly understood. In this study, we demonstrated that T134, a peptide antagonist of human CXCR4, is also a potent antagonist of mouse CXCR4. Treatment of C57BL/6 mice with T134 resulted in a rapid and time-dependent increase of white blood cells (WBC) and neutrophils, as well as hematopoietic stem and progenitor cells in peripheral blood. Interestingly, recurrent WBC and neutrophil mobilization was achieved by repeated T134 treatment, and the T134-mediated increase and subsequent retreat of WBC and neutrophils correlated with T134 activity in the peripheral blood. Kinetic analysis revealed that T134 binding to CXCR4 did not induce any significant cell-surface receptor downregulation, indicating that T134-induced WBC and neutrophil mobilization is likely due to direct blockage of the CXCR4/SDF-1 interaction. The results from this study support an important role of CXCR4/SDF-1 axis in neutrophil retention and release in the marrow.

Solid-phase synthesis of peptide–heterocycle hybrids containing a tripeptide-derived 6,6-fused bicyclic subunit

Abstract The solid-phase synthesis of peptides incorporating a tripeptide-derived 6,6-fused bicyclic subunit is described. The bicyclic moiety (3,8,10-trisubstituted 2,9-dioxo-5-thia-1,8-diazabicyclo[4.4.0]decane) can be incorporated anywhere in a peptide sequence and is formed spontaneously upon TFA cleavage through condensation of an aldehyde with a backbone amide nitrogen and side-chain thiol, resulting in a thiazinone ring. The reaction is regio- and stereoselective and also allows for the incorporation of the bicyclic moiety, which may represent a constrained β-turn mimic, into a macrocyclic peptide.

Solid-phase synthesis of a type II′ β-turn peptido-mimetic library

Abstract The solid-phase synthesis of a dipeptide derived 2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate system (IBTM) is described. The IBTM moiety is formed via a solid-phase mediated Pictet–Spengler reaction of N-terminal tryptophan and the 4-{N-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl]amino}benzyl (Dmab) ester of Fmoc protected aspartic acid β-aldehyde followed by γ-lactamization. This synthesis allows the regio- and stereoselective incorporation of a dipeptide surrogate of type II′ β-turns. The procedure is easily adaptable to combinatorial synthesis and a 576-member library was synthesized.

Repurposing a novel parathyroid hormone (PTH) analog to treat hypoparathyroidism

Human parathyroid hormone (PTH) is critical for maintaining physiological calcium homeostasis and plays an important role in the formation and maintenance of the bone. Full‐length PTH and a truncated peptide form are approved for treatment of hypoparathyroidism and osteoporosis respectively. Our initial goal was to develop an improved PTH therapy for osteoporosis, but clinical development was halted. The novel compound was then repurposed as an improved therapy for hypoparathyroidism.