Identification of cytidine-5-triphosphate synthase1-selective inhibitory peptide from random peptide library displayed on T7 phage.

Cytidine triphosphate synthase 1 (CTPS1) is an enzyme expressed in activated lymphocytes that catalyzes the conversion of uridine triphosphate (UTP) to cytidine triphosphate (CTP) with ATP-dependent amination, using either L-glutamine or ammonia as the nitrogen source. Since CTP plays an important role in DNA/RNA synthesis, phospholipid synthesis, and protein sialyation, CTPS1-inhibition is expected to control lymphocyte proliferation and size expansion in inflammatory diseases. In contrast, CTPS2, an isozyme of CTPS1 possessing 74% amino acid sequence homology, is expressed in normal lymphocytes. Thus, CTPS1-selective inhibition is important to avoid undesirable side effects. Here, we report the discovery of CTpep-3: Ac-FRLGLLKAFRRLF-OH from random peptide libraries displayed on T7 phage, which exhibited CTPS1-selective binding with a KD value of 210nM in SPR analysis and CTPS1-selective inhibition with an IC50 value of 110nM in the enzyme assay. Furthermore, two fundamentally different approaches, enzyme inhibition assay and HDX-MS, provided the same conclusion that CTpep-3 acts by binding to the amidoligase (ALase) domain on CTPS1. To our knowledge, CTpep-3 is the first CTPS1-selective inhibitor.

Discovery and Characterization of a Eukaryotic Initiation Factor 4A-3-Selective Inhibitor That Suppresses Nonsense-Mediated mRNA Decay.

Eukaryotic initiation factor 4A-3 (eIF4A3) is an Asp-Glu-Ala-Asp (DEAD) box-family adenosine triphosphate (ATP)-dependent RNA helicase. Subtypes eIF4A1 and eIF4A2 are required for translation initiation, but eIF4A3 participates in the exon junction complex (EJC) and functions in RNA metabolism including nonsense-mediated RNA decay (NMD). No small molecules for NMD inhibition via selective inhibition of eIF4A3 have been discovered. Here, we identified allosteric eIF4A3 inhibitors from a high-throughput screening campaign. Chemical optimization of the lead compounds based on ATPase activity yielded compound 2, which exhibited noncompetitive inhibition with ATP or RNA and high selectivity for eIF4A3 over other helicases. The optimized compounds suppressed the helicase activity of eIF4A3 in an ATPase-dependent manner. Hydrogen/deuterium exchange mass spectrometry demonstrated that the deuterium-incorporation pattern of compound 2 overlapped with that of an allosteric pan-eIF4A inhibitor, hippuristanol, suggesting that compound 2 binds to an allosteric region on eIF4A3. We examined NMD activity using a luciferase-based cellular reporter system and a quantitative real-time polymerase chain-reaction-based cellular system to monitor levels of endogenous NMD substrates. NMD suppression by the compounds correlated positively with their ATPase-inhibitory activity. In conclusion, we developed a novel eIF4A3 inhibitor that targets the EJC. The optimized chemical probes represent useful tools for understanding the functions of eIF4A3 in RNA homeostasis.

Design, synthesis, and biological evaluation of novel investigational nonapeptide KISS1R agonists with testosterone-suppressive activity.

Metastin/kisspeptin is a 54 amino acid peptide ligand of the KISS1R receptor and is a critical regulator of GnRH secretion. The N-terminally truncated peptide, metastin(45-54), possesses a 10-fold higher receptor-binding affinity than full-length metastin and agonistic KISS1R activity but is rapidly inactivated in rodent plasma. We have developed a decapeptide analog [D-Tyr(45),D-Trp(47),azaGly(51),Arg(Me)(53)]metastin(45-54) with improved serum stability compared with metastin(45-54) but with decreased KISS1R agonistic activity. Amino acid replacements at positions 45-47 led to an enhancement of KISS1R agonistic activity and metabolic stability. N-terminal truncation resulted in a stable nonapeptide, [D-Tyr(46),D-Pya(4)(47),azaGly(51),Arg(Me)(53)]metastin(46-54), compound 26, which displayed KISS1R binding affinities comparable to metastin(45-54) and had improved serum stability. Compound 26 reduced plasma testosterone in male rats and is the first short-length metastin analog to possess testosterone suppressive activities. Compound 26 has led to the elucidation of investigational analogs TAK-683 and TAK-448, both of which have undergone clinical evaluation for hormone-dependent diseases such as prostate cancer.

Serum stability of selected decapeptide agonists of KISS1R using pseudopeptides.

Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45-54), has 3-10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe(50)-Gly(51) and Gly(51)-Leu(52) as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, ß-(3-pyridyl)alanine, and D-tryptophan (D-Trp), produced analogs that were highly stable in mouse serum. D-Trp(47) analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution.

Trypsin resistance of a decapeptide KISS1R agonist containing an Nω-methylarginine substitution.

Metastin/kisspeptin is an amidated peptide with 54 amino acid residues isolated from human placental tissues as a ligand of the orphan G-protein-coupled receptor KISS1R that is expressed throughout the central nervous system and in a variety of endocrine and gonadal tissues. Compared to the full-length metastin protein, the N-terminal truncated peptide metastin(45-54) has 3-10 times higher receptor affinity and enhanced ability to increase intracellular calcium concentration which is essential for activation of protein kinases involved in intracellular signaling in a number of pathways that affect reproduction and cell migration. However, metastin(45-54) is rapidly inactivated in serum. In this study, we designed and synthesized a number of metastin(45-54) analogs and evaluated their agonistic activity and trypsin resistance. Among analogs with substitutions of arginine at position 53, N(ω)(-)methylarginine analog 8 showed 3-fold more potent agonistic activity compared with metastin(45-54). Furthermore, analog 8 was shown to resist trypsin cleavage between positions 53 and 54. This substitution may be useful in the development of other Arg-containing peptides for which the avoidance of cleavage is desired.

Characteristic morphology and distribution of bone marrow derived cells in the cornea.

Enhanced green fluorescence protein (eGFP)-labeled bone marrow (BM) cells were transplanted into syngeneic C57BL/6 (wild-type) mice to investigate the distribution pattern, immunohistochemical characteristics, three-dimensional structure, and ultrastructure of the BM-derived cells in the mouse cornea using a fluorescence microscope, a confocal laser scanning microscope, and a transmission electron microscope. This study provided direct evidence that two morphologically distinct types of BM-derived cells were distributed in the mouse cornea. The majority of the GFP+ cells showed a flattened polygonal form with obtuse angles and these cells were distributed in the corneal stroma. The other type was the GFP+ cells demonstrating slim cell bodies with long and extremely thin dendrites and which were distributed in the corneal epithelium. The immunohistochemical characteristics and ultrastructure of BM-derived cells suggest that most of these cells have a macrophage lineage, whereas some cells in the corneal stroma do not. Interestingly, the direct intimate contact between GFP-labeled BM derived cells and non-GFP-labeled resident cells within the corneal stroma were also clearly visualized at the fine structural level. These data provide new and more detailed insight into the nature of BM-derived cells in the cornea.

Pressure dependence of piezoelectric properties of a Pb(Mg(1/3)Nb(2/3))O(3)-PbTiO(3) binary system single crystal near a morphotropic phase boundary.

The piezoelectric properties of a Pb[(Mg(1/3)Nb(2/3))(0.68)]Ti(0.32)O(3) binary system single crystal poled along the [001] direction in the rhombohedral phase were investigated under pressures up to 400 MPa at 25 °C. For the transverse electromechanical property, the difference Δf between the resonance f(r) and antiresonance frequencies f(a), the Δf/f(r) and the electromechanical coupling coefficient k(31) value in the k(31) mode with hydrostatic pressure (p) became smaller because of the increase in f(r) and the almost constant f(a) with p. The k(31) value decreased by 16% at 400 MPa. On the other hand, for the longitudinal electromechanical property, the Δf, the Δf/f(r) and the k(33) value in the k(33) mode with p remained almost constant because of the almost constant f(r) and f(a) with p. The changes in the values of the elastic compliances s(11)(E) and s(33)(E) with p were found to be large from the changes in f(r) and f(a) with p. s(11)(E) and s(33)(E) at 400 MPa were estimated to be 35.4 and 75.1 × 10(-12) m(2) N(-1), respectively. A mechanical quality factor Q almost constant with p in the k(33) mode in contrast to the large decrease in Q in the k(31) mode with p in the pressure range up to 200 MPa was observed. A k(33) value almost constant with p is considered, on the basis of the engineered domain concept, to be due to the stable domain configuration responsible for the longitudinal k(33) mode. Furthermore, the superior piezoelectric properties of the rhombohedral [001] single crystal in the vicinity of the morphotropic phase boundary composition were recently pointed out to come from the large shear piezoelectric constant d(15) of their single domain property. The hydrostatic pressure cannot influence the piezoelectric properties from the viewpoint of the contribution of the large shear mode d(15), since the uniform pressure introduces no shearing stresses. Consequently, the k(33) value measured for the k(33) mode remained almost constant with p in the measured pressure range.

Identification of urotensin II-related peptide as the urotensin II-immunoreactive molecule in the rat brain.

Urotensin II (UII) has been reported as the most potent known vasoconstrictor. While rat and mouse orthologs of UII precursor protein have been reported, only the tentative structures of UII peptides of these animals have been demonstrated, since prepro-UII proteins lack typical processing sites for their mature peptides. In the present study, we isolated a novel peptide, UII-related peptide (URP), from the extract of the rat brain as the sole immunoreactive substance to anti-UII antibody; the amino acid sequence of the peptide was determined as ACFWKYCV. cDNAs encoding rat, mouse, and human precursor proteins for URP were cloned and revealed that the sequences of mouse and human URP peptides are the same as that for rat URP. Prepro-URP gene is expressed in several rat tissues such as those of the thymus, spleen, testis, and spinal cord, although with lower levels than the prepro-UII gene. In the human, the prepro-URP gene is expressed comparably to prepro-UII in several tissues except the spinal cord. URP was found to bind and activate the human or rat UII receptors (GPR14) and showed a hypotensive effect when administered to anesthetized rats. These results suggest that URP is the endogenous and functional ligand for UII receptor in the rat and mouse, and possibly in the human. We also describe the preparation of specific monoclonal antibodies raised against UII peptide and the establishment of a highly sensitive enzyme immunoassay system for UII peptides.

Production of recombinant human relaxin 3 in AtT20 cells.

Relaxin 3 has been reported recently as a member of the insulin/IGF/relaxin family. To clarify the function of relaxin 3, we prepared recombinant human relaxin 3 using a mouse adrenocorticotrophic hormone (ACTH)-secreting cell line, AtT20. To detect a mature form of recombinant human relaxin 3, a competitive enzyme immunoassay (EIA) was developed using a monoclonal antibody (mAb; HK4-144-10), which was raised for the N-terminal peptide of human relaxin 3 A-chain. We detected immunoreactive (ir-) relaxin 3 in the culture supernatant of AtT20 cells stably transfected with human relaxin 3 cDNA. After treatment with 5 microM forskolin for 3 days, the concentration of the ir-relaxin 3 in the culture supernatant reached 12 nM. Ir-relaxin 3 was purified from the culture supernatant by a combination of various chromatographies. By analyses of N-terminal amino acid sequence and electrospray ionization mass spectrometry (ESI-MS), we confirmed that the purified material was a mature form of human relaxin 3. The recombinant human relaxin 3 thereby obtained increased intracellular cAMP production in THP-1 cells. Our results demonstrate that the expression of relaxin 3 cDNA in AtT20 cells is a useful tool to produce a bioactive and mature form of relaxin 3.

Isolation and identification of EG-VEGF/prokineticins as cognate ligands for two orphan G-protein-coupled receptors.

Endocrine gland-derived vascular endothelial growth factor (EG-VEGF, identical to prokineticin 1) is a novel peptide recently identified as a selective mitogen for endocrine gland endothelial cells. The present study demonstrates that EG-VEGF/prokineticin 1 and a peptide closely related to EG-VEGF, prokineticin 2, are cognate ligands of two orphan G-protein-coupled receptors designated ZAQ (=EG-VEGF/PK-R1) and I5E (=EG-VEGF/PK-R2). EG-VEGF/prokineticin 1 and prokineticin 2 induced a transient increase in intracellular calcium ion concentration ([Ca(2+)](i)) with nanomolar potency in Chinese hamster ovary (CHO) cells expressing EG-VEGF/PK-R1 and -R2 and bind to these cells with high affinity and with different receptor selectivity. EG-VEGF/prokineticins provoke rapid phosphorylation of p44/42 MAP kinase and DNA synthesis in the bovine adrenal capillary endothelial cells (BACE). The mRNAs of both EG-VEGF/PK-R1 and -R2 were expressed in BACE. The identification of the receptors for EG-VEGF/prokineticins may provide a novel molecular basis for the regulation of angiogenesis in endocrine glands.