- Email: [email protected]
Development of biologically active multi-iodinated somatestatin analogs
2] 0
Abstracts from the 1 lth International Symposium on Regulatory Peptides
DEVELOPMENTOFBiOLOIRIGALLVACTIVEl l U - H l i l i K i l
81ilIATOSTAIIHANALOGS
E. A. Woltering, W. Murphy, M. S. O'Dorisio, D. Coy, G. Drouant, J. Fuselier de la Claire, F. Chen, G. Espenan, T. M. O'Dorisio. Louisiana State University, New Orleans, LA; Tulane University, New Orleans, LA; The Ohio State University, Columbus, OH. Effective in situ radiation therapy utilizing radiolabeled somatostatin analogs, requires that these compounds possess high specific activities and high biologic activity in the iodinated form. N-terminally extended, multiply-tyrosinated sometostatin analogs were solid-phase synthesized and subsequently iodinated with 1271using a chloramine-T method. Iodination methods that yielded compounds with predominantly one iodine per lyrosine were developed. All labeled analogs underwent mass spectroscopy to determine the presence of unlabeled peptide and to profile the distribution and number of iodinated species. Binding affinity and growth hormone IC~ were determined for both non-iodinated and iodinated peptides. Binding affinity (Kd, nM) was determined in IMR-32 membranes (IMR) and inhibition of growth hormone (GH) (IC5o,nM) was determined in fourto five-day-old primary rat anterior pituitary cultures. There is an excellent Not Iodinated Iodinaled(12q) correlation between an Growth Growth TheoreticSpecific analog's IMR binding affinity Activity t311 # Tyr Hormone IMR-32 Hormone IMR-32 and its GH inhibitory activity Peptide VloleculesIC= (nM/+SEIV Kd (nM)±SD IC• (nM)±SEM Kd (nM)±SD (Ci/mmol) in the non-iodinated state. ;RIF 0 0.46 ± 0.04 0.45± 0.25 NA NA NA However, iodination of these .anreotide 1 0.59±0.13 1.05±0.15 4.00,1.80 71.80 ± 20.10 1200- 2200 compounds does not alter ~/OC-Illa 3600 - 7200 3 124,0.14 1.72±0.53 0.23*0.06 21.0 + 0.25 their GH inhibition but NOC-IIIb 3600-7200 3 0.68±0.66 1.14±0.28 023±0.02 1.64±0.14 significantly alters IMR ~OC-IVa 4800 - 9600 4 0.37±0.12 0.43±0.24 0.004~ 0.006 6.68 ± 133 binding affinity, possibly by , NA = Not Applicable altering their hydrophobicity profile or their binding to SST receptor sub-types. Based on these observations, we believe that functional analysis of biologic activity may accurately predict the in vivo activity of multiply-iodinated somatostatin analogs. Multiply-tyrosinated radioiodinated somatostatin analogs demonstrating high biologic activity may be clinically useful for in vivo scintigraphy and in situ radiation therapy.
FIRST INTRACELLULAR LOOP OF CCK-A RECEPTOR IS ESSENTIAL FOR cAMP RESPONSE TO CCK IN TRANSFECTED HEK-293 CELLS S. V. Wu, M. Yang, R. Seensalu, J. McRoberts, and J. H. Walsh. CURE/ Digestive Diseases Research Center, Department of Medicine, UCLA and VA West LA, Los Angeles, CA 90073 CCK-A but not CCK-B receptors, when stably expressed in HEK-293 cells, are able to activate the cAMP signaling pathway. The aim of the present study was to determine the critical domains of the CCK-A receptor that confer the cAMP response. Human CCK-A and CCK-B receptor genes share similar exon-intron organization and each consists of five exons. A series of chimeric CCK receptor mutants were generated using combinations of CCK-A and CCK-B receptor exons. CCK-A receptor exons were systematically replaced, from the first to the fourth, with corresponding CCK-B receptor exons. Wild type CCK-A, CCK-B and chimeric receptors in mammalian expression vector pcDNA3 were transfected into the human embryonic kidney cell line (HEK-293). Stable cell lines expressing comparable numbers of receptor sites were examined for their binding to =25I-BH-CCK-8, and for intracellular calcium and cAMP responses to sulfated CCK-8. The table below summarizes the results of ligand binding and second messenger studies performed on representative cell lines. All values are in nM and NR stands for no response. Receptor~o, binding (I~, nM) calcium cAMP Ad Cyclase CCK-8 G-17 (ECs0, nM) (ECso, nM) (ECs0, nM) Ai.5 (wild type) 1.5 >1000 0.1 20 19 BI/A2_5 7.6 >1000 0.5 15 20 B 1.7JA3.5 0.1 0.2 10 NR NR BI-3/A4.5 0.3 0.7 10 NR NR BjA5 2.5 7 1 NR NR B~.5 (wild type) 0.5 0.8 0.3 NR NR All HEK-293 cells transfected with native or chimeric CCK receptors were able to bind '"I-BH-CCK-8 and to transduce Ca 2÷ signals, indicating that all constructs were functionally expressed. However, only chimeric receptor Bt/A2.5, similar to wild type CCK-AR, exhibited increased cAMP levels and adenylyl cyelase activities in response to sulfated CCK-8 stimulation, with ECs0 values about 15 and 20 nM, respectively. These constructs also were characterized by very low affinity binding to gastrin-17. The presence of the second exon (which encodes the first transmembrane domain, the first intracellular loop, the secnond transmembrane domain and the first extraeellular loop) of CCK-AR thus coincides with positive cAMP response and the absence of high affinity gastrin binding. Our data suggest the first intracellular loop of CCK-AR may be required for Gs,~coupling which, upon agonist stimulation, leads to adenylyl cyclase activation.
Abstracts from the 1 lth International Symposium on Regulatory Peptides
DEVELOPMENTOFBiOLOIRIGALLVACTIVEl l U - H l i l i K i l
81ilIATOSTAIIHANALOGS
E. A. Woltering, W. Murphy, M. S. O'Dorisio, D. Coy, G. Drouant, J. Fuselier de la Claire, F. Chen, G. Espenan, T. M. O'Dorisio. Louisiana State University, New Orleans, LA; Tulane University, New Orleans, LA; The Ohio State University, Columbus, OH. Effective in situ radiation therapy utilizing radiolabeled somatostatin analogs, requires that these compounds possess high specific activities and high biologic activity in the iodinated form. N-terminally extended, multiply-tyrosinated sometostatin analogs were solid-phase synthesized and subsequently iodinated with 1271using a chloramine-T method. Iodination methods that yielded compounds with predominantly one iodine per lyrosine were developed. All labeled analogs underwent mass spectroscopy to determine the presence of unlabeled peptide and to profile the distribution and number of iodinated species. Binding affinity and growth hormone IC~ were determined for both non-iodinated and iodinated peptides. Binding affinity (Kd, nM) was determined in IMR-32 membranes (IMR) and inhibition of growth hormone (GH) (IC5o,nM) was determined in fourto five-day-old primary rat anterior pituitary cultures. There is an excellent Not Iodinated Iodinaled(12q) correlation between an Growth Growth TheoreticSpecific analog's IMR binding affinity Activity t311 # Tyr Hormone IMR-32 Hormone IMR-32 and its GH inhibitory activity Peptide VloleculesIC= (nM/+SEIV Kd (nM)±SD IC• (nM)±SEM Kd (nM)±SD (Ci/mmol) in the non-iodinated state. ;RIF 0 0.46 ± 0.04 0.45± 0.25 NA NA NA However, iodination of these .anreotide 1 0.59±0.13 1.05±0.15 4.00,1.80 71.80 ± 20.10 1200- 2200 compounds does not alter ~/OC-Illa 3600 - 7200 3 124,0.14 1.72±0.53 0.23*0.06 21.0 + 0.25 their GH inhibition but NOC-IIIb 3600-7200 3 0.68±0.66 1.14±0.28 023±0.02 1.64±0.14 significantly alters IMR ~OC-IVa 4800 - 9600 4 0.37±0.12 0.43±0.24 0.004~ 0.006 6.68 ± 133 binding affinity, possibly by , NA = Not Applicable altering their hydrophobicity profile or their binding to SST receptor sub-types. Based on these observations, we believe that functional analysis of biologic activity may accurately predict the in vivo activity of multiply-iodinated somatostatin analogs. Multiply-tyrosinated radioiodinated somatostatin analogs demonstrating high biologic activity may be clinically useful for in vivo scintigraphy and in situ radiation therapy.
FIRST INTRACELLULAR LOOP OF CCK-A RECEPTOR IS ESSENTIAL FOR cAMP RESPONSE TO CCK IN TRANSFECTED HEK-293 CELLS S. V. Wu, M. Yang, R. Seensalu, J. McRoberts, and J. H. Walsh. CURE/ Digestive Diseases Research Center, Department of Medicine, UCLA and VA West LA, Los Angeles, CA 90073 CCK-A but not CCK-B receptors, when stably expressed in HEK-293 cells, are able to activate the cAMP signaling pathway. The aim of the present study was to determine the critical domains of the CCK-A receptor that confer the cAMP response. Human CCK-A and CCK-B receptor genes share similar exon-intron organization and each consists of five exons. A series of chimeric CCK receptor mutants were generated using combinations of CCK-A and CCK-B receptor exons. CCK-A receptor exons were systematically replaced, from the first to the fourth, with corresponding CCK-B receptor exons. Wild type CCK-A, CCK-B and chimeric receptors in mammalian expression vector pcDNA3 were transfected into the human embryonic kidney cell line (HEK-293). Stable cell lines expressing comparable numbers of receptor sites were examined for their binding to =25I-BH-CCK-8, and for intracellular calcium and cAMP responses to sulfated CCK-8. The table below summarizes the results of ligand binding and second messenger studies performed on representative cell lines. All values are in nM and NR stands for no response. Receptor~o, binding (I~, nM) calcium cAMP Ad Cyclase CCK-8 G-17 (ECs0, nM) (ECso, nM) (ECs0, nM) Ai.5 (wild type) 1.5 >1000 0.1 20 19 BI/A2_5 7.6 >1000 0.5 15 20 B 1.7JA3.5 0.1 0.2 10 NR NR BI-3/A4.5 0.3 0.7 10 NR NR BjA5 2.5 7 1 NR NR B~.5 (wild type) 0.5 0.8 0.3 NR NR All HEK-293 cells transfected with native or chimeric CCK receptors were able to bind '"I-BH-CCK-8 and to transduce Ca 2÷ signals, indicating that all constructs were functionally expressed. However, only chimeric receptor Bt/A2.5, similar to wild type CCK-AR, exhibited increased cAMP levels and adenylyl cyelase activities in response to sulfated CCK-8 stimulation, with ECs0 values about 15 and 20 nM, respectively. These constructs also were characterized by very low affinity binding to gastrin-17. The presence of the second exon (which encodes the first transmembrane domain, the first intracellular loop, the secnond transmembrane domain and the first extraeellular loop) of CCK-AR thus coincides with positive cAMP response and the absence of high affinity gastrin binding. Our data suggest the first intracellular loop of CCK-AR may be required for Gs,~coupling which, upon agonist stimulation, leads to adenylyl cyclase activation.