Evaluation of novel 111In-labeled gonadotropin-releasing hormone peptides for human prostate cancer imaging

The purpose of this study was to evaluate the tumor targeting and imaging properties of novel ¹¹¹In-labeled gonadotropin-releasing hormone (GnRH) peptides for human prostate cancer. Three new 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-linker-d-Phe-(d-Lys⁶-GnRH) peptides with different hydrocarbon linkers were designed to evaluate their effects on GnRH receptor binding affinities. The Aoc (aminooctanoic acid) linker was better than βAla (3-aminopropanoic acid) and Aun (aminoundecanoic acid) linkers in retaining strong receptor binding affinity. DOTA-Aoc-d-Phe-(d-Lys⁶-GnRH) exhibited 6.6 ± 0.1 nM GnRH receptor binding affinity. ¹¹¹In-DOTA-Aoc-d-Phe-(d-Lys⁶-GnRH) exhibited fast tumor uptake and urinary clearance in DU145 human prostate cancer-xenografted nude mice. The DU145 tumor lesions could be clearly visualized by single photon emission computed tomography (SPECT)/CT using ¹¹¹In-DOTA-Aoc-d-Phe-(d-Lys⁶-GnRH) as an imaging probe, providing an insight into the design of new GnRH peptides for prostate cancer in the future.     

Introduction of d-Phenylalanine enhanced the receptor binding affinities of gonadotropin-releasing hormone peptides

The purpose of this study was to examine whether the introduction of d-Phe could improve the GnRH receptor binding affinities of DOTA-conjugated d-Lys⁶-GnRH peptides. Building upon the construct of DOTA-Ahx-(d-Lys⁶-GnRH1) we previously reported, an aromatic amino acid of d-Phe was inserted either between the DOTA and Ahx or between the Ahx and d-Lys⁶ to generate new DOTA-d-Phe-Ahx-(d-Lys⁶-GnRH) or DOTA-Ahx-d-Phe-(d-Lys⁶-GnRH) peptides. Compared to DOTA-Ahx-(d-Lys⁶-GnRH1) (36.1 nM), the introduction of d-Phe improved the GnRH receptor binding affinities of DOTA-d-Phe-Ahx-(d-Lys⁶-GnRH) (16.3 nM) and DOTA-Ahx-d-Phe-(d-Lys⁶-GnRH) (7.6 nM). The tumor targeting and pharmacokinetic properties of ¹¹¹In-DOTA-Ahx-d-Phe-(d-Lys⁶-GnRH) was determined in MDA-MB-231 human breast cancer-xenografted nude mice. Compared to ¹¹¹In-DOTA-Ahx-(d-Lys⁶-GnRH1), ¹¹¹In-DOTA-Ahx-d-Phe-(d-Lys⁶-GnRH) exhibited comparable tumor uptake with faster renal and liver clearance. The MDA-MB-231 human breast cancer-xenografted tumors were clearly visualized by single photon emission computed tomography (SPECT) using ¹¹¹In-DOTA-Ahx-d-Phe-(d-Lys⁶-GnRH) as an imaging probe, providing a new insight into the design of new GnRH peptides in the future.     

Synthesis and evaluation of novel gonadotropin-releasing hormone receptor-targeting peptides

The purpose of this study was to develop novel radiolabeled gonadotropin-releasing hormone (GnRH) receptor-targeting peptides for breast cancer imaging. Three novel 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated GnRH peptides were designed and synthesized. The radiometal chelator DOTA was conjugated to the epsilon or alpha amino group of D-lysine, or the epsilon amino group of L-lysine via an Ahx {aminohexanoic acid} linker to generate DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3), respectively. The conjugation of the DOTA to the epsilon amino group of D-lysine (rather than alpha amino group of D-lysine nor epsilon amino group of L-lysine) maintained the nanomolar GnRH receptor binding affinity. The IC(50) values of DOTA-Ahx-(D-Lys(6)-GnRH1), DOTA-Ahx-(D-Lys(6)-GnRH2) and DOTA-Ahx-(L-Lys(6)-GnRH3) were 36.1 nM, 10.6 mM and 4.3 mM, respectively. Since only DOTA-Ahx-(D-Lys(6)-GnRH1) displayed nanomolar receptor binding affinity, the specific GnRH receptor binding of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) was determined in human GnRH receptor membrane preparations. Furthermore, the biodistribution and tumor imaging properties of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) were examined in MDA-MB-231 human breast cancer-xenografted nude mice. (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) exhibited specific GnRH receptor binding and rapid tumor uptake (1.76 ± 0.58% ID/g at 0.5 h postinjection) coupled with fast whole-body clearance through the urinary system. The MDA-MB-231 human breast cancer-xenografted tumor lesions were clearly visualized by single photon emission computed tomography (SPECT)/CT at 1 h postinjection of (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1). The profound impact of DOTA position on the binding affinity of the GnRH peptide provided a new insight into the design of novel radiolabeled GnRH peptides. The successful imaging of MDA-MB-231 human breast cancer-xenografted tumor lesions using (111)In-DOTA-Ahx-(D-Lys(6)-GnRH1) suggested its potential as a novel imaging probe for human breast cancer imaging.     

Design and synthesis of potent tyr(OMe)5-gonadotropin-releasing hormone (GnRH) analogues with modifications at positions 6, 9 and 10

We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilic D-amino acids such as D-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase

Oxime reactivators are the drugs of choice for the post-treatment of OP (organophosphorus) intoxication and used widely for mechanistic and kinetic studies of OP-inhibited cholinesterases. The purpose of the present study was to evaluate new oxime compounds to reactivate acetylcholinesterase (AChE) inhibited by the OP paraoxon. Several new bisquaternary pyridinium oximes with heterocyclic linkers along with some known bisquaternary pyridinium oximes bearing aliphatic linkers were synthesized and evaluated for their in vitro reactivation potency against paraoxon-inhibited electric eel acetylcholinesterase (EeAChE) and recombinant human acetylcholinesterase (rHuAChE). Results herein indicate that most of the compounds are better reactivators of EeAChE than of rHuAChE. The reactivation potency of two different classes of compounds with varying linker chains was compared and observed that the structure of the connecting chain is an important factor for the activity of the reactivators. At a higher concentration (10^?3 M), compounds bearing aliphatic linker showed better reactivation than compounds with heterocyclic linkers. Interestingly, oximes with a heterocyclic linker inhibited AChE at higher concentration (10^?3 M), whereas their ability to reactivate was increased at lower concentrations (10^?4 M and 10^?5 M). Compounds bearing either a thiophene linker 26, 46 or a furan linker 31 showed 59%, 49% and 52% reactivation of EeAChE, respectively, at 10^?5 M. These compounds showed 14%, 6% and 15% reactivation of rHuAChE at 10^?4 M. Amongst newly synthesized analogs with heterocyclic linkers (26–35 and 45–46), compound 31, bearing furan linker chain, was found to be the most effective reactivator with a k_r 0.042 min^?1, which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k_r 0.0041 min^?1 that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE.     

Homologous desensitization of gonadotropin-releasing hormone (GnRH) receptors in the goldfish pituitary: effects of native GnRH peptides and a synthetic GnRH antagonist.

Gonadotropin-releasing hormone (GnRH) stimulates release of gonadotropin hormone (GTH) through interaction with high affinity receptors in the goldfish pituitary. In the present study, we investigated desensitization of two native GnRH peptides, [Trp7, Leu8]-GnRH (sGnRH) and [His5, Trp7, Tyr8]-GnRH (cGnRH-II), using superfused fragments of goldfish pituitary in vitro. Pulsatile treatment with either sGnRH or cGnRH-II (2-min pulses given every 60 min) resulted in dose-dependent secretion of GTH from the goldfish pituitary; cGnRH-II had a greater GTH release potency and displayed a greater receptor binding affinity than sGnRH. Both sGnRH and cGnRH-II-induced GTH release were partially inhibited by concomitant treatment with either [D-Phe2, Pro3, D-Phe6]-GnRH or [D-pGlu1, D-Phe2, D-Trp3.6]-GnRH. These antagonists had greater receptor binding affinities than the native peptides, with no stimulatory action on GTH release in the absence of the GnRH agonists. Continuous treatment with either sGnRH or cGnRH-II (10(-7) M), rapidly desensitized pituitary GTH release in a biphasic fashion; initially there was a rapid increase in GTH release of approximately 10-20-fold (phase 1), followed by a sharp decline in GTH release, reaching a stable concentration 2-3-fold above the basal level (phase 2). Further stimulation of the pituitaries with sGnRH or cGnRH-II (10(-7) M) (second treatment) after 60 min recovery resulted in a significantly lower sGnRH or cGnRH-II-induced GTH release compared to that observed during the initial treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bean peptides have higher in silico binding affinities than ezetimibe for the N-terminal domain of cholesterol receptor Niemann-Pick C1 Like-1

Niemann-Pick C1 like-1 (NPC1L1) mediates cholesterol absorption at the apical membrane of enterocytes through a yet unknown mechanism. Bean, pea, and lentil proteins are naturally hydrolyzed during digestion to produce peptides. The potential for pulse peptides to have high binding affinities for NPC1L1 has not been determined. In this study , in silico binding affinities and interactions were determined between the N-terminal domain of NPC1L1 and 14 pulse peptides (5≥ amino acids) derived through pepsin-pancreatin digestion. Peptides were docked in triplicate to the N-terminal domain using docking program AutoDock Vina, and results were compared to those of ezetimibe, a prescribed NPC1L1 inhibitor. Three black bean peptides (−7.2 to −7.0 kcal/mol) and the cowpea bean dipeptide Lys-Asp (−7.0 kcal/mol) had higher binding affinities than ezetimibe (−6.6 kcal/mol) for the N-terminal domain of NPC1L1. Lentil and pea peptides studied did not have high binding affinities. The common bean peptide Tyr-Ala-Ala-Ala-Thr (−7.2 kcal/mol), which can be produced from black or navy bean proteins, had the highest binding affinity. Ezetimibe and peptides with high binding affinities for the N-terminal domain are expected to interact at different locations of the N-terminal domain. All high affinity black bean peptides are expected to have van der Waals interactions with SER130, PHE136, and LEU236 and a conventional hydrogen bond with GLU238 of NPC1L1. Due to their high affinity for the N-terminal domain of NPC1L1, black and cowpea bean peptides produced in the digestive track have the potential to disrupt interactions between NPC1L1 and membrane proteins that lead to cholesterol absorption.     

Design and Synthesis of Potent Tyr(OMe)5-Gonadotropin-Releasing Hormone (GnRH) Analogues with Modifications at Positions 6, 9 and 10

Summary We have recently reported the synthesis and the conformational properties of some Gonadotropin-releasing hormone (GnRH) analogues in which the tyrosine residue at position 5 is substituted with tyrosine-O-methyl (Keramida et al., Let. Pept. Sci., 3 (1996) 257/Matsoukas et al., Eur. J. Med. Chem., 32 (1997) 927). The analogue [Tyr-(OMe)⁵]-GnRH was found to exert a lower degree of desensitization than the native GnRH peptides in terms of pituitary gonadotropin (GTH) release in goldfish. Compared to GnRH, however, [Tyr-(OMe)⁵]-GnRH exerted a lower GTH-release potency in cultured goldfish pituitary fragments, and was bound with a lower binding affinity to the rat pituitary GnRH receptors. In order to increase the potency of [Tyr-(OMe)⁵]-GnRH, we have synthesized a group of GnRH peptides containing Tyr-(OMe)⁵ in combination with other substitutions at positions 6, 9 and 10 and we have estimated their binding affinity for the rat pituitary receptors and gonadotropin (GTH) release potency in the goldfish pituitary. A selected number of these analogues was also tested for their ability to induce ovulation in seabass. The important structural modifications that increased the binding and gonadotropic activity of [Tyr(OMe)⁵]-GnRH in vitro and in vivo were found to include the replacement of the proline at position 9 with azetidine, glycine amide terminus with an alkyl amide group and Gly⁶ residue with hydrophilicd-amino acids such asd-Arg⁶. Overall, the findings provide SAR information on a group of novel GnRH peptides that can be also used to induce ovulation in teleosts.     

Pentacycle derivatives as cannabinoid CB1 receptor ligands

Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of pentacycle derivatives. Five of the new compounds which displayed high in vitro rCB1 binding affinities were assayed for binding to hCB2 receptor. Noticeably, 2-(5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-(5-methyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole (16l) demonstrated good binding affinity and decent selectivity for rCB1 receptor (IC₅₀ = 1.72 nM, hCB2/rCB1 = 142).     

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A_2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A_2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A_2A adenosine receptor ligand with K_i = 0.06 μM. Similarly potent and highly A_2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs.     

Synthesis and characterization of N,N-dialkyl and N-alkyl-N-aralkyl fenpropimorph-derived compounds as high affinity ligands for sigma receptors

The sigma-1 receptor is a unique non-opioid, non-PCP binding site that has been implicated in many different pathophysiological conditions including psychosis, drug addiction, retinal degeneration and cancer. Based on the structure of fenpropimorph, a high affinity (K_i = 0.005 nM)¹ sigma-1 receptor ligand and strong inhibitor of the yeast sterol isomerase (ERG2), we previously deduced a basic sigma-1 receptor pharmacophore or chemical backbone composed of a phenyl ring attached to a di-substituted nitrogen atom via an alkyl chain.² Here, we report the design and synthesis of various N,N-dialkyl or N-alkyl-N-aralkyl derivatives based on this pharmacophore as well as their binding affinities to the sigma-1 receptor. We introduce three high affinity sigma-1 receptor compounds, N,N-dibutyl-3-(4-fluorophenyl)propylamine (9), N,N-dibutyl-3-(4-nitrophenyl)propylamine (3), and N-propyl-N′-4-aminophenylethyl-3-(4-nitrophenyl)propylamine (20) with K_i values of 17.7 nM, 0.36 nM, and 6 nM, respectively. In addition to sigma receptor affinity, we show through cytotoxicity assays that growth inhibition of various tumor cell lines occurs with our high affinity N,N-dialkyl or N-alkyl-N-aralkyl derivatives.     

Synthesis and evaluation of new N6-substituted adenosine-5′-N-methylcarboxamides as A3 adenosine receptor agonists

A number of N⁶-substituted adenosine-5′-N-methylcarboxamides were synthesised and their pharmacology, in terms of their receptor affinity, selectivity and cardioprotective effects, were explored. The first series of compounds, 4a–4f and 5a–5f, showed modest receptor affinity for the A₃AR with K_i values in the low to mid μM range. However, the incorporation of a 4-(2-aminoethyl)-2,6-di-tert-butylphenol group in the N⁶-position (in compounds 4g and 5g) significantly improved the affinity with K_i values of 30 and 9 nM, respectively. Improvements in affinity, as well as selectivity were seen when a functionalised linker was introduced. The N⁶-phenyl series, compounds 7a–7d, demonstrated low to mid nanomolar receptor affinities (K_i = 2.3–45.0 nM), with 7b displaying 109-fold selectivity for the A₃AR (vs A₁). The N⁶-benzyl series 9a–9c also proved to be potent and selective A₃AR agonists and the longer chain length linker 13 was tolerated at the A₃AR without abrogation of affinity or selectivity. Cardioprotection was demonstrated by a simulated ischaemia cell culture assay, whereby 7b, 7c, 9a, 9b and 9c all showed cardioprotective effects at 100 nM comparable or better than the benchmark A₃AR agonist IB-MECA, but which were indistinguishable by statistical analysis. For example, compound 9c reduced cell death by 68.0 ± 3.6%.     

Structure activity relationship study of benzo[d]thiazol-2(3H)one based σ receptor ligands

Herein we report the SAR study which involved structural modifications to the linker length, aryl substitution and alkylamine ring size of the benzo[d]thiazol-2(3H)one based sigma receptor (σ) ligands. Many compounds in this series displayed low nanomolar affinity for the σ receptor subtypes. In particular, 8a showed high affinity (σ-1 K_i = 4.5 nM) for σ-1 receptors and moderately high selectivity (483-fold) over σ-2 receptors.     

Synthesis and biological evaluation of 18F-labled 2-phenylindole derivatives as PET imaging probes for β-amyloid plaques

A novel series of fluorinated 2-phenylindole derivatives were synthesized and evaluated as β-amyloid imaging probes for PET. The in vitro inhibition assay demonstrated that their binding affinities for Aβ_1–42 aggregates ranged from 28.4 to 1097.8 nM. One ligand was labeled with ¹⁸F ([¹⁸F]1a) for its high affinity (K_i = 28.4 nM), which was also confirmed by in vitro autoradiography experiments on brain sections of transgenic mouse (C57BL6, APPswe/PSEN1, 11 months old, male). In vivo biodistribution experiments in normal mice showed that this radiotracer displayed high initial uptake (5.82 ± 0.51% ID/g at 2 min) into and moderate washout (2.77 ± 0.31% ID/g at 60 min) from the brain. [¹⁸F]1a could be developed as a promising new PET imaging probe for Aβ plaques although necessary modifications are still needed.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.     

Synthesis,biological activity and structure–activity relationship of endomorphin-1/substance P derivatives

Endomorphins have been shown to produce potent analgesia in various rodent models of pain. However, their central administration led to the development of tolerance and physical dependence. Conjugation of C-terminal substance P (SP) fragments to opioids and opioid peptides was previously shown to produce hybrid peptides with strong analgesic activity, with low or no propensity to develop tolerance. In this study, four peptides (2–5) comprised of endomorphin-1 (1) and C-terminal fragments of SP (four or five amino acids, SP_8–11 (2) or SP_7–11 (4), respectively), with an overlapping Phe residue, were synthesized. To overcome low metabolic stability and poor membrane permeability of the peptide, the N-terminus of 2 and 4 was further modified with a C10-carbon lipoamino acid (C10LAA) achieving 3 and 5, respectively. LAA-modification of the hybrid peptides resulted in a significant increase in metabolic stability and membrane permeability compared to peptides 1, 2 and 4. Compound 5 showed potent μ-opioid receptor binding affinity (K_iμ = 3.87 ± 0.51 nM) with dose-dependent agonist activity in the nanomolar range (IC₅₀ = 45 ± 13 nM). In silico modeling was used to investigate the binding modes and affinities of compounds 1–5 in the active site of μ-opioid receptors. The docking scores were in agreement with the K_iμ values obtained in the receptor binding affinity studies. The more active LAA-modified hybrid peptide showed a lower total interaction energy and higher negative value of MolDock score.     

Redox-sensitive stimulation of type-1 ryanodine receptors by the scorpion toxin maurocalcine

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²⁺ release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²⁺ uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent K_i for Mg²⁺ inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²⁺] from 10 μM to 0.1 μM or at cytoplasmic [Mg²⁺] ≥ 30 μM. At 0.1 μM [Ca²⁺], only channels that displayed poor activation by Ca²⁺ were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.     

Synthesis and SAR of 99mTc/Re-labeled small molecule prostate specific membrane antigen inhibitors with novel polar chelates

Prostate specific membrane antigen (PSMA) is recognized as an attractive molecular target for the development of radiopharmaceuticals to image and potentially treat metastatic prostate cancer. A series of novel ^99mTc/Re-tricarbonyl radiolabeled PSMA inhibitors were therefore synthesized by the attachment of glutamate-urea-lysine (Glu-urea-Lys) and glutamate-urea-glutamate (Glu-urea-Glu) pharmacophore to single amino acid chelate (SAAC) where the SAAC ligand was either bis(pyridin-2-ylmethyl)amino (DPA), bis((1-methyl-1H-imidazol-2-yl)methyl)amino (NMI), bis((1-(carboxymethyl)-1H-imidazol-2-yl)methyl)amino (CIM) or bis((1-(2-(bis(carboxymethyl)amino)-2-oxoethyl)-1H-imidazol-2-yl)methyl)amino (TIM). The in vitro binding affinity of the rhenium complexes was evaluated using PSMA-expressing human prostate cancer LNCaP cells. IC₅₀ values ranged from 3.8 ± 2 to >2000 nM. A linker between the SAAC chelate and pharmacophore was required for high affinity binding. However, extending the length of the linker did not substantially improve binding. PSMA binding was also influenced by the nature of the SAAC chelate. One of the most potent compounds, 23b (IC₅₀ = 4.8 ± 2.7 nM), was radiolabeled with technetium tricarbonyl ({^99mTc(CO)₃}⁺) to afford the {^99mTc(CO)₃}⁺ complex in excellent yield and high purity. This effort has led to the identification of a diverse series of promising high affinity {^99mTc(CO)₃}⁺ radiolabeled PSMA inhibitors.     

Synthesis and evaluation of 2-amino-dihydrotetrabenzine derivatives as probes for imaging vesicular monoamine transporter-2

A novel series of analogs of 2-amino-dihydrotetrabenazine derivatives, 4–6, targeting the vesicular monoamine transporter have been prepared. In vitro binding was carried out in tissue homogenates prepared from rat striatal tissue homogenates with both [¹²⁵I]-iodovinyl-TBZ and [³H]DTBZ. There was a good correlation (r² = 0.925) between the affinities of the different compounds for [¹²⁵I]-iodovinyl-TBZ and [³H]-DTBZ binding. Compound 5 exhibited a better affinity for the vesicular monoamine transporter (K_i = 8.68 ± 1.26 nM and 7.01 ± 0.07 nM, respectively), which may be a good lead compound for further structural modification to develop useful probes for VMAT2.