Functional analysis of rare variants found in schizophrenia implicates a critical role for GIT1-PAK3 signaling in neuroplasticity.

Although the pathogenesis of schizophrenia (SCZ) is proposed to involve alterations of neural circuits via synaptic dysfunction, the underlying molecular mechanisms remain poorly understood. Recent exome sequencing studies of SCZ have uncovered numerous single-nucleotide variants (SNVs); however, the majority of these SNVs have unknown functional consequences, leaving their disease relevance uncertain. Filling this knowledge gap requires systematic application of quantitative and scalable assays to assess known and novel biological functions of genes. Here we demonstrate loss-of-function effects of multiple rare coding SNVs found in SCZ subjects in the GIT1 (G protein-coupled receptor kinase interacting ArfGAP 1) gene using functional cell-based assays involving coexpression of GIT1 and PAK3 (p21 protein (Cdc42/Rac)-activated kinase 3). Most notably, a GIT1-R283W variant reported in four independent SCZ cases was defective in activating PAK3 as well as MAPK (mitogen-activated protein kinase). Similar functional deficits were found for a de novo SCZ variant GIT1-S601N. Additional assays revealed deficits in the capacity of GIT1-R283W to stimulate PAK phosphorylation in cultured hippocampal neurons. In addition, GIT1-R283W showed deficits in the induction of GAD1 (glutamate decarboxylase 1) protein expression. Extending these functional assays to 10 additional rare GIT1 variants revealed the existence of an allelic series with the majority of the SCZ case variants exhibiting loss of function toward MAPK activation in a manner correlated with loss of PAK3 activation. Taken together, we propose that rare variants in GIT1, along with other genetic and environmental factors, cause dysregulation of PAK3 leading to synaptic deficits in SCZ.

Biochemical characterization of dipeptidylcarboxypeptidase of Leishmania donovani.

The incidence of parasitic infection, leishmaniasis, has been steadily increasing worldwide. Since, the existing chemotherapy of these diseases suffers from lack of safe and effective drugs and/or the presence of widespread drug resistance, there is an urgent need for development of potent, mechanism-based anti-parasitic agents. The peptidases of protozoan parasites are becoming increasingly important for their role in parasite survival and pathogenecity. Leishmania donovani dipeptidylcarboxypeptidsae (LdDCP), an angiotensin converting enzyme (ACE) related metallopeptidase has been identified and characterized as a putative drug target for antileishmanial chemotherapy. The kinetic parameters for LdDCP with substrate, Hip-His-Leu were determined as, Km, 4 mM and Vmax, 1.173 µmole/ml/min. The enzyme was more sensitive to 1,10 phenanthroline than EDTA and was 80% inhibited in presence of NaCl. Among various protease inhibitors, pepstatin was found as potent inhibitor of LdDCP.

Thimet oligopeptidase (EC 3.4.24.15) activates CPI-0004Na, an extracellularly tumour-activated prodrug of doxorubicin.

CPI-0004Na is a tetrapeptidic extracellularly tumour-activated prodrug of doxorubicin. The tetrapeptide structure ensures blood stability and selective cleavage by unidentified peptidase(s) released by tumour cells. The purpose of this work was to identify the enzyme responsible for the first rate-limiting step of CPI-0004Na activation, initially attributed to a 70 kDa acidic (pI=5.2) metallopeptidase active at neutral pH that was subsequently purified from HeLa cell homogenates. Two electrophoretic bands were isolated and identified by matrix-assisted laser desorption ionisation-time of flight (MALDI-tof) and electrospray ionisation-quadrupole-time of flight (ESI-Q-tof) mass spectrometry as thimet oligopeptidase (TOP). The identity of the CPI-0004Na activating enzyme and TOP was further supported by the similar substrate specificity of the purified enzyme and recombinant TOP, by thiol stimulation of CPI-0004Na cleavage by cancer cell conditioned media (unique characteristic of TOP) and by the inhibition of CPI-0004Na activation by specific inhibitors or immunoprecipitation. Although other enzymes can be involved, TOP clearly appears to be a likely candidate for extracellular activation of the CPI-0004Na prodrug.

N-Succinyl-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin: an extracellularly tumor-activated prodrug devoid of intravenous acute toxicity.

Intravenous administration of N-(beta-alanyl-L-leucyl-L-alanyl-L-leucyl)doxorubicin (4) induces an acute toxic reaction, killing animals in a few minutes. This results from its positive charge at physiological pH combined with its propensity to form large aggregates in aqueous solutions. Negatively charged N-capped versions of 4 such as the succinyl derivative 5 can be administered by the iv route at more than 10 times the LD(50) of doxorubicin without inducing the acute toxic reaction, and they are active in vivo.

Dolastatin 11, a marine depsipeptide, arrests cells at cytokinesis and induces hyperpolymerization of purified actin.

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers.

In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]-enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid- and phenylpropionic acid-based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C-terminal amino acids of opioid peptides (L-Leu, [Leu5]-enkephalin; D-Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N-terminal amino acid (L-Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30-50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco-2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes-Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]-enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.

Synthesis and evaluation of the physicochemical properties of esterase-sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker.

The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]-enkephalin (Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C- and N-terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N- and C-terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes-Einstein molecular radii similar to those of [Leu5]-enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco-2 cell monolayer than [Leu5]-enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P-glycoprotein, which significantly reduced their ability to permeate across Caco-2 cell monolayers. When P-glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.

Phenylpropionic acid-based cyclic prodrugs of opioid peptides that exhibit metabolic stability to peptidases and excellent cellular permeation.

PURPOSE: To evaluate the cellular permeation characteristics and the chemical and enzymatic stability of phenylpropionic acid-based cyclic prodrugs 1 and 2 of opioid peptides [Leu5]-enkephalin (H-Tyr-Gly-Gly-Phe-Leu-OH) and DADLE (H-Tyr-D-Ala-Gly-Phe-D-Leu-OH), respectively. METHODS: The rates of conversion of cyclic prodrugs 1 and 2 to [Leu5]-enkephalin and DADLE, respectively, in HBSS, pH 7.4 (Caco-2 cell transport buffer) and in various biological media having measurable esterase activity were determined by HPLC. The cell permeation characteristics of [Leu5]-enkephalin, DADLE, and cyclic prodrugs 1 and 2 were measured using Caco-2 cell monolayers grown onto microporus membranes and monitored by HPLC. RESULTS: In HBSS, pH 7.4, cyclic prodrugs 1 and 2 degraded to [Leu5]-enkephalin and DADLE, respectively, in stoichiometric amounts. In 90% human plasma, the rates of disappearance of cyclic prodrugs 1 and 2 were slightly faster than in HBSS, pH 7.4. These accelerated rates of disappearance in 90% human plasma could be reduced to the rates observed in HBSS, pH 7.4, by pretreatment of the plasma with paraoxon, a known inhibitor of serine-dependent esterases. In homogenates of Caco-2 cells and rat liver, accelerated rates of disappearance of cyclic prodrugs 1 and 2 were not observed. When applied to the AP side of a Caco-2 cell monolayer, cyclic prodrug 1 exhibited significantly greater stability against peptidase metabolism than did [Leu5]-enkephalin. Cyclic prodrug 2 and DADLE exhibited stability similar to prodrug 1 when applied to the AP side of the Caco-2 cell monolayers. Prodrug 1 was 1680 fold more able to permeate the Caco-2 cell monolayers than was [Leu5]-enkephalin, in part because of its increased enzymatic stability. Prodrug 2 was shown to be approximately 77 fold more able to permeate a Caco-2 cell monolayer than was DADLE. CONCLUSIONS: Cyclic prodrugs 1 and 2, prepared with the phenylpropionic acid promoiety, were substantially more able to permeate Caco-2 cell monolayers than were the corresponding opioid peptides. Prodrug 1 exhibited increased stability to peptidase metabolism compared to [Leu5]-enkephalin. In 90% human plasma but not in Caco-2 cell and rat liver homogenates, the opioid peptides were released from the cyclic prodrugs by an esterase-catalyzed reaction that is sensitive to paraoxon inhibition. However, the rate of this bioconversion appears to be extremely slow.

Synthesis of an esterase-sensitive cyclic prodrug of a model hexapeptide having enhanced membrane permeability and enzymatic stability using a 3-(2'-hydroxy-4',6'-dimethylphenyl)-3,3-dimethyl propionic Acid promoiety.

One of the major obstacles to the development of biologically active peptides as clinically useful therapeutic agents has been their low permeation through biological barriers (e.g., intestinal mucosa, blood-brain barrier) and their metabolic lability (1,2). Overcoming these problems is a very contemporary issue for the development of peptide pharmaceuticals. In the preceding chapter, we have indicated that masking the C- and N-terminal polar functional groups of a peptide through cyclization with an acyloxyalkoxy linker can greatly enhance the membrane permeation and metabolic stability of the linear peptide (3). In this chapter, we wish to report a method for the preparation of esterase-sensitive cyclic prodrugs of peptides by taking advantage of a unique "trimethyl lock"-facilitated lactonization system (Fig. 1). Substituted phenol propionic acid derivatives such as 2, upon unmasking of the hydroxyl group, undergo a facile spontaneous intramolecular cyclization to release the moieties attached to the carboxyl functional group (Fig. 1) (4-6). The facile cyclization reaction is the result of the "trimethyl lock", which was shown earlier to increase the rate of the cyclization reaction in the order of 10(5-7) (4-7). The result of such facilitation is that compound 2 has a half-life of only approximately 100 s at room temperature in aqueous solution (8,9). Such systems have been used to develop prodrugs of amines and alcohols (8-10) and redox-sensitive protecting groups of amines (11). Fig. 1. The design of an esterase sensitive prodrug system for the cyclic deriva-tization of peptides.

Synthesis of an esterase-sensitive cyclic prodrug of a model hexapeptide having enhanced membrane permeability and enzymatic stability using an acyloxyalkoxy promoiety.

The clinical development of orally active peptide drugs has been limited by their unfavorable physicochemical characteristics (e.g., charge, hydrogen bonding potential, size), which prevent them from permeating biological barriers such as the intestinal mucosa, and also their lack of stability against enzymatic degradation (1-12). Unfortunately, many of the structural features of peptides (i.e., the N-terminal amino group and C-terminal carboxyl group, and side chain carboxyl, amino, and hydroxyl groups) that bestow upon the molecule affinity and specificity for its pharmacological receptor severely restrict its ability to permeate biological barriers and make the molecules substrates for peptidases. Therefore, successful oral delivery of peptides depends on strategies designed to alter the physicochemical characteristics of these potential drugs without changing their biological activity in order to circumvent the intestinal epithelial cells.

Esterase-sensitive cyclic prodrugs of peptides: evaluation of a phenylpropionic acid promoiety in a model hexapeptide.

PURPOSE: To evaluate a cyclic phenylpropionic acid prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of a peptide and stabilize it to metabolism. METHODS: Conversion to the linear hexapeptide was studied at 37 degrees C in HBSS, pH 7.4, and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. RESULTS: In aqueous buffered solution, pH 7.4, the cyclic prodrug degraded quantitatively (t1/2 = 1795 +/- 289 min) to the linear hexapeptide and the lactone. Substantially faster degradation of the cyclic prodrug was observed in 90% human plasma (t1/2 = 508 +/- 24 min), and in homogenates of Caco-2 cells (t1/2 = 940 +/- 13 min), the rat intestinal mucosa (t1/2 = 1286 +/- 32 min), and rat liver (t1/2 = 840 +/- 42 min). Pretreatment of these biological media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug was significantly more stable than the hexapeptide and at least 71-fold more able to permeate (P(app) = 1.21 +/- 0.12 X 10(-7) cm/s) than was the parent peptide (P(app) < or = 0.17 x 10(-8) cm/s). In the presence of 0.1 mM palmitoyl-DL-carnitine, the transport rate of the cyclic prodrug (P(app) = 2.19 X 10(-6) cm/s) was 1250-fold greater than that of the linear hexapeptide. CONCLUSIONS: Preparation of a cyclic peptide using a phenylpropionic acid promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.

Improvement of oral peptide bioavailability: Peptidomimetics and prodrug strategies.

Clinical development of orally active peptide drugs has been restricted by their unfavorable physicochemical properties, which limit their intestinal mucosal permeation and their lack of stability against enzymatic degradation. Successful oral delivery of peptides will depend, therefore, on strategies designed to alter the physicochemical characteristics of these potential drugs, without changing their biological activity, in order to overcome the physical and biochemical barrier properties of the intestinal cells. This manuscript will focus on the physiological limitations for oral peptide delivery and on various strategies using chemical modifications to improve oral bioavailability of peptide-based drugs.

Esterase-sensitive cyclic prodrugs of peptides: evaluation of an acyloxyalkoxy promoiety in a model hexapeptide.

PURPOSE: To evaluate a cyclic acyloxyalkoxycarbamate prodrug of a model hexapeptide (H-Trp-Ala-Gly-Gly-Asp-Ala-OH) as a novel approach to enhance the membrane permeation of the peptide and stabilize it to metabolism. METHODS: Conversion to the linear hexapeptide was studied at 37 degrees C in aqueous buffered solutions and in various biological milieus having measurable esterase activities. Transport and metabolism characteristics were assessed using the Caco-2 cell culture model. RESULTS: In buffered solutions the cyclic prodrug degraded chemically to the linear hexapeptide in stoichiometric amounts. Maximum stability was observed between pH 3-4. In 90% human plasma (t1/2 = 100 +/- 4 min) and in homogenates of the rat intestinal mucosa (t1/2 = 136 +/- 4 min) and rat liver (t1/2 = 65 +/- 3 min), the cyclic prodrug disappeared faster than in buffered solution, pH 7.4 (t1/2 = 206 +/- 11 min). Pretreatment of these media with paraoxon significantly decreased the degradation rate of the prodrug. When applied to the apical side of Caco-2 cell monolayers, the cyclic prodrug (t1/2 = 282 +/- 25 min) was significantly more stable than the hexapeptide (t1/2 = 14 min) and at least 76-fold more able to permeate (Papp = 1.30 +/- 0.15 x 10(-7) cm/s) than the parent peptide (Papp < or = 0.17 x 10(-8) cm/s). CONCLUSIONS: Preparation of a cyclic peptide using an acyloxyalkoxy promoiety reduced the lability of the peptide to peptidase metabolism and substantially increased its permeation through biological membranes. In various biological media the parent peptide was released from the prodrug by an apparent esterase-catalyzed reaction, sensitive to paraoxon inhibition.

The effect of conformation on membrane permeability of an acyloxyalkoxy-linked cyclic prodrug of a model hexapeptide.

PURPOSE: To determine the different conformations of the acyloxyalkoxy-linked cyclic prodrug 1 of the model hexapeptide 2 in solution and to investigate the relationship between these solution conformations and the cellular permeability characteristics of this prodrug. METHODS: Two-dimensional Homonuclear Hartmann-Hahn spectroscopy, Rotating-Frame Overhouser effect spectroscopy, circular dichroism and molecular dynamics simulations were used to find the solution conformers of cyclic prodrug 1. RESULTS: Our spectroscopic findings suggest that cyclic prodrug 1 exhibits a major and a minor conformer in solution. The major conformer appears to have a well-defined secondary structure, which involves a beta-turn and 4-->1 intramolecular hydrogen bond, creating a compact structure with a reduced average hydrodynamic radius compared to the model hexapeptide 2. CONCLUSIONS: The increased ability of cyclic prodrug 1 to permeate membranes compared to the model hexapeptide 2 could be due to reduction in the average hydrodynamic radius of the molecule facilitating paracellular flux and/or the reduction in the hydrogen bonding potential facilitating transcellular flux.

Tylophora compounds as Na+/K(+)-ATPase inhibitors.

1. Acetyltylophoroside (AcT) and tylogenin inhibit Na+/K(+)-ATPase in spite of having structures very different from cardiac glycosides (CGs). 2. Calculation of the lowest energy conformations of AcT and tylogenin and superpositions of these with the X-ray conformations of CGs and chlormadinone acetate led to a model for the interaction of these different types of Na+/K(+)-ATPase inhibitors with the receptor.