Rational design, engineer, and characterization of a novel pegylated single isomer human arginase for arginine depriving anti-cancer treatment.

AIMS: Arginine depleting enzymes are found effective to treat arginine-auxotrophic cancers and therapy-resistant malignancies, alone or in combination with cytotoxic agents or immune checkpoint inhibitors. We aim to select and validate a long-lasting, safe and effective PEGylated and cobalt-chelated arginase conjugated at the selective cysteine residue as a therapeutic agent against cancers. MAIN METHODS: Exploring pharmacokinetic and pharmacodynamic properties of the three arginase conjugates with different PEG modality (20 kDa linear as A20L, 20 kDa branched as A20Y, and 40 kDa branched as A40Y) by cell-based and animal studies. KEY FINDINGS: Arginase conjugates showed comparable systemic half-lives, about 20 h in rats and mice. The extended half-life of PEGylated arginase was concurrent with the integrity of conjugates of which PEG and protein moieties remain attached in bloodstream for 72 h after drug administration. Arginase modified with a linear 20 kDa PEG (A20L) was chosen as the lead candidate (PT01). In vitro assays confirmed the very potent cytotoxicity of PT01 against cancer cell lines of breast, prostate, and pancreas origin. In MIA PaCa-2 pancreatic and PC-3 prostate tumor xenograft models, weekly infusion of the PT01 at 5 and 10 mg/kg induced significant tumor growth inhibition of 44-67%. All mice experienced dose-dependent but rapidly reversible weight loss following each weekly dose, suggesting tolerable toxicity. SIGNIFICANCE: These non-clinical data support PT01 as the lead candidate for clinical development that may benefit cancer patients by providing an alternative cytotoxic mechanism.

3-Aminobenzenesulfonamides incorporating acylthiourea moieties selectively inhibit the tumor-associated carbonic anhydrase isoform IX over the off-target isoforms I, II and IV.

We describe the synthesis of a series of novel 1-aroyl/acyl-3-(3-aminosulfonylphenyl) thioureas (4a-k) acting as human carbonic anhydrase (hCA, EC 4.2.1.1) inhibitors. Reaction of alkyl/aryl isothiocyanates with 3-aminobenzenesulfonamide afforded a series of the title compounds incorporating a variety of short as well as highly lipophilic long tails. The newly synthesized sulfonamides were evaluated against 4 physiologically relevant CA isoforms (hCA I, II, IV, and IX). Several compounds showed interesting inhibitory activity. The tumor-associated hCA IX was the most sensitive isoform to inhibition with these compounds, with KIs in the range of 21.5-44.0 nM and selectivity ratios over the major cytosolic isoform hCA II in the range of 3.35-37.3. The sulfonamides incorporating the phenylacetylthioureido and pentadecanoylthioureido moieties were the most hCA IX-selective inhibitors detected in this work, making them of interest for further investigations.

New 4-[(3-cyclohexyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides, synthesis and inhibitory activity toward carbonic anhydrase I, II, IX, XII.

A series of 4-[(3-cyclohexyl-4-aryl-2,3-dihydro-1,3-thiazol-2-ylidene)amino]benzene-1-sulfonamides was synthesised and the activity of the new compounds as inhibitors of hCA I, II, IX, and XII was evaluated. These new derivatives exhibited some peculiarities with respect to previously reported sulfonamide based inhibitors of CA. We observed that the nature of the substituents in the position 3 and 4 of the dihydro-thiazole ring was relevant in determining both activity and selectivity profiles.

Fosfatase ácida resistente ao tartarato como biomarcador do metabolismo ósseo no cão / Tartrate-resistant acid phosphatase as a biomarker of bone turnover in dog

Values of serum tartrate-resistant acid phosphatase ( TRAP) activity were obtained in adult dogs and its biological variability was assessed. Nine healthy skeletally mature Portuguese Podengo dogs were used for the determination of TRAP, total and bone alkaline phosphatase serum activities, and also to study their relationship with serum minerals, namely calcium (Ca), phosphorous (P), and magnesium (Mg). The serum TRAP activity was 2.19±0.56IU/mL, with intra-individual variation of 18.3 percent and inter-individual variation of 25.6 percent. Significant correlations were observed between serum TRAP activity and Ca (r=-0.3431; P<0.05), Ca and Mg (r=-0.787; P<0.01), and TRAP and Mg (r=0.397; P<0.05). The results indicate that serum TRAP activity in dog could be of great value in research and in clinical practice, providing complementary non-invasive information on bone metabolism.

Determinaram-se os valores da atividade da fosfatase ácida resistente ao tartarato (FART) e avaliou-se a sua variabilidade biológica. Neste estudo, foram utilizados nove cães adultos e saudáveis de raça Podengo Português para as determinações das atividades da FART, da fosfatase alcalina total, da isoenzima óssea da fosfatase alcalina e da concentração dos minerais séricos - cálcio, fósforo e magnésio. A atividade sérica obtida da FART foi de 2,19±0,56 UI/mL, com uma variação intra-individual de 18,3 por cento e interindividual de 25,6 por cento. Foram observadas correlações significativas ao longo do tempo entre FART e cálcio (r=-0,3431; P<0,05), entre FART e magnésio (r=0,3974; P<0,05) e entre cálcio e magnésio (r=-0,787; P<0,01). Os resultados indicam que este marcador de reabsorção óssea pode ser de grande valor na prática clínica e na investigação e, ainda, ser utilizado como um método auxiliar não invasivo para avaliação do metabolismo ósseo.

Ca2+/calcineurin regulation of cloned vascular K ATP channels: crosstalk with the protein kinase A pathway.

BACKGROUND AND PURPOSE: Vascular ATP-sensitive potassium (K(ATP)) channels are activated by cyclic AMP elevating vasodilators through protein kinase A (PKA). Direct channel phosphorylation is a critical mechanism, though the phosphatase opposing these effects is unknown. Previously, we reported that calcineurin, a Ca(2+)-dependent phosphatase, inhibits K(ATP) channels, though neither the site nor the calcineurin isoform involved is established. Given that the type-2 regulatory (RII) subunit of PKA is a substrate for calcineurin we considered whether calcineurin regulates channel activity through interacting with PKA. EXPERIMENTAL APPROACH: Whole-cell recordings were made in HEK-293 cells stably expressing the vascular K(ATP) channel (K(IR)6.1/SUR2B). The effect of intracellular Ca(2+) and modulators of the calcineurin and PKA pathway on glibenclamide-sensitive currents were examined. KEY RESULTS: Constitutively active calcineurin A alpha but not A beta significantly attenuated K(ATP) currents activated by low intracellular Ca(2+), whereas calcineurin inhibitors had the opposite effect. PKA inhibitors reduced basal K(ATP) currents and responses to calcineurin inhibitors, consistent with the notion that some calcineurin action involves inhibition of PKA. However, raising intracellular Ca(2+) (equivalent to increasing calcineurin activity), almost completely inhibited K(ATP) channel activation induced by the catalytic subunit of PKA, whose enzymatic activity is independent of the RII subunit. In vitro phosphorylation experiments showed calcineurin could directly dephosphorylate a site in Kir6.1 that was previously phosphorylated by PKA. CONCLUSIONS AND IMPLICATIONS: Calcineurin A alpha regulates K(IR)6.1/SUR2B by inhibiting PKA-dependent phosphorylation of the channel as well as PKA itself. Such a mechanism is likely to directly oppose the action of vasodilators on the K(ATP) channel.

Carbonic anhydrase inhibitors. DNA cloning, characterization, and inhibition studies of the human secretory isoform VI, a new target for sulfonamide and sulfamate inhibitors.

The secretory isozyme of human carbonic anhydrase (hCA, EC 4.2.1.1), hCA VI, has been cloned, expressed, and purified in a bacterial expression system. The kinetic parameters for the CO2 hydration reaction proved hCA VI to possess a kcat of 3.4 x 10(5) s-1 and kcat/KM of 4.9 x 10(7) M-1 s-1 (at pH 7.5 and 20 degrees C). hCA VI has a significant catalytic activity for the physiological reaction on the same order of magnitude as the ubiquitous isoform CA I or the transmembrane, tumor-associated isozyme CA IX. A series of sulfonamides and one sulfamate have been tested for their interaction with this isozyme. Simple benzenesulfonamides were rather ineffective hCA VI inhibitors, with inhibition constants in the range of 1090-6680 nM. Better inhibitors were detected among such derivatives bearing 2- or 4-amino-, 4-aminomethyl-, or 4-hydroxymethyl moieties or among halogenated sulfanilamides (KI values of 608-955 nM). Some clinically used compounds, such as acetazolamide, methazolamide, ethoxzolamide, dichlorophenamide, dorzolamide, brinzolamide, topiramate, sulpiride, and indisulam, or the orphan drug benzolamide, showed effective hCA VI inhibitory activity, with inhibition constants of 0.8-79 nM. The best inhibitors were brinzolamide and sulpiride (KI values of 0.8-0.9 nM), the latter compound being also a CA VI-selective inhibitor. The metallic taste reported as a side effect after the treatment with systemic sulfonamides may be due to the inhibition of the salivary CA VI. Some of the compounds investigated in this study might be used as additives in toothpastes for reducing the acidification produced by the relevant CO2 hydrase activity of enamel CA VI, which leads to the formation of protons and bicarbonate and may have a role in cariogenesis.

Peptides derived from the C2 domain of protein kinase C epsilon (epsilon PKC) modulate epsilon PKC activity and identify potential protein-protein interaction surfaces.

Peptides derived from protein kinase C (PKC) modulate its activity by interfering with critical protein-protein interactions within PKC and between PKC and PKC-binding proteins (Souroujon, M. C., and Mochly-Rosen, D. (1998) Nat. Biotechnol. 16, 919-924). We previously demonstrated that the C2 domain of PKC plays a critical role in these interactions. By focusing on epsilonPKC and using a rational approach, we then identified one C2-derived peptide that acts as an isozyme-selective activator and another that acts as a selective inhibitor of epsilonPKC. These peptides were used to identify the role of epsilonPKC in protection from cardiac and brain ischemic damage, in prevention of complications from diabetes, in reducing pain, and in protecting transplanted hearts. The efficacy of these two peptides led us to search for additional C2-derived peptides with PKC-modulating activities. Here we report on the activity of a series of 5-9-residue peptides that are derived from regions that span the length of the C2 domain of epsilonPKC. These peptides were tested for their effect on PKC activity in cells in vivo and in an ex vivo model of acute ischemic heart disease. Most of the peptides acted as activators of PKC, and a few peptides acted as inhibitors. PKC-dependent myristoylated alanine-rich C kinase substrate phosphorylation in epsilonPKC knock-out cells revealed that only a subset of the peptides were selective for epsilonPKC over other PKC isozymes. These epsilonPKC-selective peptides were also protective of the myocardium from ischemic injury, an epsilonPKC-dependent function (Liu, G. S., Cohen, M. V., Mochly-Rosen, D., and Downey, J. M. (1999) J. Mol. Cell. Cardiol. 31, 1937-1948), and caused selective translocation of epsilonPKC over other isozymes when injected systemically into mice. Examination of the structure of the C2 domain from epsilonPKC revealed that peptides with similar activities clustered into discrete regions within the domain. We propose that these regions represent surfaces of protein-protein interactions within epsilonPKC and/or between epsilonPKC and other partner proteins; some of these interactions are unique to epsilonPKC, and others are common to other PKC isozymes.

A nuclear localization sequence endows human pancreatic ribonuclease with cytotoxic activity.

Some members of the ribonuclease superfamily, such as Onconase, are cytotoxic to cancer cells. This is not the case for human pancreatic ribonuclease. This lack of cytotoxicity is probably a result of the inhibition exerted by the cytosolic ribonuclease inhibitor once the protein has reached the cytosol. Until now, all cytotoxic human pancreatic ribonuclease variants have been described as being resistant to the inhibitor. Here, we report on the characterization of a cytotoxic variant of human pancreatic ribonuclease which has an Arg triplet introduced onto one of its surface-exposed loops. Despite its sensitivity to the inhibitor, this variant, called PE5, was only 5-15 times less cytotoxic than Onconase. When it was taken up by cells, it was only observed within late compartments of the endocytic pathway, probably because the number of molecules transported to the cytosol was too small to allow their visualization. Nuclear import assays showed that the Arg triplet endows PE5 with a nuclear localization signal. In these experiments, PE5 was efficiently transported to the nucleus where it was initially localized in the nucleolus. Although the Arg introduction modified the net charge of the protein and somehow impaired recognition by the cytosolic inhibitor, control variants, which had the same number of charges or were not recognized by the inhibitor, were not toxic. We concluded that targeting a ribonuclease to the nucleus results in cytotoxicity. This effect is probably due to ribonuclease interference with rRNA processing and ribosome assembly within the nucleolus.

Macrocyclic bisindolylmaleimides as inhibitors of protein kinase C and glycogen synthase kinase-3.

Efficient methods were developed to synthesize a novel series of macrocyclic bisindolylmaleimides containing linkers with multiple heteroatoms. Potent inhibitors (single digit nanomolar IC(50)) for PKC-beta and GSK-3beta were identified, and compounds showed good selectivity over PKC-alpha, -gamma, -delta, -epsilon, and -zeta. Representative compound 5a also had high selectivity in a screening panel of 10 other protein kinases. In cell-based functional assays, several compounds effectively blocked interleukin-8 release induced by PKC-betaII and increased glycogen synthase activity by inhibiting GSK-3beta.

Establishment of a binding assay for protein kinase C isozymes using synthetic C1 peptides and development of new medicinal leads with protein kinase C isozyme and C1 domain selectivity.

Conventional and novel protein kinase C (PKC) isozymes contain two cysteine-rich C1 domains (C1A and C1B), both of which are candidate phorbol-12, 13-dibutyrate (PDBu)-binding sites. We synthesized C1 peptides of 50-70 residues corresponding to all PKC isozyme C1 domains using an Fmoc solid-phase strategy. These C1 peptides were successfully folded by zinc treatment, as monitored by electrospray ionization time-of-flight mass spectrometry. We measured the K(d)'s of [3H]PDBu for all PKC C1 peptides. Most of the C1 peptides, except for delta-C1A and theta-C1A, showed strong PDBu binding affinities with K(d)'s in the nanomolar range (0.45-7.4 nM) comparable with the respective whole PKC isozymes. The resultant C1 peptide library can be used to screen for new ligands with PKC isozyme and C1 domain selectivity. Non-tumor-promoting 1-oleoyl-2-acetyl-sn-glycerol and bryostatin 1 showed relatively strong binding to all CIA peptides of novel PKCs (delta, epsilon, and eta). In contrast, the tumor promoters (-)-indolactam-V, ingenol-3-benzoate, and PDBu bound selectively to all C1B peptides of novel PKCs. The preference of tumor promoters for the domain might be related to tumorigenesis since recent investigations proposed the involvement of novel PKCs in tumor promotion in vivo using transgenic or knockout mice. Moreover, we recently have found that a new lactone analogue of benzolactams (6) shows significant selectivity in PKCeta-C1B binding.

Differential detection of rat islet and brain glutamic acid decarboxylase (GAD) isoforms with sequence-specific peptide antibodies.

We studied the distribution of the M(r) 65,000 and M(r) 67,000 isoforms of glutamic acid decarboxylase, GAD65 and GAD67, in rat islets and brain by immunocytochemistry. Synthetic peptides representing selected GAD65 or GAD67 sequences were used to produce sequence-specific antibodies, allowing differential immunocytochemical detection of the two isoforms. GAD-specific reactivity of each peptide antiserum was confirmed by ELISA, immunoblotting, and immunoprecipitation. Immunostaining specificity was verified by displacement with either immunizing or irrelevant peptide. Dual immunostaining with GAD isoform-specific antibodies and polyclonal antibodies to glucagon showed that GAD65 was primarily detected in rat pancreatic islet beta-cells, whereas alpha-cells had weak GAD65 staining. In contrast, GAD67 was detected primarily in alpha-cells. In rat brain, GAD65 and GAD67 were present in neuron cell bodies and processes. These data demonstrate that antibodies raised against the N-terminus of GAD allow differential immunocytochemical identification of GAD67 and GAD65. Differential expression of GAD isoforms within islet alpha- and beta-cells supports the role of GAD65 in autoimmune diabetes and stiff-man syndrome.

Immunolocalization of PKC zeta in rat photoreceptor inner segments.

We have utilized several peptide specific antisera directed against the C-terminals (Wetsel et al, 1992) of several protein kinase C (PKC) isozymes (alpha, beta 1, beta 11, gamma, delta, epsilon, zeta) to delineate the cellular localization of these PKC isozymes in rat retina. Antisera against PKC beta 1, beta 11, gamma, delta and epsilon were non-reactive in frozen rat retina sections, whereas, anti PKC alpha was strongly reactive with the outer plexiform, inner plexiform and nerve fiber cell layers. The most specific localization of immunoreactivity was observed with PKC zeta, which reacted strongly and exclusively with photoreceptor inner segments, but not outer segments. Immunoblot analysis of whole rat retina homogenate showed that anti-PKC alpha recognized an antigen of approximately 80kD and anti-PKC zeta recognized a approximately 72kD protein. Immunolocalization of PKC zeta to photoreceptor inner segments and possible functional significance are discussed.