Regulation of prolyl endopeptidase activity by the intracellular redox state

The activity of prolyl endopeptidase was markedly decreased during incubation of intact murine erythroleukemia cells at 45 degrees C, but not during incubation of sonicated cells or during incubation at 42 degrees C. The thermal inactivation of prolyl endopeptidase in situ required neither the synthesis of proteins and polynucleotides nor the synergistic activation of inhibitors. Moreover, inhibition of lysosomal proteinases and calpains or depletion of ATP did not affect the thermal inactivation of prolyl endopeptidase. This specific inactivation of prolyl endopeptidase was also observed following the addition to the culture medium of menadione or diamide, compounds known to increase intracellular oxidized glutathione levels. The activity of prolyl endopeptidase in the cell lysate was also dose-dependently decreased by the addition of glutathione disulfide and the decrease of the activity was prevented by coexistence of reduced glutathione. Furthermore, the level of intracellular oxidized glutathione was increased during incubation at 45 degrees C for 15 min, but not at 42 degrees C for 30 min. These results strongly suggest that the activity of prolyl endopeptidase is regulated by changes in the intracellular redox potential.     

Acid-activatable prodrug nanogels for efficient intracellular doxorubicin release

Endosomal pH-activatable doxorubicin (DOX) prodrug nanogels were designed, prepared, and investigated for triggered intracellular drug release in cancer cells. DOX prodrugs with drug grafting contents of 3.9, 5.7, and 11.7 wt % (denoted as prodrugs 1, 2, and 3, respectively) were conveniently obtained by sequential treatment of poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-co-ethyl glycinate methacrylamide) (PEG-b-P(HEMA-co-EGMA)) copolymers with hydrazine and doxorubicin hydrochloride. Notably, prodrugs 1, 2, and 3 formed monodispersed nanogels with average sizes of 114.4, 75.3, and 66.3 nm, respectively, in phosphate buffer (PB, 10 mM, pH 7.4). The in vitro release results showed that DOX was released rapidly and nearly quantitatively from DOX prodrug nanogels at endosomal pH and 37 °C in 48 h, whereas only a minor amount (ca. 20% or less) of drug was released at pH 7.4 under otherwise the same conditions. Confocal laser scanning microscope (CLSM) observations revealed that DOX prodrug nanogels delivered and released DOX into the cytosols as well as cell nuclei of RAW 264.7 cells following 24 h incubation. MTT assays demonstrated that prodrug 3 had pronounced cytotoxic effects to tumor cells following 72 h incubation with IC(50) data determined to be 2.0 and 3.4 μg DOX equiv/mL for RAW 264.7 and MCF-7 tumor cells, respectively. The corresponding polymer carrier, PEG-b-P(HEMA-co-GMA-hydrazide), was shown to be nontoxic up to a tested concentration of 1.32 mg/mL. These endosomal pH-activatable DOX prodrug nanogels uniquely combining features of water-soluble macromolecular prodrugs and nanogels offer a promising platform for targeted cancer therapy.     

Study on glycosylated prodrugs of toxoflavins for antibody-directed enzyme tumor therapy

Eight novel toxoflavin glycosides, which are potential prodrugs in antibody directed enzyme prodrug therapy (ADEPT), were synthesized. The structures of all toxoflavin glycosides were characterized by (13)C NMR spectroscopy, elemental analysis, and MS. Their enzymatic hydrolysis activities were tested against beta-glucosidase (EC.3.2.1.21).     

Porcine muscle prolyl endopeptidase: limited proteolysis of tryptic peptides from hemoglobin beta-chains at prolyl and alanyl bonds

Tryptic peptides from hemoglobin (Hb) beta-chains were used as model substrates for limited proteolysis by prolyl endopeptidase (EC 3.4.21.26) from porcine muscle. From the physicochemical and enzymatic properties of prolyl endopeptidase the conditions for routine digestion were established as follows: the molar ratio of enzyme to substrate was 1 to 100, and the reaction was carried out in sodium phosphate buffer (pH 6.4) at 37 degrees C for 4 h. Under these conditions the peptide bonds on the carboxyl terminal sides of proline and alanine residues in the tryptic peptides from Hb beta-chains (with Mr values of less than 2100) were hydrolyzed by the enzyme with the exception of the amino terminal alanyl bond and aminoacyl alanyl bond. In addition, one of five seryl bonds was cleaved by the enzyme. However, the Hb beta-chain itself, Mr 16,600, and its two CNBr-peptides with Mr 10,200 and Mr 6400, respectively, were not hydrolyzed. Under the same conditions a prolyl bond in oxidized B-chains of insulin, Mr 3400, was partially digested, and an alanyl bond was not hydrolyzed. The data indicate that the prolyl endopeptidase is useful for the limited proteolysis of peptides with relative masses of less than 3000 at both prolyl and alanyl bonds.     

Synthesis and biological evaluation of novel pyrrolo[2,1-c][1,4]benzodiazepine prodrugs for use in antibody-directed enzyme prodrug therapy

The design, synthesis and evaluation of four novel pyrrolo[2,1-c][1,4]benzodiazepine (PBD) prodrugs (1a,b and 2a,b; ) for potential use in carboxypeptidase G2 (CPG2)-based antibody-directed enzyme prodrug therapy (ADEPT) is reported. Although all four prodrugs were shown to be less cytotoxic than the released parent PBDs 3 and 4, the urea prodrugs 1b and 2b were found to be too unstable for use in ADEPT, whereas carbamates 1a and 2a are both stable in an aqueous environment and are good substrates for CPG2.     

A strategy for mapping and neutralizing conformational immunogenic sites on protein therapeutics

Antibodies are highly specific recognition molecules which are increasingly being applied to target therapy in patients. One type of developmental antibody-based therapy is antibody directed enzyme prodrug therapy (ADEPT) for the treatment of cancer. In ADEPT, an antibody specific to a tumor marker protein delivers a drug-activating enzyme to the cancer. Subsequent intravenous administration of an inactive prodrug results in drug activation and cytotoxicity only within the locale of the tumor. Pilot clinical trials with chemical conjugates of the prodrug activating enzyme carboxypeptidase G2 (CPG2) chemically conjugated with an antibody to and carcinoembryonic antigen (CEA), have shown that CPG2-mediated ADEPT is effective but limited by formation of human antibodies to CPG2 (HACA). We have developed a recombinant fusion protein (termed MFE-CP) of CPG2 with an anti-CEA single chain Fv antibody fragment and we have developed methods to address the immunogenicity of this therapeutic. A HACA-reactive discontinuous epitope on MFE-CP was identified using the crystal structure of CPG2, filamentous phage technology and surface enhanced laser desorption/ionization affinity mass spectrometry. This information was used to create a functional mutant of MFE-CP with a significant reduction (range 19.2 to 62.5%, median 38.5%) in reactivity with the sera of 11 patients with post-therapy HACA. The techniques described here are valuable tools for identifying and adapting undesirable immunogenic sites on protein therapeutics.     

Expression of active human beta-glucuronidase in Sf9 cells infected with recombinant baculovirus

Antibody directed enzyme prodrug therapy (ADEPT) using glucuronide prodrugs is an experimental approach to reduce systemic toxicity of anti-cancer agents. Bioactivation of such prodrugs is achieved by fusion proteins consisting of targeting moieties (e.g. ligands of tumor specific antigens) and human beta-glucuronidase. In order to test a large panel of possible beta-glucuronidase fusion proteins for their applicability in ADEPT, an easy, rapid and high-yield expression system like the baculovirus/insect cell expression system would be needed. A prerequisite for using such fusion proteins is functional and biochemical characterization of human beta-glucuronidase expressed in baculovirus-infected insect cells. Therefore, recombinant human beta-glucuronidase was expressed in Sf9 insect cells and characterized at the protein and functional level. As shown by Western blot analysis the recombinant enzyme consists of dimers with their monomers being linked via disulfide bonds. Posttranslational modifications of the monomers seem to be different as compared with mammalian cells or tissues. The enzyme is functionally active in cleaving the substrates 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, 4-methylumbelliferyl-beta-D-glucuronide and the glucuronide prodrug HMR 1826, respectively, with similar enzyme kinetic parameters as those found in human tissues. Our data demonstrate that beta-glucuronidase expressed in Sf9 cells displays the same enzymatic features as the protein expressed in mammalian cells. Therefore, we suggest that beta-glucuronidase fusion proteins produced in this cell line will be valuable tools for testing a large panel of various targeting moieties in human tumor xenograft models or may be used for ADEPT in man.     

Albumin-binding prodrugs of camptothecin and doxorubicin with an Ala-Leu-Ala-Leu-linker that are cleaved by cathepsin B: synthesis and antitumor efficacy

We have recently validated a macromolecular prodrug strategy for improved cancer chemotherapy based on two features: (a) rapid and selective binding of thiol-reactive prodrugs to the cysteine-34 position of endogenous albumin and (b) acid-sensitive promoted or enzymatic release of the drug at the tumor site [Kratz, F., Warnecke, A., Scheuemann, K., Stockmar, C., Schwab, J., Lazar, P., Druckes, P., Esser, N., Drevs, J., Rognan, D., Bissantz, C., Hinderling, C., Folkers, G., Fichtner, I., and Unger, C. (2002) J. Med. Chem. 45, 5523-33]. In the present work, we developed water-soluble camptothecin (CPT) and doxorubicin (DOXO) prodrugs that incorporate the peptide linker Ala-Leu-Ala-Leu that serves as a substrate for the tumor-associated protease, cathepsin B, which is overexpressed in several solid tumors. Consequently, two albumin-binding prodrugs were synthesized [EMC-Arg-Arg-Ala-Leu-Ala-Leu-Ala-CPT (1) and EMC-Arg-Arg-Ala-Leu-Ala-Leu-DOXO (2) (EMC = 6-maleimidocaproic acid)]. Both prodrugs exhibited excellent water-solubility and bound rapidly and selectively to the cysteine-34 position of endogenous albumin. Further in vitro studies showed that the albumin-bound form of the prodrugs was cleaved specifically by cathepsin B as well as in human tumor homogenates. Major cleavage products were CPT-peptide derivatives and CPT for the CPT prodrug and H-Leu-Ala-Leu-DOXO, H-Leu-DOXO, and DOXO for the doxorubicin prodrug. In vivo, 1 was superior to free camptothecin in an HT-29 human colon xenograft model; the antitumor efficacy of prodrug 2 was comparable to that of free doxorubicin in the M-3366 mamma carcinoma xenograft model at equimolar doses.     

Design and synthesis of novel pyrrolobenzodiazepine (PBD) prodrugs for ADEPT and GDEPT

Three N10-(4-nitrobenzyl)carbamate-protected PBD prodrugs (9a, 9b and 15) have been synthesized and evaluated for potential use in nitroreductase-based ADEPT and GDEPT therapies. An approximately 100-fold activation was observed for the DC-81 prodrug 9a.     

Analysis of antibody-enzyme conjugate clearance by investigation of prodrug and active drug in an ADEPT clinical study

Antibody-directed enzyme prodrug therapy (ADEPT) separates the cytotoxic function from the targeting function (5). An antibody-carboxypeptidase G2 (CPG2) enzyme is delivered prior to the nontoxic prodrug, CMDA, which is converted to a cytotoxic drug by the action of the localized conjugate at the tumor site. An indirect in vitro assay was developed to detect the presence of functional CPG2 in the plasma of patients in an ADEPT clinical trial. Compounds in the plasma of patients were characterized using liquid chromatography-mass spectrometry. Plasma at three different time points (prior to treatment, post-antibody-enzyme conjugate, and post-galactosylated anti-enzyme antibody clearing agent) was added to the CMDA prodrug and analyzed. Conversion of the CMDA prodrug to its active drug indicates that CPG2-conjugate remains in the plasma. This technique will provide essential data for the timing of prodrug administration in ADEPT.     

Systems biology and the mathematical modelling of antibody-directed enzyme prodrug therapy (ADEPT)

Antibody-directed enzyme prodrug therapy (ADEPT) can generate highly localised concentrations of cytotoxic agents directly in a tumour, thereby reducing the collateral toxicity associated with normal tissue exposure. ADEPT is a two-component approach. First, a non-toxic antibody-enzyme fusion protein is localised in the tumour matrix by binding a specific antigen expressed only on the surface of a cancer cell. Once the fusion protein is bound, an inert small molecule prodrug is administered which is the substrate for the enzyme bound to the tumour surface. When the prodrug comes into contact with the bound enzyme, an active cytotoxic agent is generated. A multiple length-scale model of ADEPT therapy in solid tumours is presented. A four-compartment pharmacokinetic (PK) model is formulated where the tumour is comprised of interstitial and cell-surface subcompartments. The macroscopic PK model which describes the biodistribution of antibody-enzyme conjugate, prodrug and active drug at the largest length scale is coupled to a reaction-diffusion tumour model. The models are qualitatively validated against current literature and experimental understanding. The relationship between tumour localisation and the affinity of the antibody-enzyme conjugate for its surface antigen is explored by simulation. The influence of pharmacokinetic and biophysical parameters such as renal elimination rate and permeability of the tumour vasculature upon tumour uptake and retention of the fusion protein are also explored. Lastly, a technique for establishing an optimal prodrug dosing schedule is formulated and initial simulation results are presented.     

Selection of catalytically active biotin ligase and trypsin mutants by phage display.

Phage display has been shown to facilitate greatly the selection of polypeptides with desired properties by establishing a direct link between the polypeptide and the gene that encodes it. However, selection for catalytic activities displayed on phage remains a challenge, since reaction products diffuse away from the enzyme and make it difficult to recover catalytically active phage-enzymes. We have recently described a selection methodology in which the reaction substrate (and eventually the reaction product) is anchored on calmodulin-tagged phage-enzymes by means of a calmodulin binding peptide. Phage displaying a catalytic activity are physically isolated by means of affinity reagents specific for the product of reaction. In this study, we investigated the efficiency of selection for catalysis by phage display, using a ligase (the Escherichia coli biotin ligase BirA) and an endopeptidase (the rat trypsin His57--> Ala mutant) as model enzymes. These enzymes could be displayed on phage as fusion proteins with calmodulin and the minor coat protein pIII. Both the display of functional enzyme and the efficiency of selection for catalysis were significantly improved by using phage vectors, rather than phagemid vectors. In model selection experiments, phage displaying BirA were consistently enriched (between 4-fold and 800-fold) per round of panning, relative to negative controls. Phage displaying the trypsin His57-->Ala mutant, a relatively inefficient endopeptidase which cleaves a specific dipeptide sequence, were enriched (between 15-fold and 2000-fold), relative to negative controls. In order to improve the catalytic properties of the trypsin His57-->Ala mutant, we constructed a combinatorial phage display library of trypsin mutants. Selection of catalytically active phage-enzymes was evidentiated by increasing phage titres at the different rounds of panning relative to negative control selections, but mutants with catalytic properties superior to those of trypsin His57-->Ala mutant could not be isolated. The results obtained provide evidence that catalytic activities can be recovered using phage display technology, but stress the importance of both library design and stringent biopanning conditions for the recovery of novel enzymes.     

Directed evolution to improve the thermostability of prolyl endopeptidase

Prolyl endopeptidase is the only endopeptidase that specifically cleaves peptides at proline residues. Although this unique specificity is advantageous for application in protein chemistry, the stability of the enzyme is lower than those of commonly used peptidases such as subtilisin and trypsin. Therefore, we attempted to apply a directed evolution system to improve the thermostability of the enzyme. First, an efficient expression system for the enzyme in Escherichia coli was established using the prolyl endopeptidase gene from Flavobacterium meningosepticum. Then, a method for screening thermostable variants was developed by combining heat treatment with active staining on membrane filters. Random mutagenesis by error-prone PCR and screening was repeated three times, and as a result the thermostability of the enzyme was increased step by step as the amino acid substitutions accumulated. The most thermostable mutant obtained after the third cycle, PEP-407, showed a half-life of 42 min at 60 degrees C, which was 60 times longer than that of the wild-type enzyme. The thermostable mutant was also more stable with a high concentration of glycerol, which is a necessary condition for in vitro amidation.     

Purification and characterization of prolyl endopeptidase from the Pacific herring, Clupea pallasi, and its role in the activation of sperm motility

Protease activities with specificity toward synthetic substrates, Suc-Gly-Pro-Leu-Gly-Pro-MCA for prolyl endopeptidase or collagenase-like peptidase, and Suc-Ala-Ala-Pro-Phe-MCA for chymotrypsin were identified in the detergent-soluble fraction of herring spermatozoa. The enzyme activities increased in the presence of herring sperm-activating protein (HSAP). Among them a prolyl endopeptidase [EC. 3. 4. 21. 26] was purified to near homogeneity from herring testis. The molecular mass of the enzyme was 79 kDa and the properties of the enzyme were quite similar to prolyl endopeptidase from other tissues or cells. Both the enzyme activation and the sperm motility activation by HSAP were inhibited by benzyloxycarbonyl-L-thioproline-thioprolinal, a specific inhibitor for prolyl endopeptidase. Furthermore, the motility activation by HSAP was inhibited by substrates of the prolyl endopeptidase. Western blotting with mouse anti-prolyl endopeptidase serum revealed the presence of 79 kDa prolyl endopeptidase in the tail fraction of herring sperm. These results suggest that prolyl endopeptidase exists on the surface of the sperm tail and interacts with the HSAP.     

A strategy for tumor-selective chemotherapy by enzymatic liberation of seco-duocarmycin SA-derivatives from nontoxic prodrugs.

Immuno-conjugates obtained by linking enzymes with appropriate monoclonal antibodies, which bind to tumor-associated antigens, can be employed in a tumor-selective antibody directed enzyme prodrug therapy (ADEPT). For this strategy the glycosides 17a--c were prepared as prodrugs of CI-TMI 14 which is a structurally simplified analogue of the highly potent antitumor agent duocarmycin SA 2. Exposure of 17a--c to cultured carcinoma cells of line A549 displayed a very low toxicity; however, after addition of the corresponding enzymes and exposure for 24 h at prodrug concentrations of <0.1 microM the proliferation of the carcinoma cells was inhibited almost completely with ED(50prodrug)/ED(50drug) of up to 270 in the presence and in the absence of the enzyme. The synthesis of 17a--c was achieved by transformation of nitroanisidine 6 into 12 which was glycosidated to give 16a--c. Removal of the silyl groups, introduction of a chlorine atom and solvolysis of the acetal groups led to 17a-c, of which 17a and 17c are promising candidates for further elaboration.     

Targeted inhibition of hedgehog signaling by cyclopamine prodrugs for advanced prostate cancer

A promising agent for use in prostate cancer therapy is the Hedgehog (Hh) signaling pathway inhibitor, cyclopamine. This compound, however, has the potential for causing serious side effects in non-tumor tissues. To minimize these bystander toxicities, we have designed and synthesized two novel peptide-cyclopamine conjugates as prostate-specific antigen (PSA)-activated prodrugs for use against prostate cancer. These prodrugs were composed of cyclopamine coupled to one of two peptides (either HSSKLQ or SSKYQ) that can be selectively cleaved by PSA, converting the mature prodrug into an active Hedgehog inhibitor within the malignant cells. Of the two prodrugs, Mu-SSKYQ-Cyclopamine was rapidly hydrolyzed, with a half-life of 3.2h, upon incubation with the PSA enzyme. Thus, modulating cyclopamine at the secondary amine with PSA-cleavable peptides is a promising strategy for developing prodrugs to target prostate cancer.     

Bioactivation of carbamate-based 20(S)-camptothecin prodrugs

Two new prodrugs of CPT were synthesized, based on carbamate linkages between the 20-hydroxy group of CPT and a linker designed to be enzymatically removed by either Penicillin-G-Amidase or catalytic antibody 38C2. Cell growth inhibition assays showed an up-to-2250-fold difference in toxicity between the prodrugs and the active drug. A significant increase in toxicity was observed upon incubation of the enzyme or the catalytic antibody with the corresponding prodrug. The described derivatives of CPT further our knowledge in the design of prodrugs for use in selective approaches for targeted chemotherapy.     

Nitrobenzocyclophosphamides as potential prodrugs for bioreductive activation: synthesis,stability, enzymatic reduction,and antiproliferative activity in cell culture

In efforts to obtain potential anticancer prodrugs for gene-directed enzyme prodrug therapy using Eschericia coli nitroreductase, a series of four benzocyclophosphamide analogues were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All four analogues were found to be stable in phosphate buffer at pH 7.4 and 37 degrees C and were good substrates of E. coli nitroreductase with half lives between 7 and 24 min at pH 7.0 and 37 degrees C. However, only two analogues 6a and 6c, both with a benzylic oxygen in the phosphorinane ring para to the nitro group, showed a modest 33-36-fold enhanced cytotoxicity in E. coli nitroreductase-expressing cells. These results suggest that good substrate activity and the para benzylic oxygen are required for activation by E. coli nitroreductase. Compounds 6a and 6c represent a new structure type for reductive activation and a lead for further modification in the development of better analogues with improved selective toxicity to be used in gene-directed enzyme prodrug therapy.     

Synthesis and biological evaluation of the suberoylanilide hydroxamic acid (SAHA) beta-glucuronide and beta-galactoside for application in selective prodrug chemotherapy

The beta-O-glucuronide and beta-O-galactoside of SAHA have been prepared and evaluated as prodrugs for selective cancer chemotherapy (ADEPT, PMT). These new compounds are stable under physiological conditions and do not exhibit any antiproliferative activity compared to the parent drug after a 48-h treatment of H661 cells. The glucuronide derivative did not lead to the release of the drug in the presence of either Escherichia coli or bovine liver beta-glucuronidase. On the other hand, under enzymatic cleavage of galactoside prodrug by the corresponding enzyme, a rapid release of SAHA was observed demonstrating that the beta-O-galactoside of SAHA is a promising candidate for in vivo investigations.     

Oxidation of acetylacetone catalyzed by horseradish peroxidase in the absence of hydrogen peroxide

Horseradish peroxidase (HRP) is a plant enzyme widely used in biotechnology, including antibody-directed enzyme prodrug therapy (ADEPT). Here, we showed that HRP is able to catalyze the autoxidation of acetylacetone in the absence of hydrogen peroxide. This autoxidation led to generation of methylglyoxal and reactive oxygen species. The production of superoxide anion was evidenced by the effect of superoxide dismutase and by the generation of oxyperoxidase during the enzyme turnover. The HRP has a high specificity for acetylacetone, since the similar beta-dicarbonyls dimedon and acetoacetate were not oxidized. As this enzyme prodrug combination was highly cytotoxic for neutrophils and only requires the presence of a non-human peroxidase and acetylacetone, it might immediately be applied to research on the ADEPT techniques. The acetylacetone could be a starting point for the design of new drugs applied in HRP-related ADEPT techniques.