Roger F. Sherwood

The bioactivation of 5-(aziridin-1-yl)2,4-dinitrobenzamide (CB1954). II: A comparison of an Escherichia coli nitroreductase and walker DT diaphorase

A nitroreductase enzyme that has been isolated from Escherichia coli B is capable of bioactivating CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] to a cytotoxic agent, a property shared with the mammalian enzyme Walker DT diaphorase [NAD(P)H dehydrogenase (quinone), EC 1.6.99.2] as isolated from Walker cells. In contrast to Walker DT diaphorase, which can only reduce the 4-nitro group of CB1954, the E. coli nitroreductase can reduce either (but not both) nitro groups of CB1954 to the corresponding hydroxylamino species. The two hydroxylamino species are formed in equal proportions and at the same rates. CB1954 is reduced much more rapidly by the E. coli nitroreductase than by Walker DT diaphorase. If the reduction of CB1954 was carried out in the presence of V79 cells (which are insensitive to CB1954) a large cytotoxic effect was evident. This cytotoxicity was only observed under conditions in which the E. coli nitroreductase or Walker DT diaphorase reduced the drug. It is proposed that E. coli B nitroreductase would be a suitable enzyme for antibody-directed enzyme prodrug therapy (ADEPT) in combination with CB1954.

Ablation of Human Choriocarcinoma Xenografts in Nude Mice by Antibody-directed Enzyme Prodrug Therapy (ADEPT) with Three Novel Compounds

Three novel prodrugs have been designed for use as anticancer agents. Each is a bifunctional alkylating agent which has been protected to form a relatively inactive prodrug. They are designed to be activated to their corresponding alkylating agents at a tumour site by prior administration of an antitumour antibody conjugated to the bacterial enzyme carboxypeptidase G2 (CPG2) in a two-phase system called antibody-directed enzyme prodrug therapy (ADEPT). The Km and Vmax values for three different antibody-CPG2 conjugates were determined in relation to each prodrug. The Km values ranged from 4.5-12 mumol/l and the Vmax from 0.5-1.6 mumol/U/min. Athymic Nu/Nu mice with palpable transplanted human choriocarcinoma xenografts, which are resistant to conventional chemotherapy, were treated with anti-human chorionic gonadotropin antibodies conjugated to CPG2. This was followed by each of the three novel prodrugs. Significant increase in survival was obtained in three of the regimens tested using only one course of treatment. This demonstrates the potential of a tumour-localised bacterial enzyme to activate protected alkylating agents in order to eradicate an established human xenograft.

Bioactivation of dinitrobenzamide mustards by an E. coli B nitroreductase

A nitroreductase isolated and purified from Escherichia coli B has been demonstrated to have potential applications in ADEPT (antibody-directed enzyme prodrug therapy) by its ability in vitro to reduce dinitrobenzamides (e.g. 5-aziridinyl 2,4-dinitrobenzamide, CB 1954 and its bischloroethylamino analogue, SN 23862) to form cytotoxic derivatives. In contrast to CB 1954, in which either nitro group is reducible to the corresponding hydroxylamine, SN 23862 is reduced by the nitroreductase to form only the 2-hydroxylamine. This hydroxylamine can react with S-acetylthiocholine to form a species capable of producing interstrand crosslinks in naked DNA. In terms of ADEPT, SN 23862 has a potential advantage over CB 1954 in that it is not reduced by mammalian DT diaphorases. Therefore, a series of compounds related to SN 23862 has been synthesized, and evaluated as potential prodrugs both by determination of kinetic parameters and by ratio of IC50 against UV4 cells when incubated in the presence of prodrug, with and without the E. coli enzyme and cofactor (NADH). Results from the two studies were generally in good agreement in that compounds showing no increase in cytotoxicity in presence of enzyme and cofactor were not substrates for the enzyme. None of the analogues were activated by DT diaphorase isolated from Walker 256 carcinoma cells. For those compounds which were substrates for the E. coli nitroreductase, there was a positive correlation between kcat and IC50 ratio. Two compounds showed advantageous properties: SN 25261 (with a dihydroxypropylcarboxamide ring substituent) which has a more than 10-fold greater aqueous solubility than SN 23862 whilst retaining similar kinetic characteristics and cytotoxic potency; and SN 25084, where a change in the position of the carboxamide group relative to the mustard resulted in an increased cytotoxicity ratio and kcat compared with SN 23862 (IC50 ratios 214 and 135; kcat values of 75 and 26.4 sec-1, respectively). An analogue (SN 25507) incorporating both these structural changes had an enhanced kcat of 576 sec-1. This study elucidates some of the structural requirements of the enzyme and aids identification of further directions in the search for suitable prodrugs for an ADEPT nitroreductase system.

The complete nucleotide sequence of the Pseudomonas gene coding for carboxypeptidase G2.

The complete nucleotide sequence of the Pseudomonas chromosomal gene coding for the enzyme carboxypeptidase G2 (CPG2) has been determined. The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that of randomly derived peptide fragments and by N-terminal sequencing of the purified protein. The gene has been shown to code for a 22 amino acid signal peptide at its N-terminus which closely resembles the signal peptides of other secreted proteins. An alternative 36 amino acid signal peptide which may function in Pseudomonas has also been identified. The codon utilisation of the gene is influenced by the high G + C (67.2%) content of the DNA and exhibits a 92.8% preference for codons ending in G or C. This unusual codon preference may contribute to the generally observed weak expression of Pseudomonas genes in Escherichia coli. A region of DNA upstream of the structural gene has also been sequenced and a ribosome binding site and two putative promoter sequences identified.

Metal ion-promoted binding of proteins to immobilized triazine dye affinity adsorbents

Low concentrations of metal ions, particularly those of the first row transition series such as Zn2+, Co2+, Mn2+, Ni2+, Cu2+, and, to a lesser extent, the group IIA ions, Ca2+ and Mg2+, promotes binding of carboxypeptidase G2, alkaline phosphatase and yeast hexokinase to immobilized Procion Red H-8BN, Procion Yellow H-A and Cibacron Blue F3G-A respectively. The binding of ovalbumin to immobilized Cibacron Blue F3G-A and Procion Orange MX-G is selectively enhanced in the presence of AI3+. With ovalbumin and alkaline phosphatase, the effect is almost totally specific for both the metal ion and dye, whereas with carboxypeptidase G2 and hexokinase, metal ions such as Co2+, Ni2+, Mn2+, Cu2+, Ca2+ and Mg2+ also promote binding to varying degrees. Almost all other monovalent and trivalent metal ions appear to be ineffective. Metal ion-bound enzymes can subsequently be eluted with appropriate chelating agents of the amine, aminocarboxylate or substituted pyridine classes.

Galactosylated antibodies and antibody‐enzyme conjugates in antibody‐directed enzyme prodrug therapy

Antibody directed enzyme prodrug therapy (ADEPT) has been studied as a two‐ and three‐phase system in which an antibody to a tumor‐associated antigen has been used to deliver an enzyme to tumor sites where it can convert a relatively nontoxic prodrug to a cytotoxic agent. In such a system, it is necessary to allow the enzyme activity to clear from the blood before prodrug injection to avoid toxicity caused by prodrug activation in plasma. To accelerate plasma clearance of enzyme activity, two approaches have been studied. The studies have been performed with a monoclonal anticarcinoembryonic‐antigen antibody fragment A5B7‐F(ab′)2 conjugated to a bacterial enzyme, carboxypeptidase G2 (CPG2), in LS174T xenografted mice. In the first approach, a monoclonal antibody (SB43), directed at CPG2, was used, which inactivates CPG2 in vitro and in vivo. SB43 was galactosylated so that it had sufficient time to form a complex with plasma CPG2, resulting in the inactivation and clearance of the complex from plasma via the carbohydrate‐specific receptors in the liver. Injection of SB43gal 19 hours after administration of the radiolabeled conjugate reduced the percentage of injected dose per gram in blood without affecting levels in the tumor.

The potential of carboxypeptidase G2-antibody conjugates as anti-tumour agents. I: Preparation of antihuman chorionic gonadotrophin-carboxypeptidase G2 and cytotoxicity of the conjugate against JAR choriocarcinoma cells in vitro

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been linked to a monoclonal antibody (W14A) raised to human chorionic gonadotrophin. The coupling efficiency and retention of antibody and enzymatic activities are compared for three separate methods of preparing 1:1 conjugates. Preliminary in vitro studies on the cytotoxicity of the free enzyme and the conjugated enzyme towards JAR choriocarcinoma cells are reported. Despite the limitations of the in vitro model, it could be demonstrated that a significant proportion of 10(6) choriocarcinoma cells lost viability when exposed to either free or conjugated enzyme for 72 hours at concentrations of carboxypeptidase G2 of 1-3 units ml-1 of medium.

Biochemical and biological properties of methotrexate analogs containing D-glutamic acid or D-erythro, threo-4-fluoroglutamic acid.

on the B-helix transition of poly-L-lysine in sodium 18. Watanabe S and Saito T, A CD study of the role of dodecyl sulfate solution. Biopolymers 14: 1841-1846. metal ions in the conformation of poly(L-Lysine). 1975. Biopolymers 26: 625-632, 1987. 17. Sarker PK and Doty P, The optical rotatory properties 19. Davidson B and Fasman GD, The conformational of the P-configuration in polypeptides and proteins. transitions of uncharged poly-L-lysine. (Y Helix-random Proc Natl Acad Sci USA 55: 981-989, 1966. coil-8 structure. Biochemistry 6: 16161629. 1967.

Crystallization and preliminary crystallographic analysis of carboxypeptidase G2 from Pseudomonas sp. strain RS-16.

Carboxypeptidase G2, a zinc metalloenzyme isolated from Pseudomonas sp. strain RS-16, which catalyses the hydrolytic cleavage of reduced and non-reduced folates to pteroates and L-glutamate, has been crystallized from polyethylene glycol (average Mr 4000) by vapour diffusion. The crystal symmetry is monoclinic C2, with unit cell dimensions a = 206 A, b = 82 A, c = 116 A and beta = 118 degrees. The molecular mass and volume of the unit cell suggest that there are two dimers of the enzyme in the asymmetric unit. The crystals diffract to at least 3.0 A and are suitable for X-ray structure analysis.

Carboxypeptidase G2 and trimetrexate cause growth delay of human colonic cancer cells in vitro

MAWI colonic cancer cells respond to sequential treatment in vitro with carboxypeptidase G2 and trimetrexate by a delay in cell growth as measured by cell numbers, but an increase in incorporation of 75-Se-selenomethionine per cell. The cells are not methionine auxotrophs.