Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs.

Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90,000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.

Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954.

Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme K(M). Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent K(M) than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent K(M) and an increased k(cat)/K(M) relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.

Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954.

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.

The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411(NTR).

Conditionally replicating adenoviruses (CRAd) 'armed' with prodrug-activating genes have the potential to augment the efficacy of virotherapy. An Escherichia coli nitroreductase (NTR) gene (nfsB) was introduced into the E3B region of the systemically active CRAd ONYX-411, to produce ONYX-411(NTR), which had single agent oncolytic activity equivalent to unarmed virus in vitro and in vivo. A fluorogenic probe (SN 29884) developed to monitor NTR expression revealed robust, durable NTR expression in ONYX-411(NTR) infected neoplastic but not primary human cell lines. NTR expression occurred >24 h post-infection in parallel with fiber and was sensitive to ara-C indicating transcriptional linkage to viral replication. A novel NTR prodrug, the 3,5-dinitrobenzamide-2-bromomustard SN 27686, was shown to be more dose potent and selective than CB 1954 and provided a superior bystander effect in 3D multicellular layer cultures. Its water-soluble phosphate ester SN 28343 was substantially more active than CB 1954 against xenografts containing a minority of stable NTR-expressing cells. A single intravenous dose of ONYX-411(NTR) (10(8) PFU) to nude mice bearing large H1299 xenografts (>350 mm(3)) resulted in tumor-specific NTR expression which increased over time. Despite extensive viral spread by day 14, this conservative virus dose and schedule was unable to control such well-established tumors. However, subsequent administration of SN 28343 resulted in the majority of mice (62.5%) being tumor-free on day 120.

Tyrosine kinase inhibitors. 18. 6-Substituted 4-anilinoquinazolines and 4-anilinopyrido[3,4-d]pyrimidines as soluble, irreversible inhibitors of the epidermal growth factor receptor.

4-Anilinoquinazoline- and 4-anilinopyrido[3,4-d]pyrimidine-6-acrylamides are potent pan-erbB tyrosine kinase inactivators, and one example (CI-1033) is in clinical trial. A series of analogues with a variety of Michael acceptor units at the 6-position were prepared to define the structural requirements for irreversible inhibition. A particular goal was to determine whether additional functions to increase solubility could be appended to the Michael acceptor. Substituted acrylamides were prepared by direct acylation of the corresponding 6-amines with the requisite acid or acid chloride. Vinylsulfonamide derivatives were obtained by acylation of the amines with chloroethylsulfonyl chloride followed by base-promoted elimination. Vinylsulfone and vinylsulfine derivatives were prepared by oxidation and base elimination of a hydroxyethylthio intermediate. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Substitution at the nitrogen of the acrylamide was tolerated only with a methyl group; larger substituents were dystherapeutic, and no substitution at all was tolerated at the acrylamide alpha-carbon. In contrast, while electron-donating groups at the acrylamide beta-carbon were not useful, even quite large electron-withdrawing groups (which increase its electrophilicity) were tolerated. A series of derivatives with solubility-enhancing substituents linked to the acrylamide beta-carbon via amides were potent irreversible inhibitors of isolated EGFR (IC50s = 0.4-1.1 nM), with weakly basic morpholine and imidazole derivatives being the best. Vinylsulfonamides were also potent and irreversible inhibitors, but vinylsulfones and vinylsulfines were reversible and only poorly active. Two compounds were evaluated against A431, H125, and MCF-7 xenografts in nude mice but were inferior in these assays to the clinical trial compound CI-1033.

Tyrosine kinase inhibitors. 17. Irreversible inhibitors of the epidermal growth factor receptor: 4-(phenylamino)quinazoline- and 4-(phenylamino)pyrido[3,2-d]pyrimidine-6-acrylamides bearing additional solubilizing functions.

4-Anilinoquinazoline- and 4-anilinopyrido[3,2-d]pyrimidine-6-acrylamides substituted with solubilizing 7-alkylamine or 7-alkoxyamine side chains were prepared by reaction of the corresponding 6-amines with acrylic acid or acrylic acid anhydrides. In the pyrido[3,2-d]pyrimidine series, the intermediate 6-amino-7-alkylamines were prepared from 7-bromo-6-fluoropyrido[3,2-d]pyrimidine via Stille coupling with the appropriate stannane under palladium(0) catalysis. This proved a versatile method for the introduction of cationic solubilizing side chains. The compounds were evaluated for their inhibition of phosphorylation of the isolated EGFR enzyme and for inhibition of EGF-stimulated autophosphorylation of EGFR in A431 cells and of heregulin-stimulated autophosphorylation of erbB2 in MDA-MB 453 cells. Quinazoline analogues with 7-alkoxyamine solubilizing groups were potent irreversible inhibitors of the isolated EGFR enzyme, with IC(50[app]) values from 2 to 4 nM, and potently inhibited both EGFR and erbB2 autophosphorylation in cells. 7-Alkylamino- and 7-alkoxyaminopyrido[3,2-d]pyrimidines were also irreversible inhibitors with equal or superior potency against the isolated enzyme but were less effective in the cellular autophosphorylation assays. Both quinazoline- and pyrido[3,2-d]pyrimidine-6-acrylamides bound at the ATP site alkylating cysteine 773, as shown by electrospray ionization mass spectrometry, and had similar rates of absorptive and secretory transport in Caco-2 cells. A comparison of two 7-propoxymorpholide analogues showed that the pyrido[3,2-d]pyrimidine-6-acrylamide had greater amide instability and higher acrylamide reactivity, being converted to glutathione adducts in cells more rapidly than the corresponding quinazoline. This difference may contribute to the observed lower cellular potency of the pyrido[3,2-d]pyrimidine-6-acrylamides. Selected compounds showed high in vivo activity against A431 xenografts on oral dosing, with the quinazolines being superior to the pyrido[3,2-d]pyrimidines. Overall, the quinazolines proved superior to previous analogues in terms of aqueous solubility, potency, and in vivo antitumor activity, and one example (CI 1033) has been selected for clinical evaluation.

Tyrosine kinase inhibitors. 15. 4-(Phenylamino)quinazoline and 4-(phenylamino)pyrido[d]pyrimidine acrylamides as irreversible inhibitors of the ATP binding site of the epidermal growth factor receptor.

A series of 6- and 7-acrylamide derivatives of the 4-(phenylamino)quinazoline and -pyridopyrimidine classes of epidermal growth factor receptor (EGFR) inhibitors were prepared from the corresponding amino compounds by reaction with either acryloyl chloride/base or acrylic acid/1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. All of the 6-acrylamides, but only the parent quinazoline 7-acrylamide, were irreversible inhibitors of the isolated enzyme, confirming that the former are better-positioned, when bound to the enzyme, to react with the critical cysteine-773. Quinazoline, pyrido[3,4-d]pyrimidine, and pyrido[3,2-d]pyrimidine 6-acrylamides were all irreversible inhibitors and showed similar high potencies in the enzyme assay (likely due to titration of the available enzyme). However the pyrido[3,2-d]pyrimidine analogues were 2-6-fold less potent than the others in a cellular autophosphorylation assay for EGFR in A431 cells. The quinazolines were generally less potent overall toward inhibition of heregulin-stimulated autophosphorylation of erbB2 (in MDA-MB-453-cells), whereas the pyridopyrimidines were equipotent. Selected compounds were evaluated in A431 epidermoid and H125 non-small-cell lung cancer human tumor xenografts. The compounds showed better activity when given orally than intraperitoneally. All showed significant tumor growth inhibition (stasis) over a dose range. The poor aqueous solubility of the compounds was a drawback, requiring formulation as fine particulate emulsions.

Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

Mono- and dysfunctional nitrogen mustard analogues of the DNA minor groove binder pibenzimol. Synthesis, cytotoxicity and interaction with DNA.

Two series of mono- and dysfunctional aniline mustards linked to a bisbenzimidazole minor groove binder have been prepared using a new method (polyphosphate ester-mediated direct coupling of appropriate mustard acids with a preformed advanced phenylenediamine intermediate). As the linker chain attaching the mustard was lengthened the binding site size of the compounds to calf thymus DNA remained essentially constant at 2.6 nucleotides, but reversible binding strength declined by a factor of 2. Analogues with longer linker chains alkylated DNA much more rapidly than those with shorter chains, consistent with the electronic factors. The short chain analogues also failed to alkylate a 120 bp HindIII to Bg/II fragment of the gpt gene, as measured by gel electrophoresis cleavage assays. The longer chain analogues (both mono- and dysfunctional mustards) showed patterns of DNA alkylation that varied with chain length. In particular, while most compounds showed substantial N7 alkylation at many guanine residues, the analogue with a (CH2)3 linker chain showed strong alkylation at adenine sites in poly-AT regions. For the longer chain analogues, the bifunctional mustards were substantially (10- to 20-fold) more cytotoxic than the corresponding monofunctional analogues.

Structure-activity relationships for 1-phenylbenzimidazoles as selective ATP site inhibitors of the platelet-derived growth factor receptor.

1-Phenylbenzimidazoles are shown to be a new class of ATP-site inhibitors of the platelet-derived growth factor receptor (PDGFR). Structure-activity relationships (SARs) are narrow, with closely related heterocycles being inactive. A systematic study of substituted 1-phenylbenzimidazoles showed clear SARs. Substituents at the 4'- and 3'-positions of the phenyl ring are tolerated but do not significantly improve activity, while substituents at the 2'-position abolish it. Substituents in the 2-, 4-, and 7-positions of the benzimidazole ring (with the exception of 4-OH) also abolish activity. Most substituents at the 5- and 6-positions maintain or increase activity, with the 5-OH, 5-OMe, 5-COMe, and 5-CO2Me analogues being >10-fold more potent than the parent 1-phenylbenzimidazole. The 5-OMe analogue was both the most potent inhibitor, and showed the highest selectivity (50-fold) between PDGFR and FGFR isolated enzymes, and also a moderately effective inhibitor (IC50 = 1.9 microM) of PDGF-stimulated PDGFR autophosphorylation in rat aorta smooth muscle cells.

DNA minor groove targeted alkylating agents based on bisbenzimidazole carriers: synthesis, cytotoxicity and sequence-specificity of DNA alkylation.

A series of bisbenzimidazoles bearing a variety of alkylating agents [ortho- and meta-mustards, imidazolebis(hydroxymethyl), imidazolebis(methylcarbamate) and pyrrolebis(hydroxymethyl)], appended by a propyl linker chain, were prepared and investigated for sequence-specificity of DNA alkylation and their cytotoxicity. Previous work has shown that, for para-aniline mustards, a propyl linker is optimal for cytotoxicity. Alkaline cleavage assays using a variety of different labelled oligonucleotides showed that the preferred sequences for adenine alkylation were 5'-TTTANANAANN and 5'-ATTANANAANN (underlined bases show the drug alkylation sites), with AT-rich sequences required on both the 5' and 3' sides of the alkylated adenine. The different aniline mustards showed little variation in alkylation pattern and similar efficiencies of DNA cross-link formation despite the changes in orientation and positioning of the mustard, suggesting that the propyl linker has some flexibility. The imidazole- and pyrrolebis(hydroxymethyl) alkylators showed no DNA strand cleavage following base treatment, indicating that no guanine or adenine N3 or N7 adducts were formed. Using the PCR-based polymerase stop assay, these alkylators showed PCR blocks at 5'-C*G sites (the * nucleotide indicates the blocked site), particularly at 5'-TAC*GA 5'-AGC*GGA, and 5'-AGCC*GGT sequences, caused by guanine 2-NH2 lesions on the opposite strand. Only the (more reactive) imidazolebis(methylcarbamoyl) and pyrrolebis(hydroxymethyl) alkylators demonstrated interstrand cross-linking ability. All of the bifunctional mustards showed large (approximately 100-fold) increases in cytotoxicity over chlorambucil, with the corresponding monofunctional mustards being 20- to 60-fold less cytotoxic. These results suggest that in the mustards the propyl linker provides sufficient flexibility to achieve delivery of the alkylator to favoured (adenine N3) sites in the minor groove, regardless of its exact geometry with respect to the bisbenzimidazole carrier. The 'targeted' bisbenzimidazole bis(hydroxymethyl)pyrrole- and imidazole analogues showed very similar patterns of alkylation to the corresponding 'untargeted' compounds, with little evidence of additional selectivity imposed by this AT-preferring carrier.