Xinghua Wu

Exploiting the Drug-Activating Properties of a Novel Trypanosomal Nitroreductase

ABSTRACT Nitroheterocyclic prodrugs have been used to treat trypanosomal diseases for more than 40 years. Recently, the key step involved in the activation of these compounds has been elucidated and shown to be catalyzed by a type I nitroreductase (NTR). This class of enzyme is normally associated with bacteria and is absent from most eukaryotes, with trypanosomes being a major exception. Here we exploit this difference by evaluating the trypanocidal activity of a library of nitrobenzylphosphoramide mustards against bloodstream-form Trypanosoma brucei parasites. Biochemical screening against the purified enzyme revealed that a subset of halogenated nitroaromatic compounds were effective substrates for T. brucei NTR (TbNTR), having apparent Kcat/Km values approximately 100 times greater than nifurtimox. When tested against T. brucei, cytotoxicity mirrored enzyme activity, with 50% inhibitory concentrations of the most potent substrates being less than 10 nM. T. brucei NTR plays a key role in parasite killing: heterozygous lines displayed resistance to the compounds, while parasites overexpressing the enzyme showed hypersensitivity. We also evaluated the cytotoxicities of substrates with the highest trypanocidal activities by using mammalian THP-1 cells. The relative toxicities of these newly identified compounds were much lower than that of nifurtimox. We conclude that halogenated nitrobenzylphosphoramide mustards represent a novel class of antitrypanosomal agents, and their efficacy validates the strategy of specifically targeting NTR activity to develop new therapeutics.

Synthesis and structure-activity relationships of nitrobenzyl phosphoramide mustards as nitroreductase-activated prodrugs.

A series of nitrobenzyl phosphoramide mustards and their analogs was designed and synthesized to explore their structure-activity relationships as substrates of nitroreductases from Escherichia coli and trypanosomes and as potential antiproliferative and antiparasitic agents. The position of the nitro group on the phenyl ring was important with the 4-nitrobenzyl phosphoramide mustard (1) offering the best combination of enzyme activity and antiproliferative effect against both mammalian and trypanosomatid cells. A preference was observed for halogen substitutions ortho to benzyl phosphoramide mustard but distinct differences were found in their SAR of substituted 4-nitrobenzyl phosphoramide mustards in E. coli nitroreductase-expressing cells and in trypanosomatids expressing endogenous nitroreductases.

A novel high-yield synthesis of aminoacyl p-nitroanilines and aminoacyl 7-amino-4-methylcoumarins: Important synthons for the synthesis of chromogenic/fluorogenic protease substrates

Summary Aminoacyl p-nitroaniline (aminoacyl-pNA) and aminoacyl 7-amino-4-methylcoumarin (aminoacyl-AMC) are important synthons for the synthesis of chromogenic/fluorogenic protease substrates. A new efficient method was developed to synthesize aminoacyl-pNA and aminoacyl-AMC derivatives in excellent yields starting from either amino acids or their corresponding commercially available N-hydroxysuccinimide esters. The method involved the in situ formation of selenocarboxylate intermediate of protected amino acids and the subsequent non-nucleophilic amidation with an azide. Common protecting groups used in amino acid/peptide chemistry were all well-tolerated. The method was also successfully applied to the synthesis of a dipeptide conjugate, indicating that the methodology is applicable to the synthesis of chromogenic substrates containing short peptides. The method has general applicability to the synthesis of chromogenic and fluorogenic peptide substrates and represents a convenient and high-yield synthesis of N α-protected-aminoacyl-pNAs/AMCs, providing easy access to these important synthons for the construction of chromogenic/fluorogenic protease substrates through fragment condensation or stepwise elongation.

3-Aminoxypropionate-based linker system for cyclization activation in prodrug design

A novel linker system based on 3-aminoxypropionate was designed and evaluated for drug release using proteolysis as an activation trigger followed by intramolecular cyclization. The hydroxylamine moiety present in the linker system enabled faster release of the parent drug from the linker-drug conjugate at lower pH as compared to an aliphatic amine moiety. Introduction of two methyl groups strategically at the alpha position to the carboxylate in the linker further improved the rate of cyclization by nearly 2-fold. The 3-aminoxypropionate linker was successfully applied to a model prodrug for protease activation using alpha-chymotrypsin as the activating enzyme; the activation of the model prodrug bearing the 3-aminoxypropionate linker was 136 times faster than the corresponding model prodrug bearing an amine linker.

Design and synthesis of peptide conjugates of phosphoramide mustard as prodrugs activated by prostate-specific antigen.

A series of Glutaryl-Hyp-Ala-Ser-Chg-Gln-4-aminobenzyl phosphoramide mustard conjugates (1a-e) was designed and synthesized as potential prodrugs for site-specific activation by PSA in prostate cancer cells. All conjugates were found to be substrates of PSA with cleavage occurring between Gln and the para-aminobenzyl (PAB) linker. Structure-activity relationship studies on these conjugates indicated that introduction of electron-withdrawing fluorine(s) on the phenyl ring in the PAB linker uniformly improved the chemical stability of the conjugates while the position of substitution affected differently the self-immolative process of conjugates upon proteolysis. Introduction of a fluorine at ortho position to benzylic phosphoramide as in 1b results in better stability of the conjugate prior to activation while maintaining its antiproliferative activity upon activation by PSA. The conjugate 1b with 2-fluoro substitution was identified as a promising lead for further evaluation and optimization in the development of prostate cancer-targeted prodrugs.