Seth Bradford

Catalytic Inactivation of Human Carbonic Anhydrase I by a Metallopeptide-Sulfonamide Conjugate is Mediated by Oxidation of Active Site Residues

Oxidation of active site residues (His and Trp), following catalytic inactivation of human carbonic anhydrase I by a copper−ATCUN conjugate of sulfanilimide, is evidenced by mass spectrometric analysis of tryptic and chymotryptic digests of the modified CA-I. Accordingly, residue oxidation rather than protein cleavage is the demonstrated mode of inactivation. An apparent second-order rate constant, k2 ∼ 7600 M-1min-1, has been determined for catalytic inactivation of CA-I.

Insight into the Recognition, Binding, and Reactivity of Catalytic Metallodrugs Targeting Stem Loop IIb of Hepatitis C IRES RNA

The complex Cu‐GGHYrFK‐amide (1‐Cu) was previously reported as a novel metallotherapeutic that catalytically inactivates stem loop IIb (SLIIb) of the hepatitis C virus (HCV) internal ribosomal entry site (IRES) RNA and demonstrates significant antiviral activity in a cellular HCV replicon assay. Herein we describe additional studies focused on understanding the cleavage mechanism as well as the relationship of catalyst configuration to structural recognition and site‐selective cleavage of the structured RNA motif. These are advanced by use of a combination of MALDI‐TOF mass spectrometry, melting temperature determinations, and computational analysis to develop a structural model for binding and reactivity toward SLIIb of the IRES RNA. In addition, the binding, reactivity, and structural chemistry of the all‐D‐amino acid form of this metallopeptide, complex 2‐Cu, are reported and compared with those of complex 1‐Cu. In vitro RNA binding and cleavage assays for complex 2‐Cu show a KD value of 76±3 nM, and Michaelis–Menten parameters of kcat=0.14±0.01 min−1 and KM=7.9±1.2 μM, with a turnover number exceeding 40. In a luciferase‐based cellular replicon assay Cu‐GGhyrfk‐amide shows activity similar to that of the 1‐Cu parent peptide, with an IC50 value of 1.9±0.4 μM and cytotoxicity exceeding 100 μM. RT‐PCR experiments confirm a significant decrease in HCV RNA levels in replicon assays for up to nine days when treated with complex 1‐Cu in three‐day dosing increments. This study shows the influence that the α‐carbon stereocenter has for this new class of compounds, while detailed mass spectrometry and computational analyses provide new insight into the mechanisms of recognition, binding, and reactivity.

Copper·Lys-Gly-His-Lys mediated cleavage of tRNAPhe: Studies of reaction mechanism and cleavage specificity

The reactivity of [Cu2+.Lys-Gly-His-Lys-NH2]2+ and [Cu2+.Lys-Gly-His-Lys]+ toward tRNA(Phe) has been evaluated. The amidated and carboxylate forms of the copper peptides display complex binding behavior with strong and weak sites evident (K(D1)(app) approximately 71 microM, K(D2)(app) approximately 211 microM for the amide form; and K(D1)(app) approximately 34 microM, K(D2)(app) approximately 240 microM for the carboxylate form), while Cu2+(aq) yielded K(D1)(app) approximately 81 microM and K(D2)(app) approximately 136 microM. The time-dependence of the reaction of [Cu2+.Lys-Gly-His-Lys]+ and [Cu2+.Lys-Gly-His-Lys-NH2]2+ with tRNA(Phe) yielded k(obs) approximately 0.075 h(-1) for both complexes. HPLC analysis of the reaction products demonstrated guanine as the sole base product. Mass spectrometric data shows a limited number of cleavage fragments with product peak masses consistent with chemistry occurring at a discrete site defined by the structurally contiguous D and TPsiC loops, and in a domain where high affinity magnesium centers have previously been observed to promote hydrolysis of the tRNA(Phe) backbone. This cleavage pattern is more selective than that previously observed by Long and coworkers for nickel complexes of a series of C-terminally amidated peptides (Gly-Gly-His, Lys-Gly-His, and Arg-Gly-His), and may reflect variations in structural recognition and a distinct reaction path by the nickel derivatives. The data emphasizes the optimal positioning of the metal-associated reactive oxygen species, relative to scissile bonds, as a major criterion for development of efficient catalytic nucleases or therapeutics.