Designed Metal-ATCUN Derivatives: Redox- and Non-redox-Based Applications Relevant for Chemistry, Biology, and Medicine

Abstract

The designed "ATCUN" motif (amino-terminal copper and nickel binding site) is a replica of naturally occurring ATCUN site found in many proteins/peptides, and an attractive platform for multiple applications, which include nucleases, proteases, spectroscopic probes, imaging, and small molecule activation. ATCUN motifs are engineered at periphery by conjugation to recombinant proteins, peptides, fluorophores, or recognition domains through chemically or genetically, fulfilling the needs of various biological relevance and a wide range of practical usages. This chemistry has witnessed significant growth over the last few decades and several interesting ATCUN derivatives have been described. The redox role of the ATCUN moieties is also an important aspect to be considered. The redox potential of designed M-ATCUN derivatives is modulated by judicious choice of amino acid (including stereochemistry, charge, and position) that ultimately leads to the catalytic efficiency. In this context, a wide range of M-ATCUN derivatives have been designed purposefully for various redox- and non-redox-based applications, including spectroscopic probes, target-based catalytic metallodrugs, inhibition of amyloid-β toxicity, and telomere shortening, enzyme inactivation, biomolecules stitching or modification, next-generation antibiotic, and small molecule activation.

Keywords: Biochemistry; Chemistry; Inorganic Chemistry; Medical Biochemistry.

Graphical abstract

Figure 1

Schematic Representation of Design Strategies of Synthesized ATCUN and Its Metal Derivatives R¹ and R²; variable amino acids at first and second positions in NH₂-R¹-R²-H sequence, H; histidine.

Figure 2

Schematic Representations of Designed M-ATCUN Derivatives in Varous Applications

Figure 3

Application of Cu-ATCUNs as Paramagnetic NMR Reporters (A–C) (A) The paramagnetic NMR probe, Cu^II-ATCUN is attached at N-terminus of ORP. The crystal structure of apo-ATCUN-ORP is derived from PDB 2WFB (Najmudin et al., 2009), (B) Paramagnetic ¹H-NMR spectrum (600 MHz) of apo-ATCUN-ORP is titrated with Cu^II salt (0–80%) in mixture of 50 mM Tris-HCl at pH 7.6/D₂O (80/20%) and only His₅₃ (b and d peaks) and His₃ (a and e peaks) amino acid redidues are represented those are highly affected, and (C) Schematically shows the protein-protein interaction in head-tail fashion (dotted black lines). Modified from , Maiti et al., 2017b.

Figure 4

Application of ATCUNs as Fluorescent Reporters Fluorescence emission of proteases models, β-secretase (A) and caspase-3 (B) is quenched by Cu^II-ATCUN. Dns; flurophore. Modified from Deng et al., 2019b.

Figure 5

Aβ_4-x Represents the ATCUN (Blue), Bis-His (Green) and Recognition Sites (Orange) In presence of copper, Aβ can produce aggregated as well produce HO⋅ but upon addition of apo-ATCUN, Cu is trapped by ATCUN and inhibit the aggregation of Aβ.

Figure 6

⁶⁴Cu-ATCUN-Octreotide Model of Somatostatin Receptor (Targeting Tumor Cell) Providing PET Imaging R¹ and R²; Y, V, N, T, G, and D amino acids.

Figure 7

Schematic Representation of M-ATCUN Induces Oxidatively DNA Strand Scission by Sugar-Hydrogen Abstraction to Yield C₁^/-, or C₂^/-, or C₃^/-, or C₄^/-, or C₅^/-, Deoxyribosyl Radical. (modified from Pogozelski and Tullius, 1998). B = Nucleobase.

Figure 8

Selectivity and Efficacy of DNA Strand Scission Are Modulated by Proper Design of ATCUN Motif (A–D) (A) Two-dimentional representation of DNA showing targeting sites, minor and major groove, (B) quadruplex structure of telomeric DNA, (C) top and bottom faces of square planar geometry of M-ATCUN (M = Cu^II, Ni^II and Co^II), and (D) introduction of positive charge amino acid (positive charge and D/L) at first, second, and or fourth positions.

Figure 9

Two-Dimentional Representation of Interaction between Ni^II-RGH and the Minor Groove of Model DNA, 5^/-d(CGCG₄A₅ATTCGC₉G)₂ through H-Bonding (Dotted Lines) (Modified from Fang et al., 2004).

Figure 10

Cleavage of Telomeric DNA by designed Cu-ATCUNs (A) Crystal structure of G4 telomeric DNA (PDB: 1KF1 (Yu et al., 2015)) shows the selective cleavage sites by (Cu-ATCUN)₂ND (black arrows with ND) and Cu-ATCUN-Acr (black arrows with Acr). ND; naphthalene diimide derivatives, ND1 and ND2 represents the two copper sites in (Cu-ATCUN)₂ND, Acr; acridine. (B and C) (B) and (C) showing two-dimensional representation of Cu-GGH-Acr, and R₂-ND derivatives (R = Cu-ATCUN = Cu-GGH, Cu-DGH and Cu-GDH) respectively (Modified from Yu et al., 2019).

Figure 11

Selective Cleavage of HIV RNA by Cu-ATCUN-Rev (A) Schematic illustration of the stem loop IIb (SL-IIb) of RRE RNA of HIV showing the cleavage site, as well as binding pocket. (B) Crystal structure of RRE RNA SL-IIb complexes with an Rev peptide (purple) (PDB: 1ETF) showing the main cleavage sites which are shown by green highlighted as well as red arrows (U₅, G₆, and C₉). The catalytic center, M-ATCUN (red star) may be conjugated with N-terminus of Rev. The structure is modified from Joyner et al. (2011).

Figure 12

Schematic Representation of the Cellular Expression of GFP (Plasmid Encoded GFP-RRE) Is Induced by Cu^II-ATCUN-Rev (Modified from Hocharoen and Cowan, 2009).

Figure 13

Selective Cleavage of HCV RNA by M-ATCUN Derivatives (A and B) (A) Two-dimensional representation of subdomain SL-IIb RNA of HCV, and (B) crystal structure of HCV SL-IIb RNA (PDB: 1P5N) showing the main cleavage sites (highlighted blue and orange color) by CuGGHYrFK or CuGGhyrfk respectively. Modified from Ross et al. (2017).

Figure 14

Inhibition of HCV Replication by La Protein (A) Two-dimensional representations of SL-IV RNA showing AUG start codon (pink circle) and 5′-GCAC-3′ sequences as a potential recognition site (orange circle). (B) NMR solution structure of the human La protein is derived from PDB 1S79. LaR2C peptide, KYKETDLLILFKDDYFAKKNEERK is highlighted as green and blue color. The truncated 7-mer, KYKETDL is highlighted as blue color. Modified from Ross et al. (2015).

Figure 15

Interaction between Zn-ACE and Inhibitor (A and B) (A) Partial crystal structure of lisinopril (inhibitor) adduct Zn-active site of ACE (PDB code: 1O86 (Natesh et al., 2003)), and (B) two-dimentional representation of natural occurring substrate, Hip-His-Leu are showing the binding sites. S1; benzene group, S1^/; His group and S2^/; dimethyl group of natural substrate sites corresponding to lisinopril.

Figure 16

Possible Reversible (Simple Inhibitor) vs. Irreversible (M-ATCUN-Inhibitor) Inactivation Mechanism of Metalloproteins-Substrate with Inhibitor

Figure 17

Crystal Structure of Human CA-I (PDB: 1CZM (Chakravarty and Kannan, 1994)) with Bound Sulfonamide Inhibitor (Stick Model and also Highlighted by Green Circle) Highlighted the possible oxidatively modified amino acids, H₆₄, H₆₇, H₂₀₀, H₂₄₃, W₉₇, W₁₂₃ (stick ball model) by Cu-GGH. Distances of these residues from Zn-center are shown by dotted double headed arrows. The Cu-GGH (red star) is assumed to coordinate to the NH₂-tail of sulfonamide inhibitor (green circle). Modified from Gokhale et al. (2008).

Figure 18

Inactivation of SrtA_ΔN59 Activity by Cu-ATCUNs Superimpose crystal structures of SrtA_ΔN59 (Wild (cyano) (PDB ID: 1T2O (Suree et al., 2009)) and mutant Cys184Ala (gray) (PDB ID: 1T2W (Suree et al., 2009)) with substrate, LPETG (blue)). Cu-GGH (red star) is assumed to bind to the L residue of substrate where close proximity amino acid residues G₁₆₇, V₁₆₈, and L₁₆₉, and G residue of substrate points toward the active-site amino acid R₁₉₇-C₁₈₄-H₁₂₀. Modified from Fidai et al. (2014).

Figure 19

Peptide-Based Inhibitor of Viral Protease Crystal structure showing oxidative damage to West Nile Virus NS2B/NS3 (PDB: 2FP7). Amino acid residues, green set (Ser135, Thr₁₃₂ and Thr₁₃₄) and orange set (Asp₇₅, Ser₇₁, Lys₇₃ and Glu₇₄) are modified by naphthoylated (Np) and benzoylated metallo-peptide (Bz) respectively. His₅₁-Asp₇₅-Ser₁₃₅ triad is active site. Modified from Pinkham et al. (2018a).

Figure 20

Possible Mechanisms of Bacteria Cell Death by M-ATCUN-AMPs vs. AMPs

Figure 21

The Formation of Tyrosine Cross-Linking Product by Ni-ATCUN in GGH-Ecotin (D137Y) The ecotin structure is derived from the PBD: 1ECZ (Shin et al., 1996). The Ni-ATCUN (red star) is assumed to bind N-terminus (green circle) of ecotin-A (gray) and distance from Tyr₁₂₇ is ~11 Å. The distance between mutant Asp₁₃₇→Tyr₁₃₇ in ecotin-B (cyano) and wild Tyr₁₂₇ in ecotin-A (gray) is 6 Å (highlighted blue box). MMPP = monoperoxyphthalate.

Figure 22

Nitration of Tyrosine in ORP by Ni-ATCUN in Presence of Na₂SO₃/NaNO₂/O₂ (Modified from Maiti et al., 2019) The drawing was adapted from the crystal structure of apo-ATCUN-ORP [PDB: 2wfb, Najmudin et al., 2009].