Regulating the Heme Active Site by Covalent Modifications: Two Case Studies of Myoglobin.

Using myoglobin (Mb) as a model protein, we herein developed a facial approach to modifying the heme active site. A cavity was first generated in the heme distal site by F46 C mutation, and the thiol group of Cys46 was then used for covalently linked to exogenous ligands, 1H-1,2,4-triazole-3-thiol and 1-(4-hydroxyphenyl)-1H-pyrrole-2,5-dione. The engineered proteins, termed F46C-triazole Mb and F46C-phenol Mb, respectively, were characterized by X-ray crystallography, spectroscopic and stopped-flow kinetic studies. The results showed that both the heme coordination state and the protein function such as H2 O2 activation and peroxidase activity could be efficiently regulated, which suggests that this approach might be generally applied to the design of functional heme proteins.

Effects of naturally occurring S47F/A mutations on the structure and function of human cytochrome c.

The sequence and structure of human cytochrome c (hCyt c) exhibit evolutionary conservations, with only a limited number of naturally occurring mutations in humans. Herein, we investigated the effects of the naturally occurring S47F/A mutations on the structure and function of hCyt c in the oxidized form. Although the naturally occurring S47F/A mutations did not largely alter the protein structure, the S47F and S47A variants exhibited a small fraction of high-spin species. Kinetic studies showed that the peroxidase activity of the variants was enhanced by ∼2.5-fold under neutral pH conditions, as well as for the rate in reaction with H2O2, when compared to those of wild-type hCyt c. In addition, we evaluated the interaction between hCyt c and human neuroglobin (hNgb) by isothermal titration calorimetry (ITC) studies, which revealed that the binding constant was reduced by ∼8-fold as result of the mutation of the hydrophilic Ser to the hydrophobic Phe/Ala. These findings provide valuable insights into the role of Ser47 in Ω-loop C in sustaining the structure and function of hCyt c.

Anti-triple-negative breast cancer metastasis efficacy and molecular mechanism of the STING agonist for innate immune pathway.

BACKGROUND: With high recurrence and metastatic rates, triple-negative breast cancer (TNBC) has few therapy choices. The innate immune stimulator of interferon genes protein (STING) pathway has emerged as a critical foundation for improving anticancer immunotherapy. Although 2',3'-cGAMP has been shown to have therapeutic potential as a STING agonist in subcutaneous solid tumour treatments in mice, the effect of cGAMP in metastatic malignancies has received less attention. METHODS: Bioluminescence imaging technology was applied to monitor TNBC tumour cell metastasis in living mice. Serum biochemical test and blood routine examination of mice were used to demonstrate cGAMP administration had no toxicity. The activation of DCs and CD8+ T cells was demonstrated by flow cytometry. The pharmacological mechanism of cGAMP for suppressing breast tumour metastasis was also explored. RESULTS: cGAMP treatment substantially suppressed tumour development and metastasis without adverse effects. cGAMP activated the cGAS-STING-IRF3 pathway, which modified the tumour immune milieu to reverse the Epithelial-Mesenchymal Transition (EMT) and PI3K/AKT pathways and prevent tumour metastasis. It was postulated and proven that cGAMP had a pharmacological mechanism for reducing breast tumour metastasis. CONCLUSION: The findings suggest that cGAMP could be useful in the immunotherapy of immune-insensitive metastatic breast cancer.

Self-oxidation of cysteine to sulfinic acid in an engineered T67C myoglobin: structure and reactivity.

Myoglobin (Mb) was found to undergo self-oxidation when a cysteine residue was engineered at position 67 in the heme distal site. Both the X-ray crystal structure and mass spectrum confirmed the formation of a sulfinic acid (Cys-SO2H). Moreover, the self-oxidation could be controlled during protein purification to yield the unmodified form (T67C Mb). Importantly, both T67C Mb and T67C Mb (Cys-SO2H) were able to be labeled by chemicals, which provided useful platforms to generate artificial proteins.

Potent Therapeutic Strategies for COVID-19 with Single-Domain Antibody Immunoliposomes Neutralizing SARS-CoV-2 and Lip/cGAMP Enhancing Protective Immunity.

The worldwide spread of COVID-19 continues to impact our lives and has led to unprecedented damage to global health and the economy. This highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We modified a single-domain antibody, SARS-CoV-2 VHH, to the surface of the liposomes. These immunoliposomes demonstrated a good neutralizing ability, but could also carry therapeutic compounds. Furthermore, we used the 2019-nCoV RBD-SD1 protein as an antigen with Lip/cGAMP as the adjuvant to immunize mice. Lip/cGAMP enhanced the immunity well. It was demonstrated that the combination of RBD-SD1 and Lip/cGAMP was an effective preventive vaccine. This work presented potent therapeutic anti-SARS-CoV-2 drugs and an effective vaccine to prevent the spread of COVID-19.

Phenoxazinone Synthase-like Activity of Rationally Designed Heme Enzymes Based on Myoglobin.

The design of functional metalloenzymes is attractive for the biosynthesis of biologically important compounds, such as phenoxazinones and phenazines catalyzed by native phenoxazinone synthase (PHS). To design functional heme enzymes, we used myoglobin (Mb) as a model protein and introduced an artificial CXXC motif into the heme distal pocket by F46C and L49C mutations, which forms a de novo disulfide bond, as confirmed by the X-ray crystal structure. We further introduced a catalytic Tyr43 into the heme distal pocket and found that the F43Y/F46C/L49C Mb triple mutant and the previously designed F43Y/F46S Mb exhibit PHS-like activity (80-98% yields in 5-15 min), with the catalytic efficiency exceeding those of natural metalloenzymes, including o-aminophenol oxidase, laccase, and dye-decolorizing peroxidase. Moreover, we showed that the oxidative coupling product of 1,6-disulfonic-2,7-diaminophenazine is a potential pH indicator, with the orange-magenta color change at pH 4-5 (pKa = 4.40). Therefore, this study indicates that functional heme enzymes can be rationally designed by structural modifications of Mb, exhibiting the functionality of the native PHS for green biosynthesis.

Encapsulation of STING Agonist cGAMP with Folic Acid-Conjugated Liposomes Significantly Enhances Antitumor Pharmacodynamic Effect.

Background: 2',3'-cGAMP (2',3'-cyclic AMP-GMP) has been reported as an agonist of the STING (stimulator of interferon genes) signaling pathway. However, cGAMP has poor membrane permeability and can be hydrolyzed by ectonucleotide pyrophosphatase/phosphodiesterase (ENPP1), limiting its ability to activate the STING-IRF3 pathway. This study aimed to investigate that the folate-targeted liposomal cGAMP could overcome the defects of free cGAMP to enhance the antitumor effect. Materials and Methods: cGAMP was encapsulated in PEGylated folic acid-targeted liposomes to construct a carrier-delivered formulation. The particle size and morphology were detected by dynamic light scattering and transmission electron microscopy. The sustained-release ability was measured by drug release and pharmacokinetics. Animal models were applied to evaluate the tumor inhibition efficiency in vivo. Flow cytometry, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction were used to detect the expression of immune cells, secreted cytokines, and target genes. The activation of the STING-IRF3 pathway was evaluated by immunofluorescence. Results: Physical characters of liposomes revealed that the prepared liposomes were stable in neutral humoral environments and released more internal drugs in acidic tumor tissues. Systemic therapy with liposomes on Colorectal 26 tumor-bearing mice in vivo effectively inhibited tumor growth via stimulating the expression of CD8+ T cells and reversed the immunosuppressed tumor microenvironment (TME). Conclusions: The study suggests that the folic acid-targeted cGAMP-loaded liposomes deliver drugs to the TME to enhance the STING agonist activity, improving the efficiency of tumor therapy via the cGAMP-STING-IRF3 pathway.

Efficient Degradation of Tetracycline Antibiotics by Engineered Myoglobin with High Peroxidase Activity.

Tetracyclines are one class of widely used antibiotics. Meanwhile, due to abuse and improper disposal, they are often detected in wastewater, which causes a series of environmental problems and poses a threat to human health and safety. As an efficient and environmentally friendly method, enzymatic catalysis has attracted much attention. In previous studies, we have designed an efficient peroxidase (F43Y/P88W/F138W Mb, termed YWW Mb) based on the protein scaffold of myoglobin (Mb), an O2 carrier, by modifying the heme active center and introducing two Trp residues. In this study, we further applied it to degrade the tetracycline antibiotics. Both UV-Vis and HPLC studies showed that the triple mutant YWW Mb was able to catalyze the degradation of tetracycline, oxytetracycline, doxycycline, and chlortetracycline effectively, with a degradation rate of ~100%, ~98%, ~94%, and ~90%, respectively, within 5 min by using H2O2 as an oxidant. These activities are much higher than those of wild-type Mb and other heme enzymes such as manganese peroxidase. As further analyzed by UPLC-ESI-MS, we identified multiple degradation products and thus proposed possible degradation mechanisms. In addition, the toxicity of the products was analyzed by using in vitro antibacterial experiments of E. coli. Therefore, this study indicates that the engineered heme enzyme has potential applications for environmental remediation by degradation of tetracycline antibiotics.

O2 Carrier Myoglobin Also Exhibits ß-Lactamase Activity That Is Regulated by the Heme Coordination State.

Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The ß-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel ß-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural ß-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the ß-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as ß-lactamases, which broadens the diversity of their catalytic functions.

Engineering Human Neuroglobin into a Cytochrome c-Like Protein with a Single Thioether Bond in Non-native State.

A double mutant of human H64M/V71C neuroglobin (Ngb) was engineered, which formed a single thioether bond as that in atypical cytochrome c, whereas the heme distal Met64 was oxidized to both sulfoxide (SO-Met) and sulfone (SO2 -Met). By contrast, no Cys-heme cross-link was formed in V71C Ngb with His64/His96 coordination, as shown by the X-ray crystal structure, which indicates that an open distal site facilitates the activation of heme iron for structural modifications.

Engineering globins for efficient biodegradation of malachite green: two case studies of myoglobin and neuroglobin.

Malachite green (MG)-contaminated wastewater resulting from industrialization causes a global problem because of its toxicity and widespread usage. Compared with traditional physical and chemical approaches, biodegradation provides a new route for the degradation of MG. As promising candidates for native enzymes, artificial enzymes have received tremendous attention for potential applications due to unlimited possibilities based on precise design. In this study, we rationally engineered artificial enzymes based on myoglobin (Mb) and neuroglobin (Ngb). We introduced an aspartic acid (H64D mutation) in the heme pocket of Mb. A distal histidine (F43H mutation) was further introduced into H64D Mb to obtain a double mutant of F43H/H64D Mb. Moreover, we used A15C/H64D Ngb as designed recently for comparison studies. The H64D Mb, F43H/H64D Mb, and A15C/H64D Ngb were found to catalyze MG degradation efficiently, with activities much higher than those of native enzymes, such as dye-decolorizing peroxidase and laccase (83-205-fold). The crystal structure of H64D Mb was solved and the interactions of MG and H64D Mb and A15C/H64D Ngb were investigated by using both experimental and molecular docking studies. The biodegradation products of MG were also revealed by ESI-MS analysis. Therefore, these artificial enzymes have potential applications in the biodegradation of MG in textile industries and fisheries.

Development of Novel Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Tumor Immunotherapy.

The cyclic guanosine monophosphate-adenosine monophosphate synthase-stimulator of interferon genes-TANK-binding kinase 1-interferon regulating factor 3 (cGAS-STING-TBK1-IRF3) axis is now acknowledged as the major signaling pathway in innate immune responses. However, 2',3'-cGAMP as a STING stimulator is easily recognized and degraded by ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), which reduces the effect of tumor immunotherapy and promotes metastatic progression. In this investigation, the structure-based virtual screening strategy was adopted to discover eight candidate compounds containing zinc-binding quinazolin-4(3H)-one scaffold as ENPP1 inhibitors. Subsequently, these novel inhibitors targeting ENPP1 were synthesized and characterized by NMR and high-resolution mass spectra (HRMS). In bioassays, 7-fluoro-2-(((5-methoxy-1H-imidazo[4,5-b]pyridin-2-yl)thio)methyl)quina-zolin-4(3H)-one(compound 4e) showed excellent activity against the ENPP1 at the molecular and cellular levels, with IC50 values of 0.188 µM and 0.732 µM, respectively. Additionally, compound 4e had superior selectivity towards metastatic breast cancer cells (4T1) than towards normal cells (LO2 and 293T) in comparison with cisplatin, indicating that compound 4e can potentially be used in metastatic breast cancer therapy. On the other hand, compound 4e upgraded the expression levels of IFN-ß in vivo by preventing the ENPP1 from hydrolyzing the cGAMP to stimulate a more potent innate immune response. Therefore, this compound might be applied to boost antitumor immunity for cancer immunotherapy. Overall, our work provides a strategy for the development of a promising drug candidate targeting ENPP1 for tumor immunotherapy.

Spiro-Oxindole Skeleton Compounds Are Efficient Inhibitors for Indoleamine 2,3-Dioxygenase 1: An Attractive Target for Tumor Immunotherapy.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an attractive heme enzyme for its significant function in cancer immunotherapy. Potent IDO1 inhibitors have been discovered for decades, whereas no clinical drugs are used for cancer treatment up to now. With the goal of developing medically valuable IDO inhibitors, we performed a systematic study of SAR405838 analogs with a spiro-oxindole skeleton in this study. Based on the expression and purification of human IDO1, the inhibitory activity of spiro-oxindole skeleton compounds to IDO1 was evaluated by IC50 and Ki values. The results demonstrated that inhibitor 3 exhibited the highest IDO1 inhibitory activity with IC50 at 7.9 µM among all inhibitors, which is ~six-fold of the positive control (4-PI). Moreover, inhibitor 3 was found to have the most effective inhibition of IDO1 in MCF-7 cancer cells without toxic effects. Molecular docking analysis revealed that the hydrophobic interaction stabilized the binding of inhibitor 3 to the IDO1 active site and made an explanation for the uncompetitive mode of inhibitors. Therefore, this study provides valuable insights into the screen of more potent IDO1 inhibitors for cancer immunotherapy.

Structural and functional regulations by a disulfide bond designed in myoglobin like human neuroglobin.

An artificial disulfide bond (Cys46-Cys61) was designed in the heme distal site of myoglobin, which regulates the conformation of the heme distal His64 and the protein reactivity, as confirmed by X-ray crystallography, EPR, and kinetic UV-vis studies. This study shows the successful design of a disulfide bond with suitable positions in globins, conferring a structure and function like those of the native human neuroglobin.

A novel insight into the molecular mechanism of human soluble guanylyl cyclase focused on catalytic domain in living cells.

Human soluble guanylate cyclase (sGC) is a heme-containing metalloprotein in NO-sGC-cGMP signaling. In this work, fluorescent proteins were employed to study the NO-induced sGC molecular mechanism via mutagenesis at the catalytic domain. The conformational change of sGC by mutant α1C595 was investigated in living cells through fluorescence lifetime imaging microscopy (FLIM). The results indicated that the NO-induced conformational change of the catalytic domain of sGC from "open to "closed" upon GTP-binding was regulated by the hydrogen (H)-bonding network of the catalytic domain. The mutation of C595 caused a big conformational change of catalytic domain with H-bond variation, which not only demonstrates the key role of the C595 site in the process of conformational change of the catalytic domain, but also reveals the regulatory mechanism of sGC at the catalytic domain. This finding would guide the design of small-molecule drugs targeting the catalytic domain to modulate sGC activity.

Functional Conversion of Acetyl-Coenzyme a Synthase to a Nickel Superoxide Dismutase via Rational Design of Coordination Microenvironment for the Nid-Site.

The Nid site coordination microenvironment of a truncated acetyl-coenzyme A synthase has been designed systematically for functional conversion to a Ni-SOD-like enzyme. To this end, the first strategy is to introduce an axial histidine ligand, using mutations F598H, S594H and S594H-GP individually. The resulting three mutants obtained Ni-SOD-like activity successfully, although the catalytic activity was about 10-fold lower than in native Ni-SOD. The second strategy is to mimic the H-bond network in the second sphere coordination microenvironment of the native Ni-SOD. Two mutations based on F598H (EFG-F598H and YGP-F598H) were designed. The successful EFG-F598H exhibited ~3-fold Ni-SOD-like activity of F598H. These designed Ni-SOD-like metalloproteins were characterized by UV/Vis, EPR and Cyclic voltammetry while F598H was also characterized by X-ray protein crystallography. The pH titrations were performed to reveal the source of the two protons required for forming H2O2 in the SOD catalytic reaction. Based on all of the results, a proposed catalytic mechanism for the Ni-SOD-like metalloproteins is presented.

Improving the cell-membrane-penetrating activity of globins by introducing positive charges on protein surface: A case study of sperm whale myoglobin.

Globins are heme proteins such as hemoglobin (Hb), myoglobin (Mb) and neuroglobin (Ngb), playing important roles in biological system. In addition to normal functions, zebrafish Ngb was able to penetrate cell membranes, whereas less was known for other globin members. In this study, to improve the cell-membrane-penetrating activity of globins, we used sperm whale Mb as a model protein and constructed a quadruple mutant of G5K/Q8K/A19K/V21K Mb (termed 4K Mb), by introduction of four positive charges on the protein surface, which was designed according to the amino acid alignment with that of zebrafish Ngb. Spectroscopic and crystallographic studies showed that the four positively charged Lys residues did not affect the protein structure. Cell-membrane-penetrating essay further showed that 4K Mb exhibited enhanced activity compared to that of native Mb. This study provides valuable information for the effect of distribution of charged residues on the protein structure and the cell-membrane-penetrating activity of globins. Therefore, it will guide the design of protein-based biomaterials for biological applications.

The X-ray crystal structure of human A15C neuroglobin reveals both native/de novo disulfide bonds and unexpected ligand-binding sites.

Human neuroglobin (Ngb) contains a heme group and three Cys residues (Cys46, Cys55, and Cys120) in the polypeptide chain. By introducing an additional Cys at position 15, the X-ray structure of A15C Ngb mutant was solved at a high resolution of 1.35 Å, which reveals the formation of both the native (C46C55) and the engineered (C15C120) disulfide bonds, likely playing a functional and structural role, respectively, according to the geometry analysis. Unexpectedly, 1,4-dioxane from the crystallization reagents was bound not only to the protein surface, but also to the heme distal pocket, providing insights into protein-ligand interactions for the globin and guiding the design of functional heme enzymes.

Inhibitory investigation of niacin derivatives on metalloenzyme indoleamine 2,3-dioxygenase 1 for its immunomodulatory function.

Inhibitors of indoleamine 2,3-dioxygenase 1 (IDO1) have received wide attention for their roles in cancer immunotherapy. It highlights the important role of metalloenzymes in performing human physiological functions. Herein, the recombinant human IDO1 was expressed and purified successfully, and the protein molecule was characterized by SDS-PAGE, MALDI-TOF mass spectrometry, and metalloenzymology. A series of niacin derivatives were investigated with regard to their inhibition on metalloenzyme IDO1, and the resulting potential anti-cancer activities in cell lines. Among the niacin derivatives, 4,4,4-trifluoro-1-(pyridin-3-yl)-butane-1,3-dione (compound 9) was found to be the most effective inhibitor to IDO1 in HepG-2 cells, with an EC50 of 11 µM with low cytotoxicity. The IC50 value of compound 9 with trifluoroethyl group in enzymatic inhibition was shown to be ∼5 times more potent than a positive control 4-phenylimidazole. The interaction between compound 9 and IDO1 was verified by isothermal titration calorimetry and molecular docking study. The most favorable molecular docking results revealed that functional groups of compound 9 contributed to the binding of 9 to IDO1 through IDO1-heme coordination, H-bond interactions and hydrophobic contacts. Our finding provides a strategy for the development of new inhibitor candidates for the therapeutic inhibition of IDO1.

Design and Engineering of an Efficient Peroxidase Using Myoglobin for Dye Decolorization and Lignin Bioconversion.

The treatment of environmental pollutants such as synthetic dyes and lignin has received much attention, especially for biotechnological treatments using both native and artificial metalloenzymes. In this study, we designed and engineered an efficient peroxidase using the O2 carrier myoglobin (Mb) as a protein scaffold by four mutations (F43Y/T67R/P88W/F138W), which combines the key structural features of natural peroxidases such as the presence of a conserved His-Arg pair and Tyr/Trp residues close to the heme active center. Kinetic studies revealed that the quadruple mutant exhibits considerably enhanced peroxidase activity, with the catalytic efficiency (kcat/Km) comparable to that of the most efficient natural enzyme, horseradish peroxidase (HRP). Moreover, the designed enzyme can effectively decolorize a variety of synthetic organic dyes and catalyze the bioconversion of lignin, such as Kraft lignin and a model compound, guaiacylglycerol-ß-guaiacyl ether (GGE). As analyzed by HPLC and ESI-MS, we identified several bioconversion products of GGE, as produced via bond cleavage followed by dimerization or trimerization, which illustrates the mechanism for lignin bioconversion. This study indicates that the designed enzyme could be exploited for the decolorization of textile wastewater contaminated with various dyes, as well as for the bioconversion of lignin to produce more value-added products.