Classical antibacterial drugs were designed to target specific bacterial properties distinct from host human cells to maximize potency and selectivity. These designs were quite effective as they could be easily derivatized to bear next... more
Classical antibacterial drugs were designed to target specific bacterial properties distinct from host human cells to maximize potency and selectivity. These designs were quite effective as they could be easily derivatized to bear next generation drugs. However, the rapid mutation of bacteria and their associated acquired drug resistance have led to the rise of highly pathogenic superbug bac-terial strains for which treatment with first line drugs is no match. More than ever, there is a dire need for antibacterial drug design that goes beyond conventional standards. Taking inspiration by the body’s innate immune response to employ its own supply of labile copper ions in a toxic attack against pathogenic bacteria, which have a very low Cu tolerance, this review article examines the feasibility of Cu-centric strategies for antibacterial preventative and therapeutic applications. Promising results are shown for the use of Cu-containing materials in the hospital setting to minimize pati...
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Vector map and the primers used in cloning. (DOCX 149Â kb)
MS/MS spectra of the recombinant Rhizopus cyt c for the peptide corresponding to K72 of yeast, to show absence of trimethylation. (DOCX 51Â kb)
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely... more
An entry from the Cambridge Structural Database, the world's repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Due to the rapid mutation of pathogenic microorganisms, drug-resistant superbugs have evolved. Antimicrobial-resistant germs may share their resistance genes with other germs, making them untreatable. The search for more combative... more
Due to the rapid mutation of pathogenic microorganisms, drug-resistant superbugs have evolved. Antimicrobial-resistant germs may share their resistance genes with other germs, making them untreatable. The search for more combative antibiotic compounds has led researchers to explore metal-based strategies centered on perturbing the bioavailability of essential metals in microbes and examining the therapeutic potential of metal complexes. Given the limited knowledge on the application of titanium(IV), in this work, eight Ti(IV) complexes and some of their corresponding ligands were screened by the Community for Open Antimicrobial Drug Discovery for antimicrobial activity. The compounds were selected for evaluation because of their low cytotoxic/antiproliferative behavior against a human non-cancer cell line. At pH 7.4, these compounds vary in terms of their solution stability and ligand exchange lability; therefore, an assessment of their solution behavior provides some insight regard...
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The recent X-ray structure of titanium(IV)-bound human serum transferrin (STf) exhibiting citrate as a synergistic anion reveals a difference in Ti(IV) coordination versus iron(III), the metal endogenously delivered by the protein to... more
The recent X-ray structure of titanium(IV)-bound human serum transferrin (STf) exhibiting citrate as a synergistic anion reveals a difference in Ti(IV) coordination versus iron(III), the metal endogenously delivered by the protein to cells. This finding enriches our bioinspired drug design strategy for Ti(IV)-based anticancer therapeutics, which applies a family of Fe(III) chelators termed chemical transferrin mimetic (cTfm) ligands to inhibit Fe bioavailability in cancer cells. Deferasirox, a drug used for iron overload disease, is a cTfm ligand that models STf coordination to Fe(III), favoring Fe(III) binding versus Ti(IV). This metal affinity preference drives deferasirox to facilitate the release of cytotoxic Ti(IV) intracellularly in exchange for Fe(III). An aqueous speciation study performed by potentiometric titration from pH 4 to 8 with micromolar levels of Ti(IV) deferasirox at a 1:2 ratio reveals exclusively Ti(deferasirox)2 in solution. The predominant complex at pH 7.4, ...
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Coordination chemistry is a major component of the undergraduate inorganic chemistry curriculum, and yet, the presentation of the material can be cumbersome due to the limitations of the course typically being taught in one semester.... more
Coordination chemistry is a major component of the undergraduate inorganic chemistry curriculum, and yet, the presentation of the material can be cumbersome due to the limitations of the course typically being taught in one semester. Also, because of the large scope of this branch of chemistry encompassing all of the elements, the course design has not been standardized. These factors result in some important coordination chemistry themes being given insufficient development. Herein, we propose a novel activity to formally introduce metal complex aqueous speciation in a holistic active-learning manner that includes a lecture component and a hands-on experience. This topic has real-world relevance and contextualizes many important coordination concepts. It would extend student comprehension about the intricate factors that affect metal complexation in an aqueous solution environment by focusing on the influence of pH. The activity explores the pH dependent speciation of the well-characterized interaction between Fe(III) and 2,3-dihydroxynapthalene-6-sulfonate and reveals the colorful changes in species throughout the pH range 0–13. Students learn how to generate speciation plots and to understand the ultraviolet–visible (UV–Vis) electronic absorption spectroscopy of transition metal compounds to be able to analyze the source of color that they observe. Assessment of the activity was conducted with 24 students who completed a Likert scale survey and responded to open-ended questions. The activity was then applied in actual course settings in which student comprehension was quantitatively evaluated. The activity can be easily adapted to students of different stages of academic development from elementary to college students.
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The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In... more
The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In the cell cytoplasm, it activates intrinsic apoptosis to terminate damaged cells. Insight into these mechanisms and the associated physicochemical properties and biomolecular interactions of Cyt c informs on the anticancer therapeutic potential of the protein, especially in its ability to subvert the current limitations of small molecule-based chemotherapy. In this review, we explore the development of Cyt c as an anticancer drug by identifying cancer types that would be receptive to the cytotoxicity of the protein and factors that can be finetuned to enhance its apoptotic potency. To this end, some information is obtained by characterizing known drugs that operate, in part, by triggering Cyt c induced apoptosis. The application of different smart drug...
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Serum transferrin (sTf) plays a pivotal role in regulating iron biodistribution and homeostasis within the body. The molecular details of sTf Fe(III) binding, blood transport, and cellular delivery through transferrin receptor-mediated... more
Serum transferrin (sTf) plays a pivotal role in regulating iron biodistribution and homeostasis within the body. The molecular details of sTf Fe(III) binding, blood transport, and cellular delivery through transferrin receptor-mediated endocytosis are generally well-understood. Emerging interest exists in exploring sTf complexation of nonferric metals as it facilitates the therapeutic potential and toxicity of several of them. This review explores recent X-ray structural and physiologically relevant metal speciation studies to understand how sTf partakes in the bioactivity of key non-redox active hard Lewis acidic metals. It challenges preconceived notions of sTf structure function correlations that were based exclusively on the Fe(III) model by revealing distinct coordination modalities that nonferric metal ions can adopt and different modes of binding to metal-free and Fe(III)-bound sTf that can directly influence how they enter into cells and, ultimately, how they may impact huma...
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Over time platinum-based anticancer drugs have dominated the market, but their side effects significantly impact the quality of life of patients. Alternative treatments are being developed all over the world. The titanocene and auranofin... more
Over time platinum-based anticancer drugs have dominated the market, but their side effects significantly impact the quality of life of patients. Alternative treatments are being developed all over the world. The titanocene and auranofin families of compounds, discovered through an empirical search for other metal-based therapeutics, hold tremendous promise to improve the outcomes of cancer treatment. Herein we present a historical perspective of these compounds and review current efforts focused on the evolution of their ligands to improve their physiological solution stability, cancer selectivity, and antiproliferative performance, guided by a clear understanding of the coordination chemistry and aqueous speciation of the metal ions, of the cytotoxic mechanism of action of the compounds, and the external factors that limit their therapeutic potential. Newer members of these families of compounds and their combination in novel bimetallic complexes are the result of years of scienti...
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Titanium is one of the most abundant elements in the earth’s crust and while there are many examples of its bioactive properties and use by living organisms, there are few studies that have probed its biochemical reactivity in... more
Titanium is one of the most abundant elements in the earth’s crust and while there are many examples of its bioactive properties and use by living organisms, there are few studies that have probed its biochemical reactivity in physiological environments. In the cosmetic industry, TiO2 nanoparticles are widely used. They are often incorporated in sunscreens as inorganic physical sun blockers, taking advantage of their semiconducting property, which facilitates absorbing ultraviolet (UV) radiation. Sunscreens are formulated to protect human skin from the redox activity of the TiO2 nanoparticles (NPs) and are mass-marketed as safe for people and the environment. By closely examining the biological use of TiO2 and the influence of biomolecules on its stability and solubility, we reassess the reactivity of the material in the presence and absence of UV energy. We also consider the alarming impact that TiO2 NP seepage into bodies of water can cause to the environment and aquatic life, and...
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A very promising direction in the development of anticancer drugs is inhibiting the molecular pathways that keep cancer cells alive and able to metastasize. Copper and iron are two essential metals that play significant roles in the rapid... more
A very promising direction in the development of anticancer drugs is inhibiting the molecular pathways that keep cancer cells alive and able to metastasize. Copper and iron are two essential metals that play significant roles in the rapid proliferation of cancer cells and several chelators have been studied to suppress the bioavailability of these metals in the cells. This review discusses the major contributions that Cu and Fe play in the progression and spreading of cancer and evaluates select Cu and Fe chelators that demonstrate great promise as anticancer drugs. Efforts to improve the cellular delivery, efficacy, and tumor responsiveness of these chelators are also presented including a transmetallation strategy for dual targeting of Cu and Fe. To elucidate the effectiveness and specificity of Cu and Fe chelators for treating cancer, analytical tools are described for measuring Cu and Fe levels and for tracking the metals in cells, tissue, and the body.
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Despite its natural abundance and widespread use as food, paint additive, and in bone implants, no specific biological function of titanium is known in the human body. High concentrations of Ti(IV) could result in cellular toxicity,... more
Despite its natural abundance and widespread use as food, paint additive, and in bone implants, no specific biological function of titanium is known in the human body. High concentrations of Ti(IV) could result in cellular toxicity, however, the absence of Ti toxicity in the blood of patients with titanium bone implants indicates the presence of one or more biological mechanisms to mitigate toxicity. Similar to Fe(III), Ti(IV) in blood binds to the iron transport protein serum transferrin (sTf), which gives credence to the possibility of its cellular uptake mechanism by transferrin-directed endocytosis. However, once inside the cell, how sTf bound Ti(IV) is released into the cytoplasm, utilized, or stored remain largely unknown. To explain the molecular mechanisms involved in Ti use in cells we have drawn parallels with those for Fe(III). Based on its chemical similarities with Fe(III), we compare the biological coordination chemistry of Fe(III) and Ti(IV) and hypothesize that Ti(IV...
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A water-soluble octanuclear cluster, [Fe], was studied with regard to its properties as a potential contrast enhancing agent in magnetic resonance imaging (MRI) in magnetic fields of 1.3, 7.2 and 11.9 T and was shown to have transverse... more
A water-soluble octanuclear cluster, [Fe], was studied with regard to its properties as a potential contrast enhancing agent in magnetic resonance imaging (MRI) in magnetic fields of 1.3, 7.2 and 11.9 T and was shown to have transverse relaxivities r = 4.01, 10.09 and 15.83 mM s, respectively. A related hydrophobic [Fe] cluster conjugated with 5 kDa hyaluronic acid (HA) was characterized by Fe-Mössbauer and MALDI-TOF mass spectroscopy, and was evaluated in aqueous solutions in vitro with regard to its contrast enhancing properties [r = 3.65 mM s (1.3 T), 26.20 mM s (7.2 T) and 52.18 mM s (11.9 T)], its in vitro cellular cytotoxicity towards A-549 cells and COS-7 cells and its in vivo enhancement of T-weighted images (4.7 T) of a human breast cancer xenografted on a nude mouse. The physiologically compatible [Fe]-HA conjugate was i.v. injected to the tumor-bearing mouse, resulting in observable, heterogeneous signal change within the tumor, evident 15 min after injection and persisti...
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The first total synthesis of -phenyl 6 fatty acids (FA) and their cytotoxicity (A549) and leishmanicidal (L. infantum) activities are described. The novel 16-phenyl-6-hexadecynoic acid (1) and the known 16-phenylhexadecanoic acid (2)... more
The first total synthesis of -phenyl 6 fatty acids (FA) and their cytotoxicity (A549) and leishmanicidal (L. infantum) activities are described. The novel 16-phenyl-6-hexadecynoic acid (1) and the known 16-phenylhexadecanoic acid (2) were synthesized in 7-8 steps with overall yields of 46 % and 41 %, respectively. The syntheses of the unprecedented 10-phenyl-6-decynoic acid (3), 10-cyclohexyl-6-decynoic acid (4) and 10-(4-methoxyphenyl)-6-decynoic acid (5) was also performed in 3 steps with 73-76 % overall yields. The use of lithium acetylide coupling enabled the 4-step synthesis of 10-phenyl-6Z-decenoic acid (6) with a 100 % cis-stereochemistry. The cytotoxicity of these novel FA was determined against A549 cells and L. infantum promastigotes and amastigotes. Among the -phenylated FA, the best cytotoxicity towards A549 was displayed by 1, with an IC50 of 18 ± 1 µM. On the other hand, among the C10 acids, the -cyclohexyl acid 4 presented the best cytotoxicity (IC50 = 40 ± 2 µM) t...
One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a... more
One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC50 value similar to the well known dru...
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Drug development is a decades-long, multibillion dollar investment that often limits itself. To decrease the time to drug approval, efforts are focused on drug targets and drug formulation for optimal biocompatibility and efficacy. X-ray... more
Drug development is a decades-long, multibillion dollar investment that often limits itself. To decrease the time to drug approval, efforts are focused on drug targets and drug formulation for optimal biocompatibility and efficacy. X-ray structural characterization approaches have catalyzed the drug discovery and design process. Single crystal X-ray diffraction (SCXRD) reveals important structural details and molecular interactions for the manifestation of a disease or for therapeutic effect. Powder X-ray diffraction (PXRD) has provided a method to determine the different phases, purity, and stability of biological drug compounds that possess crystallinity. Recently, synchrotron sources have enabled wider access to the study of noncrystalline or amorphous solids. One valuable technique employed to determine atomic arrangements and local atom ordering of amorphous materials is the pair distribution function (PDF). PDF has been used in the study of amorphous solid dispersions (ASDs). ...
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Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies... more
Human serum transferrin (sTf) is a protein that mediates the transport of iron from blood to cells. Assisted by the synergistic anion carbonate, sTf transports Fe(III) by binding the metal ion in a closed conformation. Previous studies suggest sTf's role as a potential transporter of other metals such as titanium. Ti is a widely used metal in colorants, foods, and implants. A substantial amount of Ti is leached into blood from these implants. However, the fate of the leached Ti and its transport into the cells is not known. Understanding Ti interaction with sTf assumes a greater significance with our ever increasing exposure to Ti in the form of implants. On the basis of in vitro studies, it was speculated that transferrin can bind Ti(IV) assisted by a synergistic anion. However, the role and identity of the synergistic anion(s) and the conformational state in which sTf binds Ti(IV) are not known. Here we have solved the first X-ray crystal structure of a Ti(IV)-bound sTf. We fi...
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Despite the ubiquitous nature of titanium(iv) and several examples of its beneficial behavior in different organisms, the metal remains underappreciated in biology. There is little understanding of how the metal might play an important... more
Despite the ubiquitous nature of titanium(iv) and several examples of its beneficial behavior in different organisms, the metal remains underappreciated in biology. There is little understanding of how the metal might play an important function in the human body. Nonetheless, a new insight is obtained regarding the molecular mechanisms that regulate the blood speciation of the metal to maintain it in a nontoxic and potentially bioavailable form for use in the body. This review surveys the literature on Ti(iv) application in prosthetics and in the development of anticancer therapeutics to gain an insight into soluble Ti(iv) influx in the body and its long-term impact. The limitation in analytical tools makes it difficult to depict the full picture of how Ti(iv) is transported and distributed throughout the body. An improved understanding of Ti function and its interaction with biomolecules will be helpful in developing future technologies for its imaging in the body.
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Serum albumin, the most abundant protein in human plasma (700 microM), binds diverse ligands at multiple sites. While studies have shown that serum albumin binds hard metals in chelate form, few have explored the trafficking of these... more
Serum albumin, the most abundant protein in human plasma (700 microM), binds diverse ligands at multiple sites. While studies have shown that serum albumin binds hard metals in chelate form, few have explored the trafficking of these metals by this protein. Recent work demonstrated that serum albumin may play a pivotal role in the transport and bioactivity of titanium(IV) complexes, including the anticancer drug candidate titanocene dichloride. The current work explores this interaction further by using a stable Ti(IV) complex that presents a hydrophobic surface to the protein. Ti(IV) chelation by 2,3-dihydroxynaphthalene (H2L1) and 2,3-dihydroxynaphthalene-6-sulfonate (H2L2) affords water soluble complexes that protect Ti(IV) from hydrolysis at pH 7.4 and bind to bovine serum albumin (BSA). The solution and solid Ti(IV) coordination chemistry were explored by aqueous spectropotentiometric titrations and X-ray crystallography, respectively, and with complementary electrochemistry, mass spectrometry, and IR and NMR spectroscopies. Four Ti(IV) species of L2, TiLH0, TiL2H0, TiL3H0, and TiL3H(-1), adequately represent the pH-dependent speciation. The isolation of Ti(C10H6O2)2 x 1.75 H2O at pH approximately 3 and K2[Ti(C10H6O2)3] x 3 H2O and Cs5[Ti(C10H5O5S)3] x 2.5 H2O at pH approximately 7 correlates well with the solution studies. At pH 7.4 and micromolar concentrations, the TiL3H0 species are favored. The Ti(naphthalene-2,3-diolate)3(2-) complex binds with moderate affinity to multiple sites of BSA. The primary site (K = 2.05 +/- 0.34 x 10(6) M(-1)) appears to be hydrophobic as indicated by competition studies with different ligands and a hydrophilic Ti(IV) complex. The Ti(naphthalene-2,3-diolate)3(2-) interaction with the Fe(III)-binding protein human serum transferrin (HsTf), a protein also important for Ti(IV) transport, and DNA was examined. The complex does not deliver Ti(IV) to HsTf and while it does bind to DNA, no cleavage promotion activity is observed. This investigation provides insight into the use of ligands to direct metal binding at different sites of albumin, which may facilitate transport to distinct targets.
Research Interests: Chemical Engineering, Inorganic Chemistry, Water, Titanium, Inorganic, and 13 moreMacromolecular X-Ray Crystallography, DNA, Humans, Animals, Transferrin, Cattle, Bovine Serum Albumin, Protein Binding, Chelating Agents, Hydrolysis, Organometallic Compounds, Hydrogen-Ion Concentration, and Serum albumin
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Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin,... more
Transferrins are a family of proteins that bind and transport Fe(III). Modern transferrins are typically bilobal and are believed to have evolved from an ancient gene duplication of a monolobal form. A novel monolobal transferrin, nicatransferrin (nicaTf), was identified in the primitive ascidian species Ciona intestinalis that possesses the characteristic features of the proposed ancestral Tf protein. In this work, nicaTf was expressed in Pichia pastoris. Extensive solution studies were performed on nicaTf, including UV-vis, fluorescence, CD, EPR and NMR spectroscopies, and electrospray time-of-flight mass spectrometry. The expressed protein is nonglycosylated, unlike the protein isolated from the organism. This property does not affect its ability to bind Fe(III). However, Fe(III)-bound nicaTf displays important spectral differences from other Fe(III)-bound transferrins, which are likely the consequence of differences in metal coordination. Coordination differences could also acco...
Research Interests: Molecular Evolution, Kinetics, NMR Spectroscopy, Multidisciplinary, Time-of-flight mass spectrometry, and 12 moreHigher Order Thinking, Protein Structure and Function, Animals, Transferrin, Iron, Pichia pastoris, Gene Duplication, Molecular cloning, Spectrum analysis, Human Serum, Ciona Intestinalis, and Protein Binding
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Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons.... more
Substance P (SP) is a prototypical neuropeptide with roles in pain and inflammation. Numerous mechanisms regulate endogenous SP levels, including the differential expression of SP mRNA and the controlled secretion of SP from neurons. Proteolysis has long been suspected to regulate extracellular SP concentrations but data in support of this hypothesis is scarce. Here, we provide evidence that proteolysis controls SP levels in the spinal cord. Using peptidomics to detect and quantify endogenous SP fragments, we identify the primary SP cleavage site as the C-terminal side of the ninth residue of SP. If blocking this pathway increases SP levels, then proteolysis controls SP concentration. We performed a targeted chemical screen using spinal cord lysates as a proxy for the endogenous metabolic environment and identified GM6001 (galardin, ilomastat) as a potent inhibitor of the SP(1-9)-producing activity present in the tissue. Administration of GM6001 to mice results in a greater-than-three-fold increase in the spinal cord levels of SP, which validates the hypothesis that proteolysis controls physiological SP levels.
Research Interests: Biochemistry, Chemistry, Analytical Chemistry, Metabolism, Biotechnology, and 21 moreMass Spectrometry, Biology, Neurochemistry, Proteomics, Enzyme Inhibitors, Model organisms, Chemical Biology, Multidisciplinary, Biocatalysis, Mice, Female, Animals, Spinal Cord, Hormones, Proteins, PLoS one, Small Molecules, Animal models, Substance P, Amino Acid Sequence, and Metalloproteases
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Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by... more
Evidence suggests that transferrin can bind Ti(IV) in an unhydrolyzed form (without bound hydroxide or oxide) or in a hydrolyzed form. Ti(IV) coordination by N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) at different pH values models the two forms of Ti(IV)-loaded transferrin spectrally and structurally. 13C NMR and stopped-flow kinetic experiments reveal that when the metal is delivered to the protein using an unhydrolyzed source, Ti(IV) can coordinate in the typical distorted octahedral environment with a bound synergistic anion. The crystal structure of TiHBED obtained at low pH models this type of coordination. The solution structure of the complex compares favorably with the solid state from pH 3.0 to 4.0, and the complex can be reduced with E1/2 = -641 mV vs NHE. Kinetic and thermodynamic competition studies at pH 3.0 reveal that Ti(citrate)3 reacts with HBED via a dissociative mechanism and that the stability of TiHBED (log beta = 34.024) is weaker than that of the Fe(III) complex. pH stability studies show that Ti(IV) hydrolyzes ligand waters at higher pH but still remains bound to HBED until pH 9.5. Similarly, at a pH greater than 8.0 the synergistic anion that binds Ti(IV) in transferrin is readily displaced by irreversible metal hydrolysis although the metal remains bound to the protein until pH 9.5. Thermal denaturation studies conducted optically and by differential scanning calorimetry reveal that Ti(IV)-bound transferrin experiences only minimal enhanced thermal stability unlike when Fe(III) is bound. The C- and N-lobe transition Tm values shift to a few degrees higher. The stability, competition, and redox studies performed provide insight into the possible mechanism of Ti2-Tf transport in cells.
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The trafficking of titanium(IV) by human serum transferrin (HsTf) has been implicated in the physiology of this hydrolysis-prone metal. The current work broadens to include the further interactions of Ti(IV) in serum that bear on this... more
The trafficking of titanium(IV) by human serum transferrin (HsTf) has been implicated in the physiology of this hydrolysis-prone metal. The current work broadens to include the further interactions of Ti(IV) in serum that bear on this model. Ti2HsTf (2 equiv) binds the transferrin receptor TfR1 with Kd1 = 6.3 +/- 0.4 nM and Kd2 = 410 +/- 150 nM, values that are the tightest yet measured for a metal other than iron but weaker than the corresponding ones for Fe2HsTf due to both slightly slower on rates and slightly faster off rates. Comparing the affinities of metals for HsTf with the affinities of the resulting M2HsTf species for TfR1, we speculate that the formation of an M2HsTf complex of high affinity may predict a lobe-closed conformation that leads to a favorable interaction with TfR1. Human serum albumin (HSA), an important serum competitor for metal binding, can bind up to 20 equiv of Ti(IV) supplied in several forms. With some ligands, Ti(IV) may bind to the N-terminal metal binding site of albumin, forming a ternary complex. However, the dominant type of HSA binding is via Ti(IV) in complex form, probably at surface sites. Notably, HSA greatly stabilizes the titanocene moiety of the drug candidate Cp2TiCl2 with respect to hydrolysis and precipitation. HSA binds Ti(IV) citrate supplied as a hydrolyzed or unhydrolyzed source, with 1 equiv of citrate remaining bound. Titanium(IV) monocitrate neither competes with the binding of reporter molecules known to dock at canonical drug sites I or II nor binds at the N-terminus. HsTf outcompetes HSA for soluble Ti(IV) in a direct competition, but once bound to albumin, the transfer of Ti(IV) from HSA to HsTf is quite slow. Each of these findings has implications for the metabolism of Ti(IV) in human serum.
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As an endogenous serum protein binder of Ti(IV), transferrin (Tf) serves as an excellent vehicle to stabilize the hydrolysis prone metal ion and successfully transport it into cells. This transporting role is thought to be central to... more
As an endogenous serum protein binder of Ti(IV), transferrin (Tf) serves as an excellent vehicle to stabilize the hydrolysis prone metal ion and successfully transport it into cells. This transporting role is thought to be central to Ti(IV)'s anticancer function, but efforts to synthesize Ti(IV) compounds targeting transferrin have not produced a drug. Nonetheless, the Ti(IV) transferrin complex (Ti2Tf) greatly informs on a new Ti(IV)-based anticancer drug design strategy. Ti2Tf interferes with cellular uptake of Fe(III), which is particularly detrimental to cancer cells because of their higher requirement for iron. Ti(IV) compounds of chemical transferrin mimetic (cTfm) ligands were designed to facilitate Ti(IV) activity by attenuating Fe(III) intracellular levels. In having a higher affinity for Fe(III) than Ti(IV), these ligands feature the appropriate balance between stability and lability to effectively transport Ti(IV) into cancer cells, release Ti(IV) via displacement by Fe(III), and deplete the intracellular Fe(III) levels. The cTfm ligand N,N'-di(o-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid (HBED) was selected to explore the feasibility of the design strategy. Kinetic studies on the Fe(III) displacement process revealed that Ti(IV) can be transported and released into cells by HBED on a physiologically relevant time scale. Cell viability studies using A549 cancerous and MRC5 normal human lung cells and testing the cytotoxicity of HBED and its Ti(IV), Fe(III), and Ga(III) compounds demonstrate the importance of Fe(III) depletion in the proposed drug design strategy and the specificity of the strategy for Ti(IV) activity. The readily derivatized cTfm ligands demonstrate great promise for improved Ti(IV) anticancer drugs.
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Serum albumin, the most abundant protein in human plasma (700 microM), binds diverse ligands at multiple sites. While studies have shown that serum albumin binds hard metals in chelate form, few have explored the trafficking of these... more
Serum albumin, the most abundant protein in human plasma (700 microM), binds diverse ligands at multiple sites. While studies have shown that serum albumin binds hard metals in chelate form, few have explored the trafficking of these metals by this protein. Recent work demonstrated that serum albumin may play a pivotal role in the transport and bioactivity of titanium(IV) complexes, including the anticancer drug candidate titanocene dichloride. The current work explores this interaction further by using a stable Ti(IV) complex that presents a hydrophobic surface to the protein. Ti(IV) chelation by 2,3-dihydroxynaphthalene (H2L1) and 2,3-dihydroxynaphthalene-6-sulfonate (H2L2) affords water soluble complexes that protect Ti(IV) from hydrolysis at pH 7.4 and bind to bovine serum albumin (BSA). The solution and solid Ti(IV) coordination chemistry were explored by aqueous spectropotentiometric titrations and X-ray crystallography, respectively, and with complementary electrochemistry, mass spectrometry, and IR and NMR spectroscopies. Four Ti(IV) species of L2, TiLH0, TiL2H0, TiL3H0, and TiL3H(-1), adequately represent the pH-dependent speciation. The isolation of Ti(C10H6O2)2 x 1.75 H2O at pH approximately 3 and K2[Ti(C10H6O2)3] x 3 H2O and Cs5[Ti(C10H5O5S)3] x 2.5 H2O at pH approximately 7 correlates well with the solution studies. At pH 7.4 and micromolar concentrations, the TiL3H0 species are favored. The Ti(naphthalene-2,3-diolate)3(2-) complex binds with moderate affinity to multiple sites of BSA. The primary site (K = 2.05 +/- 0.34 x 10(6) M(-1)) appears to be hydrophobic as indicated by competition studies with different ligands and a hydrophilic Ti(IV) complex. The Ti(naphthalene-2,3-diolate)3(2-) interaction with the Fe(III)-binding protein human serum transferrin (HsTf), a protein also important for Ti(IV) transport, and DNA was examined. The complex does not deliver Ti(IV) to HsTf and while it does bind to DNA, no cleavage promotion activity is observed. This investigation provides insight into the use of ligands to direct metal binding at different sites of albumin, which may facilitate transport to distinct targets.
Research Interests: Chemical Engineering, Inorganic Chemistry, Water, Titanium, Inorganic, and 13 moreMacromolecular X-Ray Crystallography, DNA, Humans, Animals, Transferrin, Cattle, Bovine Serum Albumin, Protein Binding, Chelating Agents, Hydrolysis, Organometallic Compounds, Hydrogen-Ion Concentration, and Serum albumin
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The prolyl peptidases are a family of enzymes characterized by a biochemical preference for cleaving proline-containing peptides. The members of this enzyme family include prolyl endopeptidase, prolyl endopeptidase-like, dipeptidyl... more
The prolyl peptidases are a family of enzymes characterized by a biochemical preference for cleaving proline-containing peptides. The members of this enzyme family include prolyl endopeptidase, prolyl endopeptidase-like, dipeptidyl peptidase 4 (DPP4), DPP7, DPP8, DPP9, and fibroblast activation protein. DPP4 is the best studied member of the family, due to its role in physiological glucose tolerance, exerted through the regulation of the insulinotropic peptide glucagon-like peptide-1. While other members of the prolyl peptidase family have also been implicated in various (patho)physiological processes, the underlying peptides and pathways regulated by these enzymes are less clear. The identification of endogenous substrates of the prolyl peptidases is an important step in elucidating the molecular mechanisms of these enzymes. Here, we highlight the utility of liquid chromatography-mass spectrometry-based peptidomics to enable the discovery of endogenous prolyl peptidase substrates directly from tissues, and demonstrate the utility of this information in understanding the biochemical and physiological functions of the prolyl peptidases.