Martha Sibrian-Vazquez

Homocystamides promote free-radical and oxidative damage to proteins

Elevated levels of homocysteine are associated with several major diseases. However, it is not clear whether homocysteine is a marker or a causative agent. The majority (ca. 80%) of the homocysteine present in humans is protein bound. The study of the posttranslational modification of proteins by homocysteine and its cyclic congener, homocysteine thiolactone, is emerging as an area of great current interest for unraveling the ongoing “mediator/marker controversy” [Jacobsen DW (2009) Clin Chem 55:1–2]. Interestingly, many of the pathologies associated with homocysteine are also linked to oxidative stress. In the current study, chemical evidence for a causal relationship between homocysteine-bound proteins and oxidative damage is presented. For example, a reproducible increase in protein carbonyl functionality occurs as a consequence of the reaction of human serum albumin with homocysteine thiolactone. This occurs at physiological temperature upon exposure to air without any added oxidants or free-radical initiators. Alpha-amino acid carbon-centered radicals, well-known precursors of protein carbonyls, are shown to form via a hydrogen atom transfer process involving thiolactone-derived homocystamides. Model peptides in buffer as well as native proteins in human blood plasma additionally exhibit properties in keeping with the homocystamide-facilitated hydrogen atom transfer and resultant carbon-centered radicals.

Seminaphthofluorones are a family of water-soluble, low molecular weight, NIR-emitting fluorophores

A readily accessible new class of near infrared (NIR) molecular probes has been synthesized and evaluated. Specific fluorophores in this unique xanthene based regioisomeric seminaphthofluorone dye series exhibit a combination of desirable characteristics including (i) low molecular weight (339 amu), (ii) aqueous solubility, and (iii) dual excitation and emission from their fluorescent neutral and anionic forms. Importantly, systematic changes in the regiochemistry of benzannulation and the ionizable moieties afford (iv) tunable deep-red to NIR emission from anionic species and (v) enhanced Stokes shifts. Anionic SNAFR-6, exhibiting an unusually large Stokes shift of ≈200 nm (5,014 cm−1) in aqueous buffer, embodies an unprecedented fluorophore that emits NIR fluorescence when excited in the blue/green wavelength region. The successful use of SNAFR-6 in cellular imaging studies demonstrates proof-of-concept that this class of dyes possesses photophysical characteristics that allow their use in practical applications. Notably, each of the new fluorophores described is a minimal template structure for evaluation of their basic spectral properties, which may be further functionalized and optimized yielding concomitant improvements in their photophysical properties.

Synthesis, Characterization, and Metabolic Stability of Porphyrin−Peptide Conjugates Bearing Bifunctional Signaling Sequences

A series of four porphyrin-peptide conjugates bearing one linear bifunctional sequence containing a cell penetrating peptide (CPP) and a nuclear localization signal (NLS) were synthesized and their in vitro biological and stability properties investigated. All conjugates accumulated within human HEp2 cells to a significantly higher extent than their porphyrin-PEG precursor, and the extent of their uptake and cytotoxicity depends on the nature and sequence of the amino acids. Conjugates 2 and 5 bearing a NLS-CPP accumulated the most within cells and were the most phototoxic (IC50 approximately 7 microM at 1 J/cm2). All conjugates localized preferentially within the cell lysosomes, and in addition, conjugate 2 was also found in the ER. All conjugates were highly stable under nonenzymatic conditions, but their peptide sequences were cleaved to some extent (ca. 50% after 24 h) by proteolytic enzymes, such as cathepsin B, cathepsin D, prolidase, and plasmin.

Mitochondria targeting by guanidine- and biguanidine-porphyrin photosensitizers.

We report the syntheses of three new amphiphilic porphyrin derivatives, containing a guanidine, a biguanidine, or an MLS peptide, that were designed to target the cell mitochondria. The guanidine- and biguanidine-porphyrins are poorly soluble in water, forming J-type aggregates in aqueous solutions. On the other hand, the porphyrin-MLS peptide conjugate bearing a low molecular weight PEG spacer is highly water-soluble and does not aggregate in aqueous media. The fluorescence quantum yields determined for all porphyrins were higher at low pH (<6) and the porphyrin-peptide conjugate had the highest quantum yields in aqueous media. All porphyrins showed low dark toxicity toward human carcinoma HEp2 cells, and the guanidine-porphyrin was the most phototoxic (IC 50 = 4.8 microM at 1 J cm (-2)), followed by the biguanidine-porphyrin and the porphyrin-MLS (IC50 = 8.2 microM and 9.8 microM at 1 J cm (-2), respectively). The porphyrin-MLS peptide conjugate accumulated the most within cells of all porphyrins at all times investigated and the biguanidine-porphyrin accumulated the least. Both the guanidine- and biguanidine-porphyrins localized within cell mitochondria and, in addition, were found in the lysosomes and the ER (in the case of the guanidine-porphyrin). In contrast, the porphyrin-MLS peptide conjugate localized mainly within the cell lysosomes.

Photoinduced cytotoxicity and biodistribution of prostate cancer cell-targeted porphyrins.

A series of five porphyrin-peptide conjugates bearing one or two sequences containing a cell penetrating peptide (CPP), a nuclear localization signal (NLS), or a bifunctional CPP-NLS or NLS-CPP sequences were synthesized and investigated in vitro using PC-3M human prostate cancer cells, in comparison with FDA-approved purified hematoporphyrin derivative (Porfimer Sodium) and mTHPC. The most promising porphyrin-HIV-1 Tat (48-60) conjugate 2 [lowest dark cytotoxicity (IC50 = 38.0 microM), highest phototoxicity (IC50 = 0.40 microM at 1 J/cm2)] was further evaluated in an in vivo biodistribution study using SCID mice bearing PC-3M tumors, in comparison with purified hematoporphyrin derivative. Porphyrin conjugate 2 was more tumor selective than the hematoporphyrin derivative and accumulated to a significantly greater extent in tumors. Our results show that effective photodynamic cytotoxicity can be induced in human prostate cancer cells with minimal dark toxicity and that selective accumulation in prostate tumors can be achieved in vivo with porphyrin-targeted photosensitizers.

Field Effects Induce Bathochromic Shifts in Xanthene Dyes

There is ongoing interest in near-infrared (NIR) absorbing and emitting dyes for a variety of biomedical and materials applications. Simple and efficient synthetic procedures enable the judicious tuning of through-space polar (field) effects as well as low barrier hydrogen bonding to modulate the HOMO-LUMO gap in xanthene dyes. This affords unique NIR-absorbing xanthene chromophores.

Exploring the pH dependence of viologen reduction by α-carbon radicals derived from Hcy and Cys

The colorimetric reaction of homocysteine (HCy) with a series of viologen salts suggests a linear correlation between the mid-point reduction potential of Hcy-derived alpha-carbon radicals and pH.

Influence of the number and distribution of NLS peptides on the photosensitizing activity of multimeric porphyrin–NLS

Porphyrin-peptide conjugates bearing multiple nuclear localization sequences (NLS) could show increased tumor cell uptake and affinity for nuclear receptors, and consequently increased photodynamic activity. Previous studies suggest that an increase number of NLS might enhance the nuclear uptake of proteins and other macromolecules. We report the syntheses and investigation of a series of multimeric porphyrin-NLS conjugates bearing two, three or four peptides with the minimum sequence PKKKRKV, linked via PEG or 5-carbon linkers, and with different distributions at the porphyrin periphery. Our results show that the tumor cell uptake and phototoxicity of these conjugates is mainly determined by their amphiphilic character, and not the number of NLS residues per molecule, contrary to previous studies. The mono- and di-substituted photosensitizers bearing one or two PEG linkers and up to three peptide sequences were found to be the most phototoxic toward human carcinoma HEp2 cells, while the tetra-NLS conjugates symmetrically substituted around the porphyrin ring accumulated the least within cells and were non-phototoxic. All conjugates localized intracellularly within endosomal vesicles and lysosomes, probably as a result of an endocytic mechanism of uptake; as a consequence no nuclear uptake was detected by fluorescence microscopy.

Identification of Cisplatin-Binding Proteins Using Agarose Conjugates of Platinum Compounds

Cisplatin is widely used as an antineoplastic drug, but its ototoxic and nephrotoxic side-effects, as well as the inherent or acquired resistance of some cancers to cisplatin, remain significant clinical problems. Cisplatins selectivity in killing rapidly proliferating cancer cells is largely dependent on covalent binding to DNA via cisplatins chloride sites that had been aquated. We hypothesized that cisplatins toxicity in slowly proliferating or terminally differentiated cells is primarily due to drug-protein interactions, instead of drug-DNA binding. To identify proteins that bind to cisplatin, we synthesized two different platinum-agarose conjugates, one with two amino groups and another with two chlorides attached to platinum that are available for protein binding, and conducted pull-down assays using cochlear and kidney cells. Mass spectrometric analysis on protein bands after gel electrophoresis and Coomassie blue staining identified several proteins, including myosin IIA, glucose-regulated protein 94 (GRP94), heat shock protein 90 (HSP90), calreticulin, valosin containing protein (VCP), and ribosomal protein L5, as cisplatin-binding proteins. Future studies on the interaction of these proteins with cisplatin will elucidate whether these drug-protein interactions are involved in ototoxicity and nephrotoxicity, or contribute to tumor sensitivity or resistance to cisplatin treatment.

Dimethyl Sulfoxide (DMSO) Exacerbates Cisplatin-induced Sensory Hair Cell Death in Zebrafish (Danio rerio)

Inner ear sensory hair cells die following exposure to aminoglycoside antibiotics or chemotherapeutics like cisplatin, leading to permanent auditory and/or balance deficits in humans. Zebrafish (Danio rerio) are used to study drug-induced sensory hair cell death since their hair cells are similar in structure and function to those found in humans. We developed a cisplatin dose-response curve using a transgenic line of zebrafish that expresses membrane-targeted green fluorescent protein under the control of the Brn3c promoter/enhancer. Recently, several small molecule screens have been conducted using zebrafish to identify potential pharmacological agents that could be used to protect sensory hair cells in the presence of ototoxic drugs. Dimethyl sulfoxide (DMSO) is typically used as a solvent for many pharmacological agents in sensory hair cell cytotoxicity assays. Serendipitously, we found that DMSO potentiated the effects of cisplatin and killed more sensory hair cells than treatment with cisplatin alone. Yet, DMSO alone did not kill hair cells. We did not observe the synergistic effects of DMSO with the ototoxic aminoglycoside antibiotic neomycin. Cisplatin treatment with other commonly used organic solvents (i.e. ethanol, methanol, and polyethylene glycol 400) also did not result in increased cell death compared to cisplatin treatment alone. Thus, caution should be exercised when interpreting data generated from small molecule screens since many compounds are dissolved in DMSO.