High-throughput assays for botulinum neurotoxin proteolytic activity: serotypes A, B, D, and F.

Botulinum neurotoxins (BoNT) are zinc metalloproteases that cleave and inactivate cellular proteins essential for neurotransmitter release. Because the paralytic effect of BoNT is a consequence of its enzymatic activity, selective inhibitors may be useful as drugs or as tools for further research. To expedite inhibitor discovery, we developed high-throughput, solid-phase protease activity assays for four of the seven BoNT serotypes: A, B, D, and F. Each assay consisted of a cleavable oligopeptide, based on the natural substrate sequence, labeled with fluorescein and covalently attached to maleimide-activated multiwell plates. Solutions of holotoxin or nontoxic catalytic domain of BoNT were incubated in substrate-coated wells, with or without test compounds, followed by transfer and assay of solubilized product in a multiwell fluorometer. Routine toxin concentrations ranged from 10 to 100 ng/ml, but concentrations as low as 2 ng/ml gave reproducible signals. The fluorescence assays were selective, gave very low background readings, and were stable upon prolonged storage. Using the nontoxic catalytic domain of BoNT A, we determined the relative inhibitory potencies of a family of structurally related pseudotripeptide compounds. Unlike previous methods, our assays did not employ antibodies or reverse-phase extraction steps, only well-to-well transfers, and were easily adapted to a high-throughput automated environment.

Type A botulinum neurotoxin proteolytic activity: development of competitive inhibitors and implications for substrate specificity at the S1' binding subsite.

Type A botulinum neurotoxin (botox A) is a zinc metalloprotease that cleaves only one peptide bond in the synaptosomal protein, SNAP-25. Single-residue changes in a 17-residue substrate peptide were used to develop the first specific, competitive inhibitors of its proteolytic activity. Substrate analog peptides with P4, P3, P2' or P3' cysteine were readily hydrolyzed by the toxin, but those with P1 or P2 cysteine were not cleaved and were inhibitors. Peptides with either D- or L-cysteine as the N-terminus, followed by the last six residues of the substrate, were the most effective inhibitors, each with a Ki value of 2 microM. Elimination of the cysteine sulfhydryl group yielded much less effective inhibitors, suggesting that inhibition was primarily due to binding of the active-site zinc by the sulfhydryl group. Botox A displayed an unusual requirement for arginine as the P1' inhibitor residue, demonstrating that the S1' binding subsite of botox A is dissimilar to those of most other zinc metalloproteases. This characteristic is an important element in shaping the substrate specificity of botox A.

Urinary elimination of saxitoxin after intravenous injection.

Paralytic shellfish poisoning is a serious public health concern throughout the world. An analytical method with diagnostic potential was used to isolate and measure saxitoxin, the most potent and studied paralytic shellfish poisoning toxin, in the urine of rats injected i.v. with sublethal doses (2 micrograms/kg) of saxitoxin. Urine was collected at intervals between 4 and 144 hr after injection. Saxitoxin was isolated from urine with an ion-exchange procedure, identified, and measured with a precolumn-oxidation-HPLC procedure coupled with fluorescence detection. The identity of oxidized saxitoxin was confirmed with electrospray ionization mass spectrometry. Four hours after injection, approximately 19% of the injected saxitoxin dose was excreted. By 24 hr, approximately 58% of the administered dose was excreted. Average total urinary excretion of administered saxitoxin was approximately 68% for the full study period. These results demonstrate that small quantities of unmetabolized saxitoxin can be detected in rat urine up to 144 hr after i.v. administration, and that the analytical method may have diagnostic potential for saxitoxin intoxication and paralytic shellfish poisoning.

Method for the identification of saxitoxin in rat urine.

Saxitoxin (STX) is one of several related toxins that cause paralytic shellfish poisoning. We used solid-phase extraction (SPE) and prechromatographic oxidation/HPLC with fluorescence detection to isolate, identify, and quantify STX in rat urine. STX recovery from urine with the SPE procedure was approximately 76 +/- 6.5%. The standard curve was linear between 2 and 50 ng/ml. The lower limit of quantification with the method was 2 ng STX/ml of rat urine. Preliminary results with i.v. administration of STX to rats demonstrated that this method can detect and quantify STX in urine.

Comparison of the partition coefficient and skin penetration of a marine algal toxin (lyngbyatoxin A).

Lyngbyatoxin A is produced by marine algae, and causes local cutaneous toxicity in swimmers. The purpose of this research was (1) to determine the partition coefficient of lyngbyatoxin A in octanol/water and (2) to use methods in vitro to measure the penetration and distribution of lyngbyatoxin A in guinea pig and human skin. Discs of excised guinea pig and human skin were mounted in diffusion chambers that exposed the epidermal surface to air and bathed the dermis with HEPES-buffered Hanks' balanced salt solution with gentamicin sulphate. The epidermal surfaces were dosed with 26 micrograms lyngbyatoxin A/cm2 dissolved in 13 microliters dimethyl sulphoxide/cm2. The diffusion chambers were incubated at 36 degrees C for varying periods (1.0-24 hr). HPLC was used to quantify lyngbyatoxin A. Skin penetration was calculated by summing the amount of lyngbyatoxin A recovered from the dermis and receptor fluid. The mean partition coefficient for lyngbyatoxin A was 1.53. Penetration of lyngbyatoxin A (expressed as a percentage of dose, n = 3) in guinea pig and human skin was 23 and 6.2 (respectively) after 1 hr of topical exposure. The amount of lyngbyatoxin A in the dermis and receptor fluid did not change significantly over time.

Methods for in vitro skin absorption studies of a lipophilic toxin produced by red tide.

The penetration and distribution of [3H]PbTx-3 into pig skin was determined using in vivo and in vitro methods. The dose used in each topical study was 0.3-0.4 micrograms/cm2 skin, with dimethylsulfoxide as the vehicle. In the in vivo study, mean cutaneous absorption after 48 h (expressed as percentage of the dose) was 11.5% (n = 3). In the in vitro study, mean cutaneous absorption after 48 h was 1.6% (n = 12), when based on accumulation of radioactivity in receptor fluid, or 9.9% when based on receptor fluid and dermis. [3H]PbTx-3 readily penetrated through the epidermis into the dermis, reaching maximal dermal accumulation at 4 h (9.1% in vivo and 18% in vitro). At 24 h, the amount in the dermis decreased to 2.3% and 15% in vivo and in vitro, respectively and at 48 h the amount in the dermis decreased to 8.2% in vitro. These results demonstrate the important role of the dermis as a reservoir for a lipophilic compound in both in vivo and in vitro percutaneous absorption studies.

In vitro penetration of tritium-labelled water (THO) and [3H]PbTx-3 (a red tide toxin) through monkey buccal mucosa and skin.

The permeability coefficients (Kp) for tritium-labelled water (THO) were determined in human and monkey skin, and monkey buccal mucosa. Kp of human skin (0.47 x 10(-3) cm/h) correlated favorably with previous reports. Kp of hydrated monkey skin for THO (0.77 x 10(-3) cm/h) was not significantly different (P greater than 0.05) from Kp of hydrated human skin (0.88 x 10(-3) cm/h). Kp of monkey buccal mucosa for THO (6.15 x 10(-3) cm/h) was significantly greater than that for monkey skin. Penetration and disposition of [3H]PbTx-3 into layers of monkey buccal mucosa and skin was determined. [3H]PbTx-3 (5-7 microCi) dissolved in 2 ml of water was applied to epithelial/epidermal surface (2.8 cm2) at zero time. The relative percent dose recovered from the upper layers of buccal mucosa (epithelium) and skin (epidermis) varied, but at each time interval was less than 2.5% of the dose. At most of the time intervals (2-24 h), a larger percent of the dose was recovered from the inner layer of the buccal mucosa (lamina propria) than from the inner layer of skin (dermis). After 24 h, as much as 34 or 13% of the dose was recovered from lamina propria or dermis, respectively. At each time interval studied, less than 2% of dose of [3H]PbTx-3 penetrated into the receptor fluid which bathed the inner surfaces of the lamina propria or dermis. The results of this study demonstrate that monkey buccal mucosa is more permeable than skin to THO and PbTx-3.

Photoelectron microscopy: a new approach to mapping organic and biological surfaces.

A general method of imaging organic and biological surfaces based on the photoelectric effect is reported. For the experiments, a photoelectron emission microscope was constructed. It is an ultrahigh vacuum instrument using electrostatic electron lenses, microchannel plate image intensifier, cold stage, hydrogen excitation source, and magnesium fluoride optics. The organic surfaces examined were grid patterns of acridine orange, fluorescein, and benzo(a)pyrene on a Butvar surface. A biological sample, sectioned rat epididymis, was also imaged by the new photoelectron microscope. Good contrast was obtained in these initial low magnification experiments. These data demonstrate the feasibility of mapping biological surfaces according to differences in ionization potentials of exposed molecules. A number of technical difficulties, such as the intensity of the excitation source, must be solved before high resolution experiments are practical. However, it is probable that this approach can be useful, even at low magnifications, in determination of the properties of organic and biological surfaces.