Development of a RapidFire mass spectrometry assay and a fluorescence assay for the discovery of kynurenine aminotransferase II inhibitors to treat central nervous system disorders

Kynurenine aminotransferases convert kynurenine to kynurenic acid and play an important role in the tryptophan degradation pathway. Kynurenic acid levels in brain have been hypothesized to be linked to a number of central nervous system (CNS) disorders. Kynurenine aminotransferase II (KATII) has proven to be a key modulator of kynurenic acid levels in brain and, thus, is an attractive target to treat CNS diseases. A sensitive, high-throughput, label-free RapidFire mass spectrometry assay has been developed for human KATII. Unlike other assays, this method is directly applicable to KATII enzymes from different animal species, which allows us to select proper animal model(s) to evaluate human KATII inhibitors. We also established a coupled fluorescence assay for human KATII. The short assay time and kinetic capability of the fluorescence assay provide a useful tool for orthogonal inhibitor validation and mechanistic studies.     

Cysteine sulfinate transamination activity of aspartate aminotransferases.

Aspartate aminotransferases from pig heart cytosol and mitochondria, $\text{Escherichia coli}$ B and $\text{Pseudomonas striata}$ accepted L-cysteine sulfinate as a good substrate. The mitochondrial isoenzyme and the $\text{Escherichia}$ enzyme showed higher activity toward L-cysteine sulfinate than toward the natural substrates, L-glutamate and L-aspartate. The cytosolic isoenzyme catalyzed the L-cysteine sulfinate transamination at 50% the rate of L-glutamate transamination. The $\text{Pseudomonas}$ enzyme had the same reactivity toward the three substrates. Antisera against the two isoenzymes and the $\text{Escherichia}$ enzyme inactivated almost completely cysteine sulfinate transamination activity in the crude extracts of pig heart muscle and $\text{Escherichia coli}$ B, respectively. These results indicate that cysteine sulfinate transamination is catalyzed by aspartate aminotransferase in these cells.     

Structure and mechanism of a cysteine sulfinate desulfinase engineered on the aspartate aminotransferase scaffold

The joint substitution of three active-site residues in Escherichia coli (L)-aspartate aminotransferase increases the ratio of l-cysteine sulfinate desulfinase to transaminase activity 10(5)-fold. This change in reaction specificity results from combining a tyrosine-shift double mutation (Y214Q/R280Y) with a non-conservative substitution of a substrate-binding residue (I33Q). Tyr214 hydrogen bonds with O3 of the cofactor and is close to Arg374 which binds the α-carboxylate group of the substrate; Arg280 interacts with the distal carboxylate group of the substrate; and Ile33 is part of the hydrophobic patch near the entrance to the active site, presumably participating in the domain closure essential for the transamination reaction. In the triple-mutant enzyme, k(cat)' for desulfination of l-cysteine sulfinate increased to 0.5s(-1) (from 0.05s(-1) in wild-type enzyme), whereas k(cat)' for transamination of the same substrate was reduced from 510s(-1) to 0.05s(-1). Similarly, k(cat)' for β-decarboxylation of l-aspartate increased from<0.0001s(-1) to 0.07s(-1), whereas k(cat)' for transamination was reduced from 530s(-1) to 0.13s(-1). l-Aspartate aminotransferase had thus been converted into an l-cysteine sulfinate desulfinase that catalyzes transamination and l-aspartate β-decarboxylation as side reactions. The X-ray structures of the engineered l-cysteine sulfinate desulfinase in its pyridoxal-5'-phosphate and pyridoxamine-5'-phosphate form or liganded with a covalent coenzyme-substrate adduct identified the subtle structural changes that suffice for generating desulfinase activity and concomitantly abolishing transaminase activity toward dicarboxylic amino acids. Apparently, the triple mutation impairs the domain closure thus favoring reprotonation of alternative acceptor sites in coenzyme-substrate intermediates by bulk water.     

pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I.

Human kynurenine aminotransferase I/glutamine transaminase K (hKAT-I) is an important multifunctional enzyme. This study systematically studies the substrates of hKAT-I and reassesses the effects of pH, Tris, amino acids and alpha-keto acids on the activity of the enzyme. The experiments were comprised of functional expression of the hKAT-I in an insect cell/baculovirus expression system, purification of its recombinant protein, and functional characterization of the purified enzyme. This study demonstrates that hKAT-I can catalyze kynurenine to kynurenic acid under physiological pH conditions, indicates indo-3-pyruvate and cysteine as efficient inhibitors for hKAT-I, and also provides biochemical information about the substrate specificity and cosubstrate inhibition of the enzyme. hKAT-I is inhibited by Tris under physiological pH conditions, which explains why it has been concluded that the enzyme could not efficiently catalyze kynurenine transamination. Our findings provide a biochemical basis towards understanding the overall physiological role of hKAT-I in vivo and insight into controlling the levels of endogenous kynurenic acid through modulation of the enzyme in the human brain.     

Substrate stereospecificity and active site topography of gamma-aminobutyric acid aminotransferase for beta-aryl-gamma-aminobutyric acid analogues

The substrate and inhibitory properties of (R)- and (S)-4-amino-3-phenylbutanoic acid, (R)- and (S)-4-amino-3-(4-chlorophenyl)butanoic acid (baclofens), (E)-4-amino-3-phenylbut-2-enoic acid, and (E)-4-amino-3-(4-chlorophenyl)but-2-enoic acid are determined and compared with those of 4-aminobutanoic acid, 4-aminobut-2-enoic acid (4-aminocrotonic acid), and the racemic mixtures of 4-amino-3-arylbutanoic acids. All compounds in both series were found to be substrates, except for the R-isomers, which were identified as competitive inhibitors. These results are compared with known pharmacological data regarding the appropriate isomers.     

Excitatory Amino Acid Receptor Potency and Subclass Specificity of Sulfur-Containing Amino Acids

The sulfur-containing amino acids, L- and D-cysteate, L-cysteine, L- and D-cysteine sulfinate, L- and D-cysteine-S-sulfate, L-cystine, L- and D-homocysteate, L- and D-homocysteine sulfinate, L-homocysteine, L-serine-O-sulfate, and taurine were tested in two excitatory amino acid receptor functional assays and in receptor binding assays designed to label specifically the AA1/N-methyl-D-aspartate (NMDA), AA2/quisqualate, and AA3/kainate receptor recognition sites, as well as a CaCl2-dependent L-2-amino-4-phosphonobutanoate site, and a putative glutamate uptake site. Agonist efficacies were determined by chick retinal excitotoxicity and stimulated sodium efflux from rat brain slices. D-Homocysteine sulfinate, L-homocysteate, and L-serine-O-sulfate had affinities most selective for the NMDA binding site, whereas the binding affinities of D-cysteate, D-cysteine sulfinate, D-homocysteate, and L-homocysteine sulfinate were less selective. However, the correlation of agonist activity sensitive to blockade by D-2-amino-7-phosphonoheptanoate or D-2-amino-5-phosphonopentanoate in the functional assays with affinity in the NMDA binding assay (r = 0.87, p less than 0.005 and r = 0.98, p less than 0.005 for excitotoxicity and sodium efflux, respectively) allows characterization of these sulfur-containing amino acids as acting at NMDA subclass receptors. L-Homocysteate, which has been found in the brain, and L-serine-O-sulfate are selective agonists and could serve as endogenous neurotransmitters at the NMDA receptor.     

The role of glutamine transaminase K (GTK) in sulfur and alpha-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants

Glutamine transaminase K (GTK), which is a freely reversible glutamine (methionine) aromatic amino acid aminotransferase, is present in most mammalian tissues, including brain. Quantitatively, the most important amine donor in vivo is glutamine. The product of glutamine transamination (i.e., alpha-ketoglutaramate; alphaKGM) is rapidly removed by cyclization and/or conversion to alpha-ketoglutarate. Transamination is therefore "pulled" in the direction of glutamine utilization. Major biological roles of GTK are to maintain low levels of phenylpyruvate and to close the methionine salvage pathway. GTK also catalyzes the transamination of cystathionine, lanthionine, and thialysine to the corresponding alpha-keto acids, which cyclize to ketimines. The cyclic ketimines and several metabolites derived therefrom are found in brain. It is not clear whether these compounds have a biological function or are metabolic dead-ends. However, high-affinity binding of lanthionine ketimine (LK) to brain membranes has been reported. Mammalian tissues possess several enzymes capable of catalyzing transamination of kynurenine in vitro. Two of these kynurenine aminotransferases (KATs), namely KAT I and KAT II, are present in brain and have been extensively studied. KAT I and KAT II are identical to GTK and alpha-aminoadipate aminotransferase, respectively. GTK/KAT I is largely cytosolic in kidney, but mostly mitochondrial in brain. The same gene codes for both forms, but alternative splicing dictates whether a 32-amino acid mitochondrial-targeting sequence is present in the expressed protein. The activity of KAT I is altered by a missense mutation (E61G) in the spontaneously hypertensive rat. The symptoms may be due in part to alteration of kynurenine transamination. However, owing to strong competition from other amino acid substrates, the turnover of kynurenine to kynurenate by GTK/KAT I in nervous tissue must be slow unless kynurenine and GTK are sequestered in a compartment distinct from the major amino acid pools. The possibility is discussed that the spontaneous hypertension in rats carrying the GTK/KAT I mutation may be due in part to disruption of glutamine transamination. GTK is one of several pyridoxal 5'-phosphate (PLP)-containing enzymes that can catalyze non-physiological beta-elimination reactions with cysteine S-conjugates containing a good leaving group attached at the sulfur. These elimination reactions may contribute to the bioactivation of certain electrophiles, resulting in toxicity to kidney, liver, brain, and possibly other organs. On the other hand, the beta-lyase reaction catalyzed by GTK may be useful in the conversion of some cysteine S-conjugate prodrugs to active components in vivo. The roles of GTK in (a) brain nitrogen, sulfur, and aromatic amino acid/kynurenine metabolism, (b) brain alpha-keto acid metabolism, (c) bioactivation of certain electrophiles in brain, (d) prodrug targeting, and (e) maintenance of normal blood pressure deserve further study.     

Enantiomers of 4-amino-3-fluorobutanoic acid as substrates for gamma-aminobutyric acid aminotransferase. Conformational probes for GABA binding

Gamma-aminobutyric acid aminotransferase (GABA-AT), a pyridoxal 5'-phosphate dependent enzyme, catalyzes the degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) to succinic semialdehyde with concomitant conversion of pyridoxal 5'-phosphate (PLP) to pyridoxamine 5'-phosphate (PMP). The enzyme then catalyzes the conversion of alpha-ketoglutarate to the excitatory neurotransmitter L-glutamate. Racemic 4-amino-3-fluorobutanoic acid (3-F-GABA) was shown previously to act as a substrate for GABA-AT, not for transamination, but for HF elimination. Here we report studies of the reaction catalyzed by GABA-AT on (R)- and (S)-3-F-GABA. Neither enantiomer is a substrate for transamination. Very little elimination from the (S)-enantiomer was detected using a coupled enzyme assay; The rate of elimination of HF from the (R)-enantiomer is at least 10 times greater than that for the (S)-enantiomer. The (R)-enantiomer is about 20 times more efficient as a substrate for GABA-AT catalyzed HF elimination than GABA is a substrate for transamination. The (R)-enantiomer also inhibits the transamination of GABA 10 times more effectively than the (S)-enantiomer. Using a combination of computer modeling and the knowledge that vicinal C-F and C-NH3+ bonds have a strong preference to align gauche rather than anti to each other, it is concluded that on binding of free 3-F-GABA to GABA-AT the optimal conformation places the C-NH3+ and C-F bonds gauche in the (R)-enantiomer but anti in the (S)-enantiomer. Furthermore, the dynamic binding process and the bioactive conformation of GABA bound to GABA-AT have been inferred on the basis of the different biological behavior of the two enantiomers of 3-F-GABA when they bind to the enzyme. The present study suggests that the C-F bond can be utilized as a conformational probe to explore the dynamic binding process and provide insight into the bioactive conformation of substrates, which cannot be easily determined by other biophysical approaches.     

Substrate specificity of human glutamine transaminase K as an aminotransferase and as a cysteine S-conjugate beta-lyase

Rat kidney glutamine transaminase K (GTK) exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. The β-lyase reaction products are pyruvate, ammonium and a sulfhydryl-containing fragment. We show here that recombinant human GTK (rhGTK) also exhibits broad specificity both as an aminotransferase and as a cysteine S-conjugate β-lyase. S-(1,1,2,2-Tetrafluoroethyl)-l-cysteine is an excellent aminotransferase and β-lyase substrate of rhGTK. Moderate aminotransferase and β-lyase activities occur with the chemopreventive agent Se-methyl-l-selenocysteine. l-3-(2-Naphthyl)alanine, l-3-(1-naphthyl)alanine, 5-S-l-cysteinyldopamine and 5-S-l-cysteinyl-l-DOPA are measurable aminotransferase substrates, indicating that the active site can accommodate large aromatic amino acids. The α-keto acids generated by transamination/l-amino acid oxidase activity of the two catechol cysteine S-conjugates are unstable. A slow rhGTK-catalyzed β-elimination reaction, as measured by pyruvate formation, was demonstrated with 5-S-l-cysteinyldopamine, but not with 5-S-l-cysteinyl-l-DOPA. The importance of transamination, oxidation and β-elimination reactions involving 5-S-l-cysteinyldopamine, 5-S-l-cysteinyl-l-DOPA and Se-methyl-l-selenocysteine in human tissues and their biological relevance are discussed.     

A general mechanism of polypeptide cross-linking by 3-hydroxykynurenine

Abstract

The human lens contains a group of fluorescent compounds, derived from tryptophan, which act to absorb UV light in the 300–400 nm region of the spectrum.¹ The major component is the glucoside of 3-hydroxykynurenine (3HK), 3-hydroxykynurenine glucoside (3HKG).²In the lens, 3HKG represents a unique pathway of tryptophan metabolism. Smaller amounts of kynurenine and 3HK have been detected in human lens extracts.^3,4 . More recently, a new UV-filter compound derived from tryptophan, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside (AHBG), was identified, and constitutes the second most abundant UV-filter in the human lens.⁵     

Transamination of LTE4 by cysteine conjugate aminotransferase

Leukotriene E4 (LTE4) was transaminated to 5(S)-hydroxy-6(R)-S-(2-keto-3-thiopropionyl)-7,9-trans-11,14-cis- eicosatetraenoic acid (tentatively designated as LTG4) by cysteine conjugate aminotransferase I purified from rat liver supernatant in the presence of alpha-ketoglutaric acid or alpha-keto-gamma-methiolbutyric acid. The transamination activity was present in the kidney as well as in the liver, but not in the lung or leukocytes.     

Biochemical and mass spectrometric characterization of human N-acylethanolamine-hydrolyzing Acid amidase inhibition

The mechanism of inactivation of human enzyme N-acylethanolamine-hydrolyzing acid amidase (hNAAA), with selected inhibitors identified in a novel fluorescent based assay developed for characterization of both reversible and irreversible inhibitors, was investigated kinetically and using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). 1-Isothiocyanatopentadecane (AM9023) was found to be a potent, selective and reversible hNAAA inhibitor, while two others, 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701) and N-Benzyloxycarbonyl-L-serine β-lactone (N-Cbz-serine β-lactone), inhibited hNAAA in a covalent and irreversible manner. MS analysis of the hNAAA/covalent inhibitor complexes identified modification only of the N-terminal cysteine (Cys126) of the β-subunit, confirming a suggested mechanism of hNAAA inactivation by the β-lactone containing inhibitors. These experiments provide direct evidence of the key role of Cys126 in hNAAA inactivation by different classes of covalent inhibitors, confirming the essential role of cysteine for catalysis and inhibition in this cysteine N-terminal nucleophile hydrolase enzyme. They also provide a methodology for the rapid screening and characterization of large libraries of compounds as potential inhibitors of NAAA, and subsequent characterization or their mechanism through MALDI-TOF MS based bottom up-proteomics.     

Comparative study of miscellaneous properties of cysteine sulfinate transaminase and glutamate oxaloacetate transminase in chick retina homogenate

The activity, properties, and developmental pattern of cysteine sulfinate transaminase (CSA-T) were studied in chick retina and compared with the activity, properties, and developmental pattern of glutamate oxaloacetate transaminase (GOT). Their optimum pH is identical whereas the effect of pyridoxal phosphate seems to be different. Developmental patterns are also different. TheK _m andV _m of CSA-T and GOT were determined in chick retina homogenate. These results suggest that two different enzymes are responsible for the transamination of cysteine sulfinate (CSA) and aspartate.     

Effect of various analogues of D-glutamic acid on the D-glutamate-adding enzyme from Escherichis coli

Abstract Twenty-four analogues of D-glutamic acid were tested as substrates or inhibitors of the D-glutamate-adding enzyme from Escherichia coli . The best substrates were, in decreasing order of specific activity, D- erythro -4-methylglutamic acid, D- erythro - methylglutamic acid, DL-homocysteic acid, (±)- trans -1-amino-3-carboxy-cyclopentanecarboxylic acid and (±)- trans -1-amino-3-carboxy-cyclohexanecarboxylic acid. Among the different stereoisomers, only the D- erythro isomers for methylglutamic acids, and the trans isomers for the cyclic analogs, were substrates. Apart from the D- erythro -3 and 4-methylglutamic acids and DL-homocysteic acid, none of the examined compounds significantly inhibited the addition of radioactive D-glutamic acid to UDP-N-acetylmuramyl-L-alanine.     

Spectroscopic characterization of true enzyme-substrate intermediates of aspartate aminotransferase trapped at subzero temperatures

Absorption and circular dichroism spectra of stable enzyme-substrate intermediates of aspartate aminotransferase were recorded at subzero temperatures (down to -65 degrees C) in the cryosolvent water/methanol. The intermediates were formed either between the pyridoxal form of the enzyme and its amino acid substrates, or between the pyridoxamine form and its oxo acid substrates. Kd values determined by spectroscopic titration were very close to the Km values reported for the different substrates. The adsorption complex of the pyridoxal form was probably obtained on addition of cysteine sulfinate. This complex is characterized by an increased absorption at 430 nm together with a positive Cotton effect, as also observed in the case of the complex with the competitive inhibitor maleate indicating protonation of the internal aldimine. Addition of the substrates aspartate or glutamate to the pyridoxal form seemed to result in the direct accumulation of the external aldimine which showed a slight decrease in both the absorbance and the Cotton effect at 360 nm. Additionally, a bathochromic shift of 5 nm was observed in the case of glutamate. At 430 nm, only a minor increase in absorbance, but not in circular dichroism, was observed with aspartate, and no changes were found with glutamate and the substrate analog 2-methylaspartate, indicating a deprotonated external aldimine. Presumably, the ketimine intermediate was obtained on addition of the oxo acids 2-oxoglutarate or oxalacetate to the pyridoxamine form. The intermediate showed a slight bathochromic shift (2 nm) of the absorption band and decreased circular dichroism. On formation of the ketimine, a tyrosine residue, probably active-site Tyr225, becomes partly ionized. The finding that the external aldimine can probably be accumulated in the conversion of the pyridoxal to the pyridoxamine form with the natural substrates would confirm the proton abstraction at C alpha to be the rate-limiting step in the tautomerization, although with cysteine sulfinate, the formation of the external aldimine might contribute to the rate limitation. Accumulation of the ketimine in the reverse direction would indicate that the proton abstraction at C4' is rate-limiting in this half-reaction. The results demonstrate the feasibility of further structural investigations of true enzyme-substrate intermediates.     

Structural insight into the mechanism of substrate specificity of aedes kynurenine aminotransferase

Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I-phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.     

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida.

Urocanase is inactivated in intact cells of Pseudomonas putida and photoactivated by brief exposure of the cells to the UV radiation in sunlight. The dark reversion (inactivation) in vitro is explained by the formation of a sulfite-NAD adduct. Our objective was to investigate the dark reversion in vivo. Various compounds were added to P. putida cells, and the reversion was measured, after sonication, by comparison of the activity before and after UV irradiation. Sulfite, cysteine sulfinate, and hypotaurine enhanced the reversion of urocanase in resting cells. The reversion was time and concentration dependent. Sulfite modified the purified enzyme, but cysteine sulfinate and hypotaurine could not, indicating that those two substances had to be metabolized to support the reversion. Both of those compounds yielded sulfite when they were incubated with cells. Transaminases form sulfite from cysteine sulfinate. P. putida extract contained a transaminase whose activity involved as alpha-keto acid and either cysteine sulfinate or hypotaurine for (i) production of sulfite, (ii) disappearance of substrates, (iii) formation of corresponding amino acids, and (iv) urocanase reversion. Porcine crystalline transaminase caused reversion of highly purified P. putida urocanase with cysteine sulfinate and alpha-ketoglutarate. We conclude that in P. putida cysteine sulfinate or hypotaurine is catabolized in vivo by a transaminase reaction to sulfite, which modifies urocanase to a form that can be photoactivated. We suggest that this photoregulatory process is natural because it occurs in cells with the aid of sunlight and cellular metabolism.     

Extracellular Acidic Sulfur-Containing Amino Acids and γ-Glutamyl Peptides in Global Ischemia: Postischemic Recovery of Neuronal Activity Is Paralleled by a Tetrodotoxin-Sensitive Increase in Cysteine Sulfinate in the CA1 of the Rat Hippocampus

An excessive activation of the excitatory amino acid system has been proposed as one possible mediator of the ischemia-induced delayed death of CA1 pyramidal cells in the hippocampus. Using dialytrodes in the CA1 of the rat, we have investigated multiple-unit activity and extracellular changes in acidic sulfur-containing amino acids and gamma-glutamyl peptides during ischemia (20-min, four-vessel occlusion) and during 8 h of reflow. Multiple-unit activity was abolished during ischemia and for the following 1 h, but then recovered, gradually reaching preischemic levels after 8 h of reflow. Extracellular cysteate, cysteine sulfinate, and gamma-glutamyltaurine increased (1.5- to threefold) during ischemia, and extracellular glutathione and gamma-glutamylaspartate plus gamma-glutamylglutamine increased during early reflow (two- to threefold). The recovery of neuronal activity at 4-8 h was paralleled by an increase in extracellular cysteine sulfinate (2.5-fold at 8 h of reflow). Perfusion with 10 microM tetrodotoxin at 8 h of reflow abolished the multiple-unit activity and reduced extracellular cysteine sulfinate. Considering the glutamate-like properties of cysteine sulfinate, the observed postischemic increase may be involved in the development of the delayed neuronal death.     

The Sulfated Triphenyl Methane Derivative Acid Fuchsin Is a Potent Inhibitor of Amyloid Formation by Human Islet Amyloid Polypeptide and Protects against the Toxic Effects of Amyloid Formation

Islet amyloid polypeptide (IAPP), also known as amylin, is responsible for amyloid formation in type 2 diabetes. The formation of islet amyloid is believed to contribute to the pathology of the disease by killing β-cells, and it may also contribute to islet transplant failure. The design of inhibitors of amyloid formation is an active area of research, but comparatively little attention has been paid to inhibitors of IAPP in contrast to the large body of work on β-amyloid, and most small-molecule inhibitors of IAPP amyloid are generally effective only when used at a significant molar excess. Here we show that the simple sulfonated triphenyl methane derivative acid fuchsin, 3-(1-(4-amino-3-methyl-5-sulfonatophenyl)-1-(4-amino-3-sulfonatophenyl) methylene) cyclohexa-1,4-dienesulfonic acid, is a potent inhibitor of in vitro amyloid formation by IAPP at substoichiometric levels and protects cultured rat INS-1 cells against the toxic effects of human IAPP. Fluorescence-detected thioflavin-T binding assays, light-scattering, circular dichroism, two-dimensional IR, and transmission electron microscopy measurements confirm that the compound prevents amyloid fibril formation. Ionic-strength-dependent studies show that the effects are mediated in part by electrostatic interactions. Experiments in which the compound is added at different time points during the lag phase after amyloid formation has commenced reveal that it arrests amyloid formation by trapping intermediate species. The compound is less effective against the β-amyloid peptide, indicating specificity in its ability to inhibit amyloid formation by IAPP. The work reported here provides a new structural class of IAPP amyloid inhibitors and demonstrates the power of two-dimensional infrared spectroscopy for characterizing amyloid inhibitor interactions.