Structure–activity relationships of peptidomimetics that inhibit PPI of HER2‐HER3

Human epidermal growth factor receptor‐2 (HER2) is a tyrosine kinase family protein receptor that is known to undergo heterodimerization with other members of the family of epidermal growth factor receptors (EGFR) for cell signaling. Overexpression of HER2 and deregulation of signaling has implications in breast, ovarian, and lung cancers. We have designed several peptidomimetics to block the HER2‐mediated dimerization, resulting in antiproliferative activity for cancer cells. In this work, we have investigated the structure–activity relationships of peptidomimetic analogs of Compound 5. Compound 5 was conformationally constrained by N‐ and C‐terminal modification and cyclization as well as by substitution with d ‐amino acids at the N‐and C‐termini. Among the compounds studied in this work, a peptidomimetic Compound 21 with d ‐amino acid substitution and its N‐ and C‐termini capped with acetyl and amide functional groups and a reversed sequence compared to that of Compound 5 exhibited better antiproliferative activity in HER2‐overexpressed breast, ovarian, and lung cancer cell lines. Compound 21 was further evaluated for its protein–protein interaction (PPI) inhibition ability using enzyme fragment complementation assay, proximity ligation assay, and Western blot analysis. Results suggested that Compound 21 is able to block HER2:HER3 interaction and inhibit phosphorylation of the kinase domain of HER2. The mode of binding of Compound 21 to HER2 protein was modeled using a docking method. Compound 21 seems to bind to domain IV of HER2 near the PPI site of EGFR:HER2, and HER:HER3 and inhibit PPI. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 693–702, 2014.     

Novel insights into the dynamics behavior of glucagon-like peptide-1 receptor with its small molecule agonists

Abstract

The glucagon-like peptide-1 receptor (GLP-1R) is a well-known target of therapeutics industries for the treatment of various metabolic diseases like type 2 diabetes and obesity. The structural–functional relationships of small molecule agonists and GLP-1R are yet to be understood. Therefore, an attempt was made on structurally known GLP-1R agonists (Compound 1, Compound 2, Compound A, Compound B, and (S)-8) to study their interaction with the extracellular domain of GLP-1R. In this study, we explored the dynamics, intrinsic stability, and binding mechanisms of these molecules through computational modeling, docking, molecular dynamics (MD) simulations and molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) binding free energy estimation. Molecular docking study depicted that hydrophobic interaction (pi–pi stacking) plays a crucial role in maintaining the stability of the complex, which was also supported by intermolecular analysis from MD simulation study. Principal component analysis suggested that the terminal ends along with the turns/loops connecting adjacent helix and strands exhibit a comparatively higher movement of main chain atoms in most of the complexes. MM/PBSA binding free energy study revealed that non-polar solvation (van der Waals and electrostatic) energy subsidizes significantly to the total binding energy, and the polar solvation energy opposes the binding agonists to GLP-1R. Overall, we provide structural features information about GLP-1R complexes that would be conducive for the discovery of new GLP-1R agonists in the future for the treatment of various metabolic diseases.

Communicated by Ramaswamy H. Sarma     

Pre-steady-state kinetic study on the formation of Compound I and II of ligninase

The reaction between ligninase and hydrogen peroxide yielding Compound I has been investigated using a stopped-flow rapid-scan spectrophotometer. The optical absorption spectrum of Compound I appears different to that reported by Andrawis, A. et al. (1987) and Renganathan, V. and Gold, M.H. (1986), in that the Soret-maximum is at 401 nm rather than 408 nm. The second-order rate constant (4.2·10⁵ M⁻¹·s⁻¹) for the formation of Compound I was independent of pH (pH 3.0–6.0). In the absence of external electron donors, Compound I decayed to Compound II with a half-life of 5–10 s at pH 3.1. The rate of this reaction was not affected by the H₂O₂ concentration used. In the presence of either veratryl alcohol or ferrocyanide, Compound II was rapidly generated. With ferrocyanide, the second-order rate constant increased from 1.9·10⁴ M⁻¹·s⁻¹ to 6.8·10⁶ M⁻¹·s⁻¹ when the pH was lowered from 6.0 to 3.1. With veratryl alcohol as an electron donor, the second-order rate constant for the formation of Compound II increased from 7.0·10³ M⁻¹·s⁻¹ at pH 6.0 to 1.0·10⁵ M⁻¹·s⁻¹ at pH 4.5. At lower pH values the rate of Compound II formation no longer followed an exponential relationship and the steady-state spectral properties differed to those recorded in the presence of ferrocyanide. Our data support a model of enzyme catalysis in which veratryl alcohol is oxidized in one-electron steps and strengthen the view that veratryl alcohol oxidation involves a substrate-modified Compound II intermediate which is rapidly reduced to the native enzyme.     

abeo‐Taxane Diterpenoids from the Taiwanese Yew Taxus sumatrana

Chemical investigation of the acetonic extract of the leaves and twigs of Taxus sumatrana (Taxaceae) led to the isolation of four new taxane diterpene esters, taiwantaxins A–D ( 1 – 4 , resp.). Their structures were determined primarily on the basis of 1D‐ and 2D‐NMR techniques as well as MS. Compound 1 is a rearranged taxane diterpenoid possessing an opened oxetane ring moiety containing C(4), C(5), and C(20). The metabolites 2 and 3 belong to a 5/6/6 taxene system having a rare five‐membered γ‐lactone ring comprising C(8), C(9), C(10), and C(19). Compound 4 is an example of a taxane diterpene containing a 6/8/6 ring system with a tetrahydrofuran ring comprising C(2), C(3), C(4), and C(20). The 11(15→1)abeo‐taxane diterpenoids, taiwantaxins A–C ( 1 – 3 , resp.) are lacking an O‐bearing functionality at either C(13) or C(14). Compound 2 showed significant cytotoxic activity against human PC‐3 tumor cells.     

Volatile compounds of Asphodelus microcarpus Salzm. et Viv. Honey obtained by HS-SPME and USE analyzed by GC/MS

Chemical analysis of Asphodelus microcarpus Salzm. et Viv. honey is of great importance, since melissopalynology does not allow the unambiguous determination of its botanical origin. Therefore, the volatile compounds of eight unifloral asphodel honeys have been investigated for the first time. The honey extracts were obtained by headspace solid-phase microextraction (HS-SPME) and ultrasonicsolvent extraction (USE) and analyzed by GC and GC/MS. In the honey headspace, 31 volatile compounds were identified with high percentages of 2-phenylacetaldehyde (2; 14.8–34.7%), followed by somewhat lower percentages of methyl syringate (1; 10.5–11.5%). Compound 2 is not a specific marker of the botanical origin of the honey, but its high percentage can be emphasized as headspace characteristic of asphodel honey. The extraction solvent for all the samples was selected after extracting a representative sample with pentane, Et(2)O, pentane/Et(2)O 1:2 (v/v), and CH(2)Cl(2) . Compound 1 was the major constituent of all the USE extracts (46.8–87.0%). According to these preliminary results, all the honey samples were extracted by USE with the solvent pentane/Et(2)O 1:2. A total of 60 volatile compounds were identified with 1 as predominant compound (69.4–87.0%), pointing out 1 as Asphodelus honey volatile marker.     

Chemical Constituents of Primulina eburnea (Gesneriaceae) and Their Cytotoxic Activities

Two new ursane-type triterpenes, eburnealactones A and B ( 1 and 2 ), one new flavonoid, eburneatin A ( 6 ), and one new phenylethanoid glycoside, chiritoside D ( 7 ), along with 9 known compounds ( 3–5 , 8–13 ) were isolated from the whole plant of Primulina eburnea. Their structures were elucidated by comprehensive spectroscopic data analysis (IR, UV, NMR, and HR-ESI-MS). All the compounds were evaluated for their cytotoxic activities. Compound 1 showed significant cytotoxic activities against MKN-45 cell lines and 5637 cell lines with the IC₅₀ values of 9.57 μM and 8.30 μM, respectively. Compound 1 exhibited moderate cytotoxic activities against A549 and PATU8988T cell lines with the IC₅₀ values of 30.70 μM and 38.22 μM, respectively. Compound 6 exhibited moderate cytotoxic activities against MKN-45, HCT116, PATU8988T, 5637 and A-673 cell lines with the IC₅₀ values of 19.69 μM, 16.44 μM, 18.07 μM, 11.51 μM and 18.15 μM, respectively. Compound 5 showed moderate cytotoxic activities against A549 cell lines with the IC₅₀ values of 24.06 μM.     

A new diterpene from the fungal mycelia of Hericium erinaceus

One new diterpene (1) and new compound (2), along with four known compounds (3–6) were isolated from the fungal mycelia of Hericium erinaceus by the tracking method of antibacterial activity. The structures of compounds (1–6) were elucidated on the basis of spectral data. Compound (3) showed strong antibacterial activity against Helicobacter pylori (ATCC43504) and compounds (1), (2) and (4) showed moderate antibacterial activity. Compound (1) exhibited good cytotoxicity against three human cancer cell lines (K562, LANCAP, HEP2).     

Design,synthesis, in vitro anticancer evaluation,kinase inhibitory effects,and pharmacokinetic profile of new 1,3,4-triarylpyrazole derivatives possessing terminal sulfonamide moiety

The present work describes the design and synthesis of a novel series of 1,3-diaryl-4-sulfonamidoarylpyrazole derivatives 1a–q and 2a–q and their in vitro biological activities. The target compounds were evaluated for antiproliferative activity against NCI-60 cell line panel. Compounds 1c, 1g, 1k–m, 1o, 2g, 2h, 2k–m, 2o, and 2q showed the highest mean inhibition percentages at 10 µM single-dose testing and were selected to be tested at 5-dose mode. The ICs₅₀ of the most potent compounds were determined over the 60 cell lines. Compound 2l exhibited the strongest activity against different cell lines with IC₅₀ 0.33 µM against A498 renal cancer cell line. Compound 2l was tested over a panel of 20 kinases to determine its molecular target(s), and its IC₅₀ values over the most sensitive kinases were defined. In vitro stability and in vivo pharmacokinetic profile of compound 2l was also investigated.     

New Sulphonamide and Carboxamide Derivatives of Acyclic C-Nucleosides of Triazolo-Thiadiazole and the Thiadiazine Analogues. Synthesis,Anti-HIV,and Antitumor Activities. Part 2

A new series of acyclic C-nucleosides 1′,2′-O-isopropylidene-D-ribo-tetritol-1-yl)[1,2,4] triazolo[3,4-b][1,3,4]thiadiazoles bearing arylsulfonamide (5–8) and arylcarboxamide (9–12) residues have been synthesized under microwave irradiation. Thiadiazines 13–15 have been analogously prepared, and upon acid hydrolysis, afforded the free nucleosides 16–18. The new synthesized compounds were assayed against HIV-1 and HIV-2 in MT-4 cells. Compound 7 was also screened against a panel of tumor cell lines consisting of CD4 human T-cells.     

Efficient biotransformation for preparation of pharmaceutically active ginsenoside Compound K by Penicillium oxalicum sp. 68

Pathogenic fungus Penicillium oxalicum sp. 68 was screened from soil and identified by ITS sequencing. The strain was found to be able to transform protopanaxadiol-type ginsenosides to produce a series of bioactive metabolites. Glycosidase from the culture of P. oxalicum sp. 68 was partially purified with a simple two-step procedure consisting of DEAE-cellulose chromatography and ammonium sulfate precipitation. Bioactive ginsenoside Compound K was prepared selectively and efficiently by biotransformation of ginsenosides Rb1, Rb2, Rc and Rd using the partially purified glycosidase. The optimal conditions for transforming Rb1 into Compound K were pH 4.0, 55 °C and 0.5 mg mL^?1 Rb1. The sole product is Compound K and the maximum yield reached 87.7 % (molar ratio). The transformation pathways of Rb1, Rb2, Rc and Rd are Rb1→Rd→F2→Compound K, Rb2→CO→CY→Compound K, Rc→Mb→Mc→Compound K and Rd→F2→Compound K, respectively. This biotransformation method showed great potential for preparing minor bioactive ginsenosides, especially Compound K, in the pharmaceutical industry because of its high specificity and favorable environmental compatibility.     

Three New Selaginellin Derivatives from Selaginella pulvinata and Their α-Glucosidase Inhibitory Activity

Three new selaginellin derivatives, selaginpulvilins V-X ( 1–3 ), together with seven known analogs ( 4–10 ) were isolated from whole plants of Selaginella pulvinata. Their structures were determined by extensive spectroscopic methods including 1D and 2D NMR, HR-ESI-MS and chemical derivatization method. Compound 1 represents a rare example of naturally occurring selaginellin with an alkynylphenol-trimmed skeleton. Biological evaluation showed that compounds 2 , 6 and 8 displayed moderate inhibition against α-glucosidase with IC₅₀ values of 3.71, 2.04 and 4.00 μM, respectively.     

Synthesis of Zidovudine Derivatives with Anti-HIV-1 and Antibacterial Activities

Twelve novel zidovudine derivatives were prepared by modifying 5 ′-hydroxyl group of sugar moiety (1–8) and 5-methyl group of thymidine nucleus (9–12) and characterized spectrally. The compounds were evaluated for anti-HIV-1, antitubercular and antibacterial activities. Compound (3-azido-tetrahydro-5- (3,4-dihydro-5-methyl-2,4-dioxopyrimidin- 1 (2H)-yl) furan-2-yl)methyl 7- (4- (2-phenylacetoyloxy) -3,5- dimethylpiperazin-1-yl) -5- (2-phenylacetoyloxyamino) -1-cyclopropyl-6,8-difluoro-1,4-dihydro-4-oxoquinoline-3-carboxylate (5) was found to be the most potent anti-HIV-1 agent with EC₅₀ of 0.0012 μM against HIV-1_IIIB and CC₅₀ of 34.05 μM against MT-4 with selectivity index of 28,375. Compound 5 inhibited Mycobacterium tuberculosis with MIC of 1.72 μM and inhibited four pathogenic bacteria with MIC of less than 1 μM.     

A specific antagonist of vasopressin produced plasma hyperosmolality and reduced ischemic-induced cerebral edema in rats

Water diuresis was produced in rats after s.c. injections of d(CH2)5,D-Ile2,Ile4-AVP (compound 1) and d(CH2)5,Tyr(OEt)2Val4-AVP (compound 2). Compound 1 is known to be a more potent antagonist against the antidiuretic effect, while Compound 2 is a more potent antagonist against the vasopressor effect of vasopressin. Compound 1 (but not compound 2) also increased plasma osmolality significantly at the diuretic doses. In rats rendered ischemic of the forebrain by 4-hour occlusion of both common carotid arteries, the resulting increases in brain water were significantly reversed by the injection of compound 1. Compound 2 did not reduce the edema. The results suggests a novel approach to the treatment of cerebral edema.     

A steady-state study on the formation of Compounds II and III of myeloperoxidase

The reaction between native myeloperoxidase and hydrogen peroxide, yielding Compound II, was investigated using the stopped-flow technique. The pH dependence of the apparent second-order rate constant showed the existence of a protonatable group on the enzyme with a pKa of 4.9. This group is ascribed to the distal histidine imidazole, which must be deprotonated to enable the reaction of Compound I with hydrogen peroxidase to take place. The rate constant for the formation of Compound II by hydrogen peroxide was 3.5.10(4) M-1.s-1. During the reaction of myeloperoxidase with H2O2, rapid reduction of added cytochrome c was observed. This reduction was inhibitable by superoxide dismutase, and this demonstrates that superoxide anion radicals are generated. When potassium ferrocyanide was used as an electron donor to generate Compound II from Compound I, the pH dependence of the apparent second-order rate constant indicated involvement of a group with a pKa of 4.5. However, with ferrocyanide as an electron donor, protonation of the group was necessary to enable the reaction to take place. The rate constant for the generation of Compound II by ferrocyanide was 1.6.10(7) M-1.s-1. We also investigated the reaction of Compound II with hydrogen peroxide, yielding Compound III. Formation of Compound III (k = 50 M-1.s-1) proceeded via two different pathways, one of which was inhibitable by tetranitromethane. We further investigated the stability of Compound II and Compound III as a function of pH, ionic strength and enzyme concentration. The half-life values of both Compound II and Compound III were independent of the enzyme concentration and ionic strength. The half-life value of Compound III was pH-dependent, showing a decreasing stability with increasing pH, whereas the stability of Compound II was independent of pH over the range 3-11.     

Biosynthesis of riboflavin: reductase and deaminase of Ashbya gossypii.

Two enzymes involved in the biosynthesis of riboflavin have been found in $\text{Ashbya gossypii}$, an organism that produces and excretes riboflavin in large quantities. The first of these (called “reductase”) functions to reduce the ribose group of 2,5-diamino-6-oxy-4-(5′-phosphoriboyslamino)pyrimidine (PRP, Compound II, Fig. 1) to the ribityl group of Compound IV (Fig. 1). The second enzyme (called “deaminase”) catalyzes the deamination of Compound IV to Compound V. The evidence indicates that in $\text{A. gossypii}$ the reductase functions before the deaminase, in contrast to the reaction sequence known to operate in $\text{Escherichia coli}$ in which deamination takes place before reduction (Burrows, R.B. and Brown, G.M. (1978) J. Bacteriol., $\text{136}$, 657–667).     

Reactions of purified hog thyroid peroxidase with H2O2, tyrosine, and methylmercaptoimidazole (goitrogen) in comparison with bovine lactoperoxidase

Stopped flow experiments were carried out with purified hog thyroid peroxidase (A413 nm/A280 nm = 0.42). It reacted with H2O2 to form Compound I with a rate constant of 7.8 X 10(6) M-1 s-1. Compound I was reduced to Compound II by endogeneous donor with a half-life of 0.36 s. Compound I was reduced by tyrosine directly to the ferric enzyme with a rate constant of 7.5 X 10(4) M-1 s-1. Tyrosine could also reduce Compound II to the ferric enzyme with a rate constant of 4.3 X 10(2) M-1 s-1. Methylmercaptoimidazole accelerated the conversion of Compound I to Compound II and reacted with Compound II to form an inactivated form, which was discernible spectrophotometrically. The reactions of thyroid peroxidase with methylmercaptoimidazole quite resembled those of lactoperoxidase, but occurred at higher speeds. The absorption spectra of thyroid peroxidase were similar to those of lactoperoxidase and intestinal peroxidase, but obviously different from those of metmyoglobin, horseradish peroxidase, and chloroperoxidase. Similarity and dissimilarity between thyroid peroxidase and lactoperoxidase are discussed.     

Suicide inhibition of canine myocardial cytosolic calcium-independent phospholipase A2. Mechanism-based discrimination between calcium-dependent and -independent phospholipases A2

The majority of phospholipase A2 activity in myocardium is calcium-independent and selective for hydrolysis of plasmalogen substrate (Wolf, R. A., and Gross, R. W. (1985) J. Biol. Chem. 260, 7295-7303; Hazen, S. L., Stuppy, R. J., and Gross, R. W. (1990) J. Biol. Chem. 265, 10622-10630). Accordingly, identification of an inhibitor which selectively targets calcium-independent phospholipases A2 would facilitate elucidation of the biologic significance of this class of intracellular phospholipases. We now report that the haloenol lactone, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (Compound 1), is a potent, irreversible, mechanism-based inhibitor of myocardial calcium-independent phospholipase A2 which is greater than 1000-fold specific for inhibition of myocardial calcium-independent phospholipase A2 in comparisons with multiple calcium-dependent phospholipases A2. Mechanism-based inhibition of myocardial cytosolic calcium-independent phospholipase A2 by Compound 1 was established by demonstrating: 1) time-dependent irreversible inactivation; 2) covalent binding of [3H]Compound 1 to the purified phospholipase A2; 3) ablation of covalent binding of [3H]Compound 1 after chemical inactivation of phospholipase A2 enzymic activity; 4) identical inhibition of myocardial phospholipase A2 by Compound 1 in the absence or presence of nucleophilic scavengers; 5) Compound 1 is a substrate for myocardial calcium-independent phospholipase A2 resulting in the generation of the electrophilic alpha-bromomethyl ketone; 6) phospholipase A2 inhibition requires the in situ generation of the reactive electrophile (i.e. neither the alpha-bromomethyl ketone nor the diproteoenol lactone analog are inhibitory); and 7) concomitant attenuation of the inhibitory potency and the extent of covalent adduct formation in the presence of saturating substrate. Collectively, these results demonstrate that the haloenol lactone, Compound 1, is a substrate for, covalently binds to, and irreversibly inhibits canine myocardial cytosolic calcium-independent phospholipase A2.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Three new cyclopiane-type diterpenes from a deep-sea derived fungus Penicillium sp. YPGA11 and their effects against human esophageal carcinoma cells

Cyclopianes, featuring a highly rigid 6/5/5/5-fused tetracyclic framework, are structurally unique and biologically significant and belong to a rarely reported diterpenoid family. Chemical investigation of an EtOAc extract of a deep-sea-derived Penicillium sp. led to the isolation of three new cyclopiane diterpenes, namely, conidiogenols C–D (1–2) and conidiogenone L (3). The structures were determined by extensive analyses of the spectroscopic data in association with ECD calculations and chemical conversion for configurational assignments. Compound 1 represents the second example of cyclopianes bearing a hydroxyl group at C-13. Compound 2, the third example of conidiogenols, possesses a distinct α-oriented 1-hydroxy group relative to other analogues. The bioassay study demonstrated that compounds 2 and 4–6 exhibited moderate inhibitory effects against five esophageal cancer cell lines with IC₅₀ values ranging from 25 to 55 μM. The cytotoxicities of all compounds toward esophageal cancer cell lines were evaluated for the first time.     

Enzymes of vitamin B6 degradation. Purification and properties of two N-acetylamidohydrolases

alpha-(N-Acetylaminomethylene)succinic acid hydrolase (Compound A hydrolase, EC 3.5.1-) and alpha-hydroxymethyl-alpha'-(N-acetylaminomethylene)succinic acid hydrolase (Compound B hydrolase, EC 3.5.1-) were purified to homogeneity from Pseudomonas MA-1 and Arthrobacter Cr-7, respectively. The two inducible enzymes catalyze Reactions 1 and 2, respectively, which release the first generally useful anabolic intermediates during growth of these organisms with (formula; see text) pyridoxine as a sole source of carbon and nitrogen. Compound A hydrolase is a monomeric protein of Mr 32,500 with aspartic acid as its NH2-terminal residue. Compound B hydrolase (Mr congruent to 205,000) is a multimer containing probably six identical subunits with glycine as the NH2 terminus. The two enzymes have quite different amino acid analyses, although both are high in Asx and Glx, lack tryptophan, and show similar stabilities to pH and temperature. Compound A hydrolase has a pI of 4.4, a Km of 3.3 microM, and a Vmax of 3.1 mumol X min-1 X mg-1 at pH 6.5 and 25 degrees C; no analogue substrates were found. Compound B hydrolase has a pI of 4.2, a Km of 25 microM, and a Vmax of 3.8 mumol X min-1 X mg-1 at 25 degrees C and pH 7.0; it also hydrolyzes Compound A slowly. Both enzymes are inhibited competitively by di- and tricarboxylic acids, itaconic acid being among the most effective. Sulfite inhibits both enzymes by a time-dependent mechanism not yet understood. The two amidases appear to differ greatly in architecture despite the similarity in properties and in the overall reactions they catalyze.