Structural and functional studies on a variant of cystatin purified from brain of Capra hircus.

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40-60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10-7 cm2 s-1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (Ki = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters - ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (-5.78 kcal mol-1) and positive ΔS (11.01 cal mol-1 deg-1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (-9.19 kcal mol-1) value implies a spontaneous CBC-papain interaction.

Functional proteomics-aided selection of protease inhibitors for herbivore insect control.

Studies have reported the potential of protease inhibitors to engineer insect resistance in transgenic plants but the general usefulness of this approach in crop protection still remains to be established. Insects have evolved strategies to cope with dietary protease inhibitors, such as the use of proteases recalcitrant to inhibition, that often make the selection of effective inhibitors very challenging. Here, we used a functional proteomics approach for the 'capture' of Cys protease targets in crude protein extracts as a tool to identify promising cystatins for plant improvement. Two cystatins found to differ in their efficiency to capture Cys proteases of the coleopteran pest Leptinotarsa decemlineata also differed in their usefulness to produce transgenic potato lines resistant to this insect. Plants expressing the most potent cystatin at high level had a strong repressing effect on larval growth and leaf intake, while plants expressing the weakest cystatin showed no effect on both two parameters compared to untransformed parental line used for genetic transformation. Our data underline the relevance of considering the whole range of possible protease targets when selecting an inhibitor for plant pest control. They also confirm the feasibility of developing cystatin-expressing transgenics resistant to a major pest of potato.

Purification and biochemical characterization of phytocystatin from Brassica alba.

Phytocystatins belong to the family of cysteine proteinases inhibitors. They are ubiquitously found in plants and carry out various significant physiological functions. These plant derived inhibitors are gaining wide consideration as potential candidate in engineering transgenic crops and in drug designing. Hence it is crucial to identify these inhibitors from various plant sources. In the present study a phytocystatin has been isolated and purified by a simple two-step procedure using ammonium sulfate saturation and gel filtration chromatography on Sephacryl S-100HR from Brassica alba seeds (yellow mustard seeds).The protein was purified to homogeneity with 60.3% yield and 180-fold of purification. The molecular mass of the mustard seed cystatin was estimated to be nearly 26,000 Da by sodium dodecyl sulfate polyacrylamide gel electrophoresis as well as by gel filtration chromatography. The stokes radius and diffusion coefficient of the mustard cystatin were found to be 23A° and 9.4 × 10(-7) cm(2) s(-1) respectively. The isolated phytocystatin was found to be stable in the pH range of 6-8 and is thermostable up to 60 °C. Kinetic analysis revealed that the phytocystatin exhibited non-competitive type of inhibition and inhibited papain more efficiently (K(i) = 3 × 10(-7) M) than ficin (K(i) = 6.6 × 10(-7) M) and bromelain (K(i) = 7.7 × 10(-7) M respectively). CD spectral analysis shows that it possesses 17.11% alpha helical content.

Potential Broad Spectrum Inhibitors of the Coronavirus 3CLpro: A Virtual Screening and Structure-Based Drug Design Study.

Human coronaviruses represent a significant disease burden; however, there is currently no antiviral strategy to combat infection. The outbreak of severe acute respiratory syndrome (SARS) in 2003 and Middle East respiratory syndrome (MERS) less than 10 years later demonstrates the potential of coronaviruses to cross species boundaries and further highlights the importance of identifying novel lead compounds with broad spectrum activity. The coronavirus 3CL(pro) provides a highly validated drug target and as there is a high degree of sequence homology and conservation in main chain architecture the design of broad spectrum inhibitors is viable. The ZINC drugs-now library was screened in a consensus high-throughput pharmacophore modeling and molecular docking approach by Vina, Glide, GOLD and MM-GBSA. Molecular dynamics further confirmed results obtained from structure-based techniques. A highly defined hit-list of 19 compounds was identified by the structure-based drug design methodologies. As these compounds were extensively validated by a consensus approach and by molecular dynamics, the likelihood that at least one of these compounds is bioactive is excellent. Additionally, the compounds segregate into 15 significantly dissimilar (p < 0.05) clusters based on shape and features, which represent valuable scaffolds that can be used as a basis for future anti-coronaviral inhibitor discovery experiments. Importantly though, the enriched subset of 19 compounds identified from the larger library has to be validated experimentally.

Papain-like protease (PLpro) inhibitory effects of cinnamic amides from Tribulus terrestris fruits.

Tribulus terrestris fruits are well known for their usage in pharmaceutical preparations and food supplements. The methanol extract of T. terrestris fruits showed potent inhibition against the papain-like protease (PLpro), an essential proteolylic enzyme for protection to pathogenic virus and bacteria. Subsequent bioactivity-guided fractionation of this extract led to six cinnamic amides (1-6) and ferulic acid (7). Compound 6 emerged as new compound possessing the very rare carbinolamide motif. These compounds (1-7) were evaluated for severe acute respiratory syndrome coronavirus (SARS-CoV) PLpro inhibitory activity to identify their potencies and kinetic behavior. Compounds (1-6) displayed significant inhibitory activity with IC50 values in the range 15.8-70.1 µM. The new cinnamic amide 6 was found to be most potent inhibitor with an IC50 of 15.8 µM. In kinetic studies, all inhibitors exhibited mixed type inhibition. Furthermore, the most active PLpro inhibitors (1-6) were proven to be present in the native fruits in high quantities by HPLC chromatogram and liquid chromatography with diode array detection and electrospray ionization mass spectrometry (LC-DAD-ESI/MS).

Biochemical, immunological and kinetic characterisation of thiol protease inhibitor (cystatin) from liver.

Regulation of the cysteine protease activity is imperative for proper functioning of the various organ systems. Elevated activities of cysteine proteinases due to impaired regulation by the endogenous cysteine proteinase inhibitors (cystatins) have been linked to liver malignancies. To gain an insight into these regulatory processes, it is essential to purify and characterise the inhibitors, cystatins. Present study was undertaken to purify the inhibitor from the liver. The purification was accomplished in four steps: alkaline treatment, ammonium sulphate fractionation, acetone precipitation and gel filtration column (Sephacryl S-100 HR). The eluted protein exhibited inhibitory activity towards papain, and its purity was further reaffirmed using western blotting and immunodiffusion. The purified inhibitor (liver cystatin (LC)) was stable in the pH range of 6-8 and temperature up to 45 °C. In view of the significance of kinetics parameters for drug delivery, the kinetic parameters of liver cystatin were also determined. LC showed the greatest affinity for papain followed by ficin and bromelain. UV and fluorescence spectroscopy results showed that binding of LC with thiol proteases induced changes in the environment of aromatic residues. Recent advances in the field of proteinase inhibitors have drawn attention to the possible use of this collected knowledge to control pathologies.

Potential value of a rice protein extract, containing proteinaceous inhibitors against cysteine proteinases from Porphyromonas gingivalis, for managing periodontal diseases.

Arg-specific gingipain (Rgp) is a major pathogenic determinant of Porphyromonas gingivalis which is a major pathogen in periodontal disease. We prepared protein extracts with Rgp-inhibitory activity from polished rice (Oryza sativa) and evaluated the effects of these extracts on the growth and pathogenicity of P. gingivalis. The extracts inhibited the proteolytic degradation of human proteins by P. gingivalis proteinases, and repressed the growth and homotypic biofilm formation of P. gingivalis. The disruption of adhesion of epithelial cells by P. gingivalis was also restricted by the rice protein extracts. Our results suggested that the rice protein extracts suppressed the pathogenicity and growth of P. gingivalis by inhibiting the bacterial proteinase activities, implying that the Rgp-inhibitory proteins prepared from rice may be potentially valuable as nutraceutical agents for preventing periodontal diseases.

A novel transglutaminase substrate from Streptomyces mobaraensis inhibiting papain-like cysteine proteases.

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at 42 degrees C, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the 28 degrees C culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower Ki (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.

Recombinant amaranth cystatin (AhCPI) inhibits the growth of phytopathogenic fungi.

Phytocystatins are cysteine proteinase inhibitors from plants implicated in defense mechanisms against insects and plant pathogens. We have previously characterized an amaranth cystatin cDNA and analyzed its response to different kinds of abiotic stress [37]. In order to characterize amaranth cystatin, the coding sequence was expressed in Escherichia coli using the pQE-2 vector. Recombinant cystatin was predominantly found in the soluble fraction of the cell extract. Large amounts (266 mgL(-1)) of pure recombinant protein were obtained by affinity chromatography in a single step of purification. The amaranth cystatin with a pI 6.8 and an apparent 28 kDa molecular mass inhibited papain (E.C.3.4.22.2) (Ki 115 nM), ficin (E.C.3.4.22.3) (Ki 325 nM) and cathepsin L (E.C.3.4.22.15) (Ki 12.7 nM) but not stem bromelain (E.C.3.4.22.32), and cathepsin B (E.C.3.4.22.1) activities, in colorimetric assays. Furthermore, it was able to arrest the fungal growth of Fusarium oxysporum, Sclerotium cepivorum and Rhyzoctonia solani. It was further demonstrated that recombinant AhCPI is a weak inhibitor of the endogenous cysteine proteinase activities in the fungal mycelium. These findings contribute to a better understanding of the amaranth cystatin activity and encourage further studies of this protein.

Characterisation of adult green lacewing (Chrysoperla carnea) digestive physiology: impact of a cysteine protease inhibitor and a synthetic pyrethroid.

BACKGROUND: In spite of concern regarding potential non-target effects of GM crops, few studies have compared GM pest control with conventional methods. The impacts of cypermethrin and oilseed rape expressing oryzacystatin-1 (OC-1) were compared in this study on the predator Chrysoperla carnea (Stephens). RESULTS: Adults fed purified rOC-1 showed a subtle shift in digestive protease profile, with an increasing reliance on serine proteases (chymotrypsin), increase in aspartic proteases and a slight reduction in elastase activity. Although there were no effects on mortality, onset of oviposition was delayed; however, once egg production commenced, egg laying and hatching success rates were comparable with those of controls. Oryzacystatin-1 expressed in pollen showed no detrimental effects. Cypermethrin had no effect on mortality owing to high levels of non-specific esterase activity resulting in partial breakdown of the insecticide. In spite of this, there was a significant delay in onset of oviposition and a significant reduction in egg production and viability. CONCLUSION: This study demonstrates the potential for pest management to impact on predators, but importantly it highlights the ability of the predator to detoxify/respond to treatments with different modes of action. In this case, exposure to an insecticide carried a greater fitness cost than exposure to a protease inhibitor expressed in transgenic crops.

Purification, characterization and kinetics of thiol protease inhibitor from goat (Capra hircus) lung.

In the present study, two molecular forms of goat lung cystatin (GLC), I and II, were purified to homogeneity by a two-step procedure including ammonium sulfate precipitation (40-60%) and ion exchange chromatography. The inhibitor forms migrated as single bands under native and SDS-PAGE with and without reducing agent giving molecular mass of 66.4 and 76.4 kDa, respectively. GLC-I possesses 0.07% and GLC-II 2.3% carbohydrate content and no -SH groups. GLC-I showed greater affinity for papain than for ficin and bromelain. Immunological studies showed that the inhibitor was pure and there was cross reactivity between anti-GLC-I serum and goat brain cystatin. Both inhibitor forms were stable in the pH range of 3-10 and up to 75 degrees C. GLC-I was found to possess 49% alpha-helical structure by CD spectroscopy. The inhibitor-papain complexes showed conformational changes as invoked by UV and fluorescence spectroscopic studies.

Calpain inhibitory flavonoids isolated from Orostachys japonicus.

The n-butanol (n-BuOH) fraction of Orostachys japonicus A. Berger (Crassulaceae) significantly inhibited calpain activity. Through the activity-guided isolation from the n-BuOH fraction, herbacetin 8-O-alpha-D-ribopyranoside (1), kaempferol (2), quercetin (3), afzelin (4), astragalin (5), isoquercetin (6) and quercitrin (7) were obtained. Their structures were determined by spectroscopic techniques. Among them, compound 3 and 5 had significant calpain inhibitory activities.

[Protein inhibitors of fibrin stabilizing factor FXIII].

The ability of cysteine proteinase inhibitors (CPIs) isolated from tubers of potato (Solanum tuberosum) to suppress transpeptidase activity of fibrin stabilizing factor (FXIIIa) through the direct effect on the essential SH group of the enzyme active site has been studied. The formation of fibrin clots soluble in 5 M urea and 2% acetic acid as well as spectrophotometric turbidity analysis of the stabilization and resistance of fibrin clots formed in the presence of FXIIIa and CPIs from potato tubers to plasmin, and electrophoresis of reduced fibrin samples indicate the decrease or absence of covalent crosslinking of fibrin chains. In addition, CPIs added to the substrate proved to decelerate fibrinogen polymerization almost twice relative to control. It is concluded that natural CPIs can both take part in the regulation of FXIIIa transpeptidase activity in vitro and modify the substrate.

Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp.

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.

Isolation, characterization and kinetics of goat cystatins.

Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3-10 and up to 95 degrees C. Immunological identity with the sheep LMW kininogen was obtained suggesting that the inhibitor is closely related to kininogens. Spectral studies confirm that the inhibitors have predominantly an alpha-helical structure and undergo major conformational changes during complex formation with papain. The inhibitors had similar inhibitory activities on cysteine proteinases. Both inhibitors inhibited papain, ficin and bromelain competitively, with maximum affinity for papain. The overall lower affinity of these inhibitors to cysteine proteinases compared to other known cystatins can be attributed to the unusual N-terminal sequence where Leu is substituted by Ile. Furthermore, N-terminal sequence analysis revealed maximum homology to mammalian LMW kininogen.

Neoadjuvant chemotherapy for epithelial ovarian cancer--role of apoptosis.

BACKGROUND: Ovarian cancer is one of the most frequently fatal gynecological cancers because most cases are diagnosed at an advanced stage. Loss of growth control and a marked resistance to apoptosis are considered major mechanisms driving tumor progression. Little is known about the effect of various treatment regimens on the distribution of molecular markers of apoptosis in epithelial ovarian cancer. The objective of this study was to compare the expression levels of both proapoptotic and antiapoptotic proteins p53, p73, Bcl-2, Bcl-XL and survivin in the ascitic cells and tumor samples of patients undergoing treatment with two different regimens. METHODS: A total of 24 patients with untreated epithelial ovarian cancer were randomized into two groups of 12 each. Group 1 patients received three cycles of chemotherapy prior to surgery and three cycles after surgery and group 2 patients received six cycles of chemotherapy prior to surgery. The expression of apoptosis-related proteins was analyzed in ascitic fluid and tumor samples by Western blotting and immunohistochemistry. The apoptotic index was also determined in these samples by the TUNEL assay. RESULTS: Significant decreases in antiapoptotic bcl-2 and survivin were seen, accompanied by increases in apoptotic index in tumors that had undergone chemotherapy as compared to the baseline ascites samples. No significant change in bcl-XL was observed. A significant decrease in proapoptotic p53 was also seen. No expression of p73 was observed in tumors or ascites. The findings were similar in groups 1 and 2 patients and were not statistically significantly different, perhaps due to the small sample size (n=12) of each group. CONCLUSIONS: The above findings indicate that chemotherapy in ovarian carcinoma leads to an increase in apoptosis by a p53-independent pathway, which involves the downregulation of antiapoptotic Bcl-2 and survivin but not Bcl-XL. Furthermore, administering neoadjuvant chemotherapy (six cycles) as an alternative form of therapy for advanced epithelial ovarian cancer is more effective in inducing apoptosis than three cycles. However, the findings of this study need to be corroborated using a larger sample.

Cystatins may confer viral resistance in plants by inhibition of a virus-induced cell death phenomenon in which cysteine proteinases are active: cloning and molecular characterization of a cDNA encoding cysteine-proteinase inhibitor (celostatin) from Celosia cristata (crested cock's comb).

Cystatins (cysteine proteinase inhibitors) have been recently used in plants as antiviral strategy against those viruses whose replication involves cysteine proteinase activity. We proposed an idea that cystatins may confer resistance by inhibition of a virus-induced cell-death phenomenon in which cysteine proteinases are active. To test this idea, a full-length cDNA library was constructed from the preflowering stage of Celosia cristata (crested cock's comb) leaves, and a cDNA clone with cystatin domain was isolated using an oligonucleotide probe designed on the basis of the conserved peptide of plant cystatins. It was expressed in an Escherichia coli expression system as a fusion protein. The purified recombinant product, termed 'celostatin' (Celosia cystatin), inhibited the enzymatic activity of papain indicating its cystatin activity and prevented TMV (tobacco mosaic virus)-induced hypersensitive-response cell death in Nicotiana glutinosa (a wild species of tobacco) leaves by 65-70% at the concentration of approx. 50 ng/ml. It also offered resistance against TMV and caused normal growth of the test plant. Since the activity of cysteine proteinases is not involved in the TMV replication process, we speculated that inhibition of the hypersensitive response by celostatin may be due to the inactivation of proteolysis involved in the plant cell death programme, a phenomenon that has already been reported in animal systems.

Inhibition of proteasome activity by various fruits and vegetables is associated with cancer cell death.

There is a large amount of scientific evidence showing that fruits and vegetables lower the risk of cancer. However, the responsible molecular mechanisms remain poorly understood. Our previous studies have demonstrated that inhibition of proteasomal chymotrypsin-like activity is associated with cancer cell apoptosis, which may also be the major mechanism responsible for the anticancer effects of green tea polyphenols. In the current study, we tested the hypothesis that some fruits and vegetables inhibit tumor cell proteasome activity and that this inhibition contributes to their cancer-preventative activities. We report that the extracts of apple and grape are more potent than onion, tomato and celery in: (i) inhibiting the proteasomal chymotrypsin-like activity in leukemia Jurkat T cell extract; (ii) accumulating the polyubiquitinated proteins in intact Jurkat T cells; (iii) inducing activation of caspase-3/-7 and cleavage of poly(ADP-ribose) polymerase in intact Jurkat T cells; and (iv) inducing the appearance of spherical cells preferentially in prostate cancer PC-3 over the normal NIH 3T3 cell line. We also found that strawberry extract had some effect on Jurkat T cell extract and the prostate PC-3 cell line but not on intact Jurkat T cells. Our findings suggest that the proteasome is a cancer-related molecular target for, at least, the extracts of apple, grape and onion, and that the inhibition of proteasome activity by these fruits or vegetable may contribute to their cancer-preventative effects, although other molecular mechanisms may also be involved.

Purification and characterization of phytocystatins from kiwifruit cortex and seeds.

Kiwifruit cysteine proteinase inhibitors (KCPIs) were purified from the cortex and seeds of kiwifruit after inactivation of the abundant cortex cysteine proteinase actinidain. One major (KCPI1) and four minor cystatins were identified from Actinidia deliciosa ripe mature kiwifruit cortex as well as a seed KCPI from A. chinensis. The predominant cortex cystatin, KCPI1, inhibited clan CA, family C1 (papain family) cysteine proteinases (papain, chymopapain, bromelain, ficin, human cathepsins B, H and L, actinidain and the house dust mite endopeptidase 1), while cysteine proteinases belonging to other families, [clostripain (C11), streptopain (C10) and calpain (C2)] were not inhibited. Inhibition constants (K(I)) ranged between 0.001 nM for cathepsin L and 0.98 nM for endopeptidase 1. The K(I) (14 nM) for KCPI1 inhibiting actinidain is at least 2 orders of magnitude higher than for other plant proteinases measured. The cortex KCPI1 and a seed KCPI purified from seeds had the same N-terminal sequence (VAAGGWRPIESLNSAEVQDV). BLAST-matching the peptide sequence against an in-house generated Actinidia EST database, identified 81 cDNAs that exactly matched the measured KCPI1 peptide sequence. Peptide sequences of two other cortex KCPIs each exactly matched a predicted peptide sequence of a cDNA from kiwifruit. The predicted peptide sequence of KCPI1 of 116 amino acids encodes a signal peptide and does not contain cysteine. Without the signal peptide (mature protein), KCPI1 has a molecular mass of approximately 11 kDa, possesses the consensus sequence characteristic for the phytocystatins and shows the highest homology to a cystatin from Citrusxparadisi (52% identity). This is the first report of phytocystatins from the Ericales.

A new dimeric dihydrochalcone and a new prenylated flavone from the bud covers of Artocarpus altilis: potent inhibitors of cathepsin K.

A MeOH/CH(2)Cl(2) extract of the bud covers of Artocarpus altilis collected in Micronesia showed activity in a cathepsin K inhibition assay. In addition to the three known flavonoids isolated from the bud covers of this species, two new compounds have been identified whose structures were determined on the basis of spectral data. These compounds include a dimeric dihydrochalcone, cycloaltilisin 6 (2), and a new prenylated flavone, cycloaltilisin 7 (3). Novel compounds 2 and 3 have IC(50) values of 98 and 840 nM, respectively, in cathepsin inhibition.