Peptide-based inhibitors of the hepatitis C virus serine protease

Hexapeptide DDIVPC-OH is a competitive inhibitor of the hepatitis C virus (HCV) NS3 protease complexed with NS4A cofactor peptide. This hexapeptide corresponds to the N-terminal cleavage product of an HCV dodecapeptide substrate derived from the NS5A/5B cleavage site. Structure-activity studies on Ac-DDIVPC-OH revealed that side chains of the P4, P3 and P1 residues contribute the most to binding and that the introduction of a D-amino acid at the P5 position improves potency considerably. Furthermore, there is a strong preference for cysteine at the P1 position and conservative replacements, such as serine, are not well tolerated.     

Cleavage activity of hepatitis C virus serine proteinase

To study the character of the hepatitis C virus (HCV) encoding serine proteinase and to search for inhibitors, a practical in vitro assay system using the purified enzyme and synthetic peptide substrates was established. The enzyme used was expressed in Escherichia coli as a fusion form with protein tags and purified to apparent homogeneity by single-step affinity chromatography. The purified enzyme exhibited proteolytic activity with pH optima of around eight, and the addition of NS4A fragments increased the activity as well as the thermal stability of the enzyme. The activity was inhibited by EDTA and some divalent ions, i.e., copper and zinc, though calcium, magnesium, and manganese were stimulative both in the presence and absence of the NS4A fragment. None of the common protease inhibitors, including serine protease inhibitors, effectively inhibited the activity. Based on the kinetic parameters of the cleavage reaction of the synthetic 20 mer peptides corresponding to the three cleavage sites, NS4A/4B, NS4B/5A, and NS5A/5B, the peptide with the NS5A/5B junction was found to be the most efficient substrate. Analysis of the minimal peptide substrate of NS5A/5B indicated that 5 to 7 amino acids on both sides of the junction were required for efficient cleavage. These findings are expected to contribute to the search for a proteinase inhibitor.     

Potent peptide inhibitors of human hepatitis C virus NS3 protease are obtained by optimizing the cleavage products

In the absence of a broadly effective cure for hepatitis caused by hepatitis C virus (HCV), much effort is currently devoted to the search for inhibitors of the virally encoded protease NS3. This chymotrypsin-like serine protease is required for the maturation of the viral polyprotein, cleaving it at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites. In the course of our studies on the substrate specificity of NS3, we found that the products of cleavage corresponding to the P6-P1 region of the substrates act as competitive inhibitors of the enzyme, with IC50s ranging from 360 to 1 microM. A detailed study of product inhibition by the natural NS3 substrates is described in the preceding paper [Steinkühler, C., et al. (1997) Biochemistry 37, 8899-8905]. Here we report the results of a study of the structure-activity relationship of the NS3 product inhibitors, which suggest that the mode of binding of the P region-derived products is similar to the ground-state binding of the corresponding substrates, with additional binding energy provided by the C-terminal carboxylate. Optimal binding requires a dual anchor: an "acid anchor" at the N terminus and a "P1 anchor" at the C-terminal part of the molecule. We have then optimized the sequence of the product inhibitors by using single mutations and combinatorial peptide libraries based on the most potent natural product, Ac-Asp-Glu-Met-Glu-Glu-Cys-OH (Ki = 0.6 microM), derived from cleavage at the NS4A-NS4B junction. By sequentially optimizing positions P2, P4, P3, and P5, we obtained several nanomolar inhibitors of the enzyme. These compounds are useful both as a starting point for the development of peptidomimetic drugs and as structural probes for investigating the substrate binding site of NS3 by modeling, NMR, and crystallography.     

Mutagenesis of the coxsackie B3 virus 2B/2C cleavage site: determinants of processing efficiency and effects on viral replication

The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.     

cis-acting RNA elements required for replication of bovine viral diarrhea virus-hepatitis C virus 5'' nontranslated region chimeras

Pestiviruses, such as bovine viral diarrhea virus (BVDV), share many similarities with hepatitis C virus (HCV) yet are more amenable to virologic and genetic analysis. For both BVDV and HCV, translation is initiated via an internal ribosome entry site (IRES). Besides IRES function, the viral 5' nontranslated regions (NTRs) may also contain cis-acting RNA elements important for viral replication. A series of chimeric RNAs were used to examine the function of the BVDV 5' NTR. Our results show that: (1) the HCV and the encephalomyocarditis virus (EMCV) IRES element can functionally replace that of BVDV; (2) two 5' terminal hairpins in BVDV genomic RNA are important for efficient replication; (3) replacement of the entire BVDV 5' NTR with those of HCV or EMCV leads to severely impaired replication; (4) such replacement chimeras are unstable and efficiently replicating pseudorevertants arise; (5) pseudorevertant mutations involve deletion of 5' sequences and/or acquisition of novel 5' sequences such that the 5' terminal 3-4 bases of BVDV genome RNA are restored. Besides providing new insight into functional elements in the BVDV 5' NTR, these chimeras may prove useful as pestivirus vaccines and for screening and evaluation of anti-HCV IRES antivirals.     

Bovine viral diarrhea virus strain Oregon: a novel mechanism for processing of NS2-3 based on point mutations

Bovine viral diarrhea virus (BVDV) isolates can either be cytopathogenic (cp) or noncytopathogenic (noncp). While both biotypes express the nonstructural protein NS2-3, generation of NS3 strictly correlates with the cp phenotype. The production of NS3 is usually caused by cp specific genome alterations, which were found to be due to RNA recombination. Molecular analyses of the cp BVDV strain Oregon revealed that it does not possess such genome alterations but nevertheless is able to generate NS3 via processing of NS2-3. The NS3 serine protease is not involved in this cleavage, which, according to protein sequencing, occurs between amino acids 1589 and 1590 of the BVDV Oregon polyprotein. Transient-expression studies indicated that important information for the cleavage of NS2-3 is located within NS2. This was verified by expression of chimeric constructs containing cDNA fragments derived from BVDV Oregon and a noncp BVDV. It could be shown that the C-terminal part of NS2 plays a crucial role in NS2-3 cleavage. These data, together with results obtained by site-specific exchanges in this region, revealed a new mechanism for NS2-3 processing which is based on point mutations within NS2.     

Ribosomal S27a coding sequences upstream of ubiquitin coding sequences in the genome of a pestivirus

Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.     

Product inhibition of the hepatitis C virus NS3 protease

The nonstructural protein NS3 of the hepatitis C virus (HCV) harbors a serine protease domain that is responsible for most of the processing events of the nonstructural region of the polyprotein. Its inhibition is presently regarded as a promising strategy for coping with the disease caused by HCV. In this work, we show that the NS3 protease undergoes inhibition by the N-terminal cleavage products of substrate peptides corresponding to the NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B cleavage sites, whereas no inhibition is observed with a cleavage product of the intramolecular NS3-NS4A junction. The Ki values of the hexamer inhibitory products [Ki(NS4A) = 0.6 microM, Ki(NS5A) = 1.4 microM, and Ki(NS4B) = 180 microM] are lower than the Km values of the respective substrate peptides [Km(NS4A-NS4B) = 10 microM, Km(NS5A-NS5B) = 3.8 microM, and Km(NS4B-NS5A) > 1000 microM]. Mutagenesis experiments have identified Lys136 as an important determinant for product binding. The phenomenon of product inhibition can be exploited to optimize peptide inhibitors of NS3 protease activity that may be useful in drug development.     

Identification and characterization of an RNA-dependent RNA polymerase activity within the nonstructural protein 5B region of bovine viral diarrhea virus

Nonstructural protein 5B (NS5B) of bovine viral diarrhea virus (BVDV) contains sequence motifs that are predictive of an RNA-dependent RNA polymerase activity. We describe the expression and purification of the BVDV NS5B protein derived from an infectious cDNA clone of BVDV (NADL strain). BVDV NS5B protein was active in an in vitro RNA polymerase assay using homopolymeric RNA or BVDV minigenomic RNA templates. The major product was a covalently linked double-stranded molecule generated by a "copy-back" mechanism from the input template RNA. In addition, a nucleotide-nonspecific and template-independent terminal nucleotidyl transferase activity was observed with the BVDV NS5B preparation.     

Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion

Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains are generated in cattle persistently infected with noncytopathogenic (noncp) BVDV.cp BVDV strains are considered crucial for the development of fatal mucosal disease. Comparative analysis of cp and noncp BVDV strains isolated from one animal suffering from mucosal disease revealed that the genomes of the cp BVDV strain (CP7) and the corresponding noncp BVDV strain (NCP7) are highly homologous. However, only the genome of CP7 contains an insertion of 27 nucleotides in the NS2 coding region. The inserted sequence represents a duplication of bases 4064 to 4090 of the viral genome, located between the formerly neighboring nucleotides 4353 and 4354. Parts of the viral polyproteins of CP7 and NCP7 were expressed in the T7 vaccinia virus system. These studies revealed that the insertion identified in the CP7 genome is necessary and sufficient for the induction of NS2-3 cleavage. Since the expression of NS3 is strictly correlated to cp BVDV, the insertion identified in the genome of BVDV CP7 represents most likely the relevant mutation leading to the evolvement of CP7 from NCP7.     

Autoprocessing and peptide substrates for human herpesvirus 6 proteinase

Autoprocessing of the precursor form of human herpesvirus 6 (HHV-6) proteinase at two sites (termed M and R) is required to generate the mature enzyme. Kinetic constants were determined for the hydrolysis of a series of synthetic peptide substrates by mature HHV-6 proteinase, purified to homogeneity. Truncation or replacement of individual residues in peptides mimicking the R-site sequence, indicated that the minimum length for effective hydrolysis by the viral enzyme was P4-P3-P2-Ala*Ser-P2'-P3'-P4' and revealed the importance of the P1 Ala and P4 Tyr residues. Consequently, relevant (P1 or P4) mutations were introduced into the precursor form of the proteinase and the ability of these altered proteins to autoprocess was examined. Introduction of Val in place of the P1 Ala at the M-site essentially abrogated cleavage but mature HHV-6 proteinase was still generated by cleavage at the R-site, indicating that processing of the M-site is not a prerequisite for cleavage of the R-site in the precursor. At the R-site, mutation of the P1 Ala, or of the preceding P4 Tyr residue, prevented processing at the R-site in the precursor so that the mature form of HHV-6 proteinase was not generated. The accumulated data suggest a possible new approach to the design of inhibitors for therapeutic intervention in the life cycle of herpesviruses.     

The effect of chemical anti-inhibitors on fibrinolytic enzymes and inhibitors

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.     

Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex

Hepatitis C virus (HCV) protease NS3 and its protein activator NS4A participate in the processing of the viral polyprotein into its constituent nonstructural proteins. The NS3/4A complex is thus an attractive target for antiviral therapy against HCV. We expressed the full-length NS3 and NS4A in insect cells as a soluble fusion protein with an N-terminal polyhistidine tag and purified the two proteins to homogeneity. Cleavage at the junction between HisNS3 and NS4A occurs during expression, producing a noncovalent complex between HisNS3 and NS4A with a subnanomolar dissociation constant. We purified the HisNS3/4A complex by detergent extraction of cell lysate and by metal chelate chromatography. We removed the His tag by thrombin cleavage and then further purified the complex by gel filtration. The purified NS3/4A complex is active in a protease assay using a synthetic peptide substrate derived from the NS5A-NS5B junction, with kcat/K(m) of 3700 (+/- 600) M-1 s-1, an order of magnitude above those previously reported for NS3 expressed by other strategies. This high protease activity implies that the full-length sequences of NS3 and NS4A are required for optimal activity of the NS3 protease domain. We examined the dependence of the NS3/4A protease activity on buffer conditions, temperature, and the presence of detergents. We find that, under most conditions, NS3 protease activity is dependent on the aggregation state of the NS3/4A complex. The monodisperse, soluble form of the NS3/4A complex is associated with the highest protease activity.     

The arterivirus nsp4 protease is the prototype of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases

The replicase of equine arteritis virus, an arterivirus, is processed by at least three viral proteases. Comparative sequence analysis suggested that nonstructural protein 4 (Nsp4) is a serine protease (SP) that shares properties with chymotrypsin-like enzymes belonging to two different groups. The SP was predicted to utilize the canonical His-Asp-Ser catalytic triad found in classical chymotrypsin-like proteases. On the other hand, its putative substrate-binding region contains Thr and His residues, which are conserved in viral 3C-like cysteine proteases and determine their specificity for (Gln/Glu) downward arrow(Gly/Ala/Ser) cleavage sites. The replacement of the members of the predicted catalytic triad (His-1103, Asp-1129, and Ser-1184) confirmed their indispensability. The putative role of Thr-1179 and His-1199 in substrate recognition was also supported by the results of mutagenesis. A set of conserved candidate cleavage sites, strikingly similar to junctions cleaved by 3C-like cysteine proteases, was identified. These were tested by mutagenesis and expression of truncated replicase proteins. The results support a replicase processing model in which the SP cleaves multiple Glu downward arrow(Gly/Ser/Ala) sites. Collectively, our data characterize the arterivirus SP as a representative of a novel group of chymotrypsin-like enzymes, the 3C-like serine proteases.     

An experimental study of a concurrent primary infection with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) in calves

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 degrees C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response.     

Mutagenesis of the NS3 protease of dengue virus type 2

The flavivirus protease is composed of two viral proteins, NS2B and NS3. The amino-terminal portion of NS3 contains sequence and structural motifs characteristic of bacterial and cellular trypsin-like proteases. We have undertaken a mutational analysis of the region of NS3 which contains the catalytic serine, five putative substrate binding residues, and several residues that are highly conserved among flavivirus proteases and among all serine proteases. In all, 46 single-amino-acid substitutions were created in a cloned NS2B-NS3 cDNA fragment of dengue virus type 2, and the effect of each mutation on the extent of self-cleavage of the NS2B-NS3 precursor at the NS2B-NS3 junction was assayed in vivo. Twelve mutations almost completely or completely inhibited protease activity, 9 significantly reduced it, 14 decreased cleavage, and 11 yielded wild-type levels of activity. Substitution of alanine at ultraconserved residues abolished NS3 protease activity. Cleavage was also inhibited by substituting some residues that are conserved among flavivirus NS3 proteins. Two (Y150 and G153) of the five putative substrate binding residues could not be replaced by alanine, and only Y150 and N152 could be replaced by a conservative change. The two other putative substrate binding residues, D129 and F130, were more freely substitutable. By analogy with the trypsin model, it was proposed that D129 is located at the bottom of the substrate binding pocket so as to directly interact with the basic amino acid at the substrate cleavage site. Interestingly, we found that significant cleavage activity was displayed by mutants in which D129 was replaced by E, S, or A and that low but detectable protease activity was exhibited by mutants in which D129 was replaced by K, R, or L. Contrary to the proposed model, these results indicate that D129 is not a major determinant of substrate binding and that its interaction with the substrate, if it occurs at all, is not essential. This mutagenesis study provided us with an array of mutations that alter the cleavage efficiency of the dengue virus protease. Mutations that decrease protease activity without abolishing it are candidates for introduction into the dengue virus infectious full-length cDNA clone with the aim of creating potentially attenuated virus stocks.     

Poliovirus/Hepatitis C virus (internal ribosomal entry site-core) chimeric viruses: improved growth properties through modification of a proteolytic cleavage site and requirement for core RNA sequences but not for core-related polypeptides

H.-H. Lu and E. Wimmer (Proc. Natl. Acad. Sci. USA 93:1412-1417, 1996) have demonstrated that the internal ribosomal entry site (IRES) of poliovirus (PV) can be functionally replaced by the related genetic element from hepatitis C virus (HCV). One important finding of this study was that open reading frame sequences 3' of the initiating AUG, corresponding to the open reading frame of the HCV core polypeptide, are required to create a viable chimeric virus. This made necessary the inclusion of a PV 3C protease (3Cpro) cleavage site for proper polyprotein processing to create the authentic N terminus of the PV capsid precursor. Chimeric PV/HCV (P/H) viruses, however, grew poorly relative to PV. The goal of this study was to determine the molecular basis of impaired replication and enhance the growth properties of this chimeric virus. Genetic modifications leading to a different proteinase (PV 2Apro) cleavage site between the HCV core sequence and the PV polyprotein (P/H701-2A) proved far superior with respect to viral protein expression, core-PV fusion polyprotein processing, plaque phenotype, and viral titer than the original prototype PV/HCV chimera containing the PV 3Cpro-specific cleavage site (P/H701). We have used this new virus model to answer two questions concerning the role of the HCV core protein in P/H chimeric viral proliferation. First, a derivative of P/H701-2A with frameshifts in the core-encoding sequence was used to demonstrate that production of the core protein was not necessary for the translation and replication of the P/H chimera. Second, a viral construct with a C-terminal truncation of 23 amino acids of the core gene was used to show that a signal sequence for signal peptidase processing, when present in the viral construct, is detrimental to P/H virus growth. The novel P/H chimera described here are suitable models for analyzing the function(s) of the HCV elements by genetic analyses in vivo and for antiviral drug discovery.     

Class specific inhibition of house dust mite proteinases which cleave cell adhesion, induce cell death and which increase the permeability of lung epithelium

1. House dust mite (HDM) allergens with cysteine and serine proteinase activity are risk factors for allergic sensitization and asthma. A simple method to fractionate proteinase activity from HDM faecal pellets into cysteine and serine class activity is described. 2. Both proteinase fractions increased the permeability of epithelial cell monolayers. The effects of the serine proteinase fraction were inhibited by 4-(2-aminoethyl)-benzenesulphonyl fluoride hydrochloride (AEBSF) and soybean trypsin inhibitor (SBTI). The effects of the cysteine proteinase fraction could be inhibited by E-64. No reciprocity of action was found. 3. Treatment of epithelial monolayers with either proteinase fraction caused breakdown of tight junctions (TJs). AEBSF inhibited TJ breakdown caused by the serine proteinase fraction, whereas E-64 inhibited the cysteine proteinase fraction. 4. Agarose gel electrophoresis revealed that the proteinases induced DNA cleavage which was inhibited by the matrix metalloproteinase inhibitor BB-250. Compound E-64 inhibited DNA fragmentation caused by the cysteine proteinase fraction, but was without effect on the serine proteinase fraction. Staining of proteinase-treated cells with annexin V (AV) and propidium iodide (PI) revealed a diversity of cellular responses. Some cells stained only with AV indicating early apoptosis, whilst others were dead and stained with both AV and PI. 5. HDM proteinases exert profound effects on epithelial cells which will promote allergic sensitization; namely disruption of intercellular adhesion, increased paracellular permeability and initiation of cell death. Attenuation of these actions by proteinase inhibitors leads to the conclusion that compounds designed to be selective for the HDM enzymes may represent a novel therapy for asthma.