Cysteine protease inhibitors for use in treatment of IGE mediated allergic diseases

The invention provides compounds for use in the treatment of allergic diseases including juvenile asthma and eczema. The compounds can inhibit IgE mediated reaction to major environmental and occupational allergens and can also have a prophylactic effect against allergic disease by preventing allergic sensitization to environmental and occupational allergens when administered to at-risk individuals (e.g., those at genetic risk of asthma and those exposed to occupational allergens in the workplace). The compounds are also useful for inactivation or attenuation of the allergenicity of allergens in situ. The invention provides compounds and ligands per se, pharmaceutical compositions containing the compounds, processes for producing the compounds and pharmaceutical compositions, and methods for using the compounds and compositions in treatment or prophylaxis of IgE mediated allergic diseases and in inactivation or attenuation of allergens in situ. The invention also enables the reduction or destruction of the viability of allergy-causing organisms.

FIELD OF THE INVENTION 
The invention relates to compounds for use in the treatment of allergic 
diseases including juvenile asthma and eczema. 
Compounds of the invention can inhibit IgE mediated reaction to major 
environmental and occupational allergens. They can also have a 
prophylactic effect against allergic disease by preventing allergic 
sensitisation to environmental and occupational antigens when administered 
to at-risk individuals (e.g. those at genetic risk of asthma, and those 
exposed to occupational allergens in the workplace). The compounds of the 
invention can also be useful for inactivation or attenuation of the 
allergenicity of allergens in situ. The invention provides novel compounds 
and ligands per se, pharmaceutical compositions containing the compounds, 
processes for producing the compounds and pharmaceutical compositions, and 
methods for using the compounds and compositions in treatment or 
prophylaxis of IgE mediated allergic diseases and in inactivation or 
attenuation of allergens in situ. The invention also provides means for 
the reducing or destroying the viability of allergy-causing organisms. 
The invention is made possible by our new understanding of the role of the 
low-affinity receptor for IgE (FceRII), also known as CD23, in IgE 
mediated allergic diseases. 
DESCRIPTION OF THE RELATED ART 
The Multiple Roles of CD23 CD23 plays an important role in the regulation 
of immune responses--particularly the regulation of IgE responses. CD23 is 
a cell surface protein which extends from the plasma membrane via a stalk 
which is cleaved proteolytically during immune responses. We have 
demonstrated that CD 23 is cleaved by Der p I; a protease which is the 
major allergen of the house dust mite, although the endogenous proteases 
responsible or cleaving CD 23 have not so far been identified. In its 
membrane bound form, CD23 acts as a cellular receptor for IgE and is found 
on various cell types including B cells, T cells, platelets, eosinophils, 
keratinocytes and also on antigen presenting cells (including follicular 
dendritic cells) which present antigens to T and B lymphocytes. The level 
of expression of CD23 at the cell surface determines its functionality and 
is regulated by cytokines, notably IL4. 
In its membrane bound form, CD23 allows eosinophils to attach to parasites 
via antigen-specific IgE. It also plays an important regulatory role on B 
lymphocytes (which produce antibodies). In the presence of soluble IgE, 
probably in the form so immune complexes with the allergen, cell surface 
CD23 becomes occupied by IgE, conveying an inhibitory signal to the 
B-lymphocyte. This is believed to be an important negative feedback loop 
in the regulation of IgE synthesis. Occupancy of membrane bound CD23 by 
IgE protects CD23 from proteolytic cleavage, preventing the release of 
"cytokine active" forms of soluble CD23 (see below) which favour the 
production of IgE as opposed to other classes of immunoglobulin. CD23 also 
interacts with ligands (or "counterstructures") other than IgE. By 
association of CD23 on an activated B cell with CD21 (the type two 
complement receptor "CR2") on a follicular dendritic cell of the lymph 
node, cell-surface CD23 functions as an intercellular adhesion molecule. 
This function of CD23 is believed to be important in the rescue of 
germinal centre B lymphocytes from "apoptosis" (i.e. programmed 
cell-death), allowing the survival of antibody producing clones which 
would otherwise have been destined to die. There is also evidence that 
CD23 associates with the cell-surface molecules responsible for presenting 
antigenic peptides to T lymphocytes (i.e. the HLA class-II molecules) and 
may thereby influence antigen presentation to T lymphocytes. Moreover, the 
presence and degree of expression of CD23 on Langerhans cells (a type of 
antigen presenting cell), and its affinity for immune complexes comprised 
of allergen and IgE, will also determine to what extent such complexes are 
processed and presented to T lymphocytes. CD23 may therefore influence 
antigen presentation to both B and T lymphocytes, processes which 
determine the degree and nature of immune responsiveness to foreign 
antigens. 
Proteolytic cleavage of CD23 Native CD23 (45 ka) can be cleaved from the 
cell surface by proteolytic digestion at several sites within the stalk 
region to generate soluble CD23 (sCD23). The largest soluble fragment is 
of 37 kDa. Cleavage nearer the membrane-distal lectin domain gives soluble 
fragments of 33, 29 and 25 kDa containing the lectin domain and a 
C-terminal tail. Some forms of sCD23 (notably the 37 kDa form) are active 
upon ocher cells. Thus, Ghadieri et al and Bonnefoy et al have 
demonstrated that sCD23 (37 kDa) is a potent stimulator of mast 
cells--eliciting degranulation at nanograms per ml concentrations. 
Moreover, the larger forms of sCD23 also have cytokine activities which 
favour the production of IgE and IgG4 subclass antibodies associated with 
allergic, anti-parasitic and chronic immune responses. Indeed in vitro 
experiments have shown that in the presence of IL4 sCD23 induces IgE 
producing B cells to differentiate into plasma cells (Liu; Gordon). The 
regulatory role of CD23 upon IgE synthesis has also been confirmed in vivo 
using antibodies to CD23 (which inhibit antigen-specific IgE responses), 
and using CD23 gene-knockout mice, in which antigen-specific IgE responses 
are exaggerated. 
In addition to these data from experiments in animals elevated levels of 
sCD23 and of CD23 positive peripheral blood lymphocytes have been reported 
in atopic individuals (Gordon et al) (Ghadieri et al) implicating CD23 as 
an important regulatory factor in IgE immune responses. 
From these considerations it its evident that CD23 has important regulatory 
functions determining the quality and quantity of an immune response, 
particularly affecting humoral immunity (i.e. the production of specific 
antibodies). Moreover, the physical form of CD23 (i.e. cellular versus 
soluble) has a major influence on its regulatory function, particularly in 
the case of the IgE responses of B lymphocytes. Thus, CD23 in its cellular 
form participates in the negative feedback inhibition of IgE synthesis. By 
contrast, in its soluble forms CD23 stimulates the production of IgE via 
its cytokine activities. The balance between cellular and soluble forms of 
CD23 is therefore seen to have a pivotal role in determining the character 
of an immune response, in particular whether IgE is produced against a 
particular antigen, and also how much IgE is produced. However, the nature 
of proteases which bring about the cleavage of CD23 and which determine 
the balance between membrane bound and soluble forms has not so-far been 
established, although current theory, supported only by circumstantial 
evidence, has it that CD23 is autocatalytic, and brings about its own 
cleavage from the plasma membrane. 
Proteolytic activity of certain environmental and occupational allergens. 
Studies in mice and in man suggest that allergic sensitising potency of 
environmental allergens is, in some cases, related to their proteolytic 
activity. Thus, papain (a cysteinyl protease of papaya) is a potent 
allergen in man. Also, inhaled bromelain (a cysteinyl protease of 
pineapple) causes occupational allergies and asthma (Gailhoffer 1988). 
Also, the major allergen of house dust mite (Der p I), to which many 
asthmatic individuals are sensitive, has proteolytic activity. Proteolytic 
enzymes of environmental antigens and parasites may influence the quality 
of T lymphocyte responses to favour IgE production (reviewed by Finkelmann 
1992) although how they do so has not been established. Thus, subcutaneous 
daily injections of certain strains of mice with papain result in a 
dramatic increase in non-specific IgE which is markedly attenuated by 
prior inactivation of the catalytic activity of the enzyme. The elevation 
of total IgE by active papain is associated with the production of 
cytokines characteristic of the T.sub.k 2 subset of T-helper lymphocytes 
which are involved in allergic and anti-parasitic responses. However, the 
substrate of this proteolytic mechanism whereby papain elevates IgE 
production has not been identified. 
SUMMARY OF THE INVENTION 
Current teaching has it that CD23 is cleaved from the plasma membrane by a 
putative autoproteolytic activity (i.e. a proteolytic activity of CD23 
itself upon CD23 as a substrate). No candidate protease (either endogenous 
or exogenous) other than CD23 has been proposed. Moreover, the putative 
"autoproteolytic" activity of CD23 has never been demonstrated. 
Surprisingly therefore, it has now been found that the exogenous protease 
and allergen Der p I, in highly purified form, is very effective and 
specific at cleaving CD23 from the plasma membrane of cultured B 
lymphocytes. Since the cleavage of CD23 is an important regulatory step 
governing IgE synthesis, it follows that the potent allergic sensitising 
activity of Der p I (i.e. its allergenicity) resides, in part, in its 
ability to cleave CD23 from the cell surface. Although it had been 
speculated previously that the proteolytic activity of Der p I might be 
related to its allergenicity, no explanation had been offered for the 
mechanism of the putative proteolytic event, nor has any candidate 
previously been proposed as a substrate of this proteolytic activity. 
In a first aspect of the invention, we have demonstrated that the cleavage 
of CD23 by (Gordon et al) is: i) stimulated by cysteine; ii) inhibited by 
the specific cysteinyl protease inhibitor E64; and iii) not inhibited by 
the trypsin protease inhibitor alpha-1-antitrypsin (which inhibits various 
trypsin-like proteases as well as trypsin). The compound E64 is 
L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (Sigma, Poole, UK). 
These findings demonstrate that Der p I is indeed a cysteinyl protease as 
suggested tentitively by earlier studies, and moreover that it is the 
cysteinyl protease activity of Der p I which is responsible for CD23 
cleavage. 
From these considerations, it follows that compounds of this invention 
other than E64, yet capable (like E64) of inhibiting cysteinyl proteases, 
would also prevent the cleavage of CD23 by Der p I. This would include the 
peptide sequence comprising the cleavage sites of CD23 which are cleaved 
by Der p I, and analogues thereof. The latter would include D-amino acid 
analogues, including "reverse-D" peptides made exclusively of D-amino 
acids but of the reverse sequence of the natural cleavage site--as 
described recently by Van Regenmortal et al for biologically active 
analogues of CD4. 
In a second aspect of the invention, we have now also demonstrated that the 
protease inhibitor human alpha-1-antitrypsin, rather than inhibiting Der p 
I, is a substrate for Der p I becoming cleaved at a specific site. Since 
Jul. 17, 1995, this site has been identified as "QVS/SGF" (Kalsheker N. et 
al 1996). It follows that peptide analogues (as described above for CD23 
cleavage sites) and non-peptide mimetics of this site may also be specific 
inhibitors of Der p I. Such compounds of this invention may therefore have 
uses as described above for inhibitors of Der p I (i.e. prevention of in 
vivo cleavage of CD23 by Der p I). Moreover, since Der p I is an 
extracorporeal digestive enzyme of the house dust mite, it follows that 
inhibitors of Der p I may cause the dust mite to have "indigestion" (i.e. 
nutritional deprivation) due to the failure of this enzyme. Indeed, since 
the food of the house dust mites is comprised mainly of human skin flakes 
(which contain alpha-1-antitrypsin) it may be necessary to Der p I to 
destroy or inactivate alpha-1-antitrypsin in order to digest the skin 
flakes. Thus, in a third aspect of the invention inhibitors of Der p I are 
predicted to have a "toxic" effect (via nutritional deprivation) on house 
dust mites. 
Inhibitors of Der p I may be useful for killing house dust mites in situ, 
in addition to attenuating the allergenicity (i.e. sensitising activity) 
of Der p I. We believe that these effects would synergise resulting in a 
highly effective anti-asthma agent for application to furnishings (beds, 
carpets etc.) which are the natural habitat for house dust mites. 
We have demonstrated that the principal cleavage fragment of the native 45 
kDa form of CD23 released by Der p I is indistinguishable (by SDS 
electrophoresis) from the major naturally occurring cleavage fragment of 
CD23: i.e. the 25 kDa fragment. However, sequence analysis of the 
N-terminal of the fragment liberated by Der p I demonstrates that the 
cleavage site "QVS/SGF" recognised by Der p I is distinct from the natural 
cleavage site that generates the 25 kda fragment. Smaller amounts of 
larger (presumably "cytokine active") forms of CD23 were also generated by 
Der p I, indicating the existence of additional, more membrane proximal 
cleavage sites in the stalk region. A further cleavage site has also been 
identified by us in the C terminal tail region as SAE/SMG. 
Since inhibitors of the CD23 cleavage activity of Der p I (such as E64 and 
analogues) may also inhibit the endogenous protease(s) that cleave CD23, 
whether or not these proteases are identical in specificity to Der p I the 
invention therefore includes inhibitors of endogenous proteases that 
cleave CD23 in addition to exogenous proteases such as Der p I and 
bromelain and certain other environmental allergens with proteolytic 
activity. Where legally permissible, the invention includes the use of 
inhibitors of the enzymatic cleavage of CD23 (whether by endogenous or 
exogenous proteases) for the treatment of allergic diseases such as 
juvenile asthma and eczema, and the use of such inhibitors to inactivate 
the proteolytic activities of environmental sensitising agents or 
allergens such as Der p I and bromelain. 
In a third aspect the invention provides novel compounds which have 
cysteinyl protease inhibitor activity and are capable of inhibiting 
proteolytic cleavage of membrane bound CD23 in vivo excluding 
L-trans-epoxysuccinyl-leucylamido (4-guanidino) butane (E64). 
In a forth aspect the invention provides cysteinyl protease inhibitor 
compounds which include a chemical composition capable of adopting a 
structure essentially equivalent to an inhibitor of the enzyme Der p I, 
excluding E64, optionally together with a pharmaceutically acceptable 
carrier or excipient for use in the treatment of allergic diseases. 
In a fifth aspect the invention provides cysteinyl protease inhibitor 
compounds capable of adopting a structure having a pharmacophoric pattern 
essentially equivalent to the pharmacophoric pattern of a section of an 
inhibitor of Der p I, excluding E64. 
In a sixth aspect the invention provides a ligand which cross reacts with a 
cysteinyl protease inhibitor compound which inhibits the enzyme Der p I, 
excluding E64, which compound includes 1 or more copies of a motif which 
comprises: 
i) a hydrogen bond donor; 
ii) three hydrophobes; and 
iii) a hydrogen bond acceptor. 
In a seventh aspect the invention provides compounds or ligands of the 
general formula (I): 
##STR1## 
wherein X, Y and Z are N or CH; 
R.sub.1 is a blocking group for the N-terminal nitrogen; 
R.sub.2, R.sub.3, and R.sub.4 are side-chains on X, Y, and Z; and 
W is a group that reacts irreversibly with an active cysteine thiol of Der 
p I. 
In a eighth aspect the invention provides an agent for treatment of IgE 
mediated allergic disease which includes as active ingredient an effective 
amount of a compound selected from the group consisting of: a cysteinyl 
protease inhibitor; a substrate for Der p I which reacts with Der p I at a 
specific site; and a Der p I inhibitor capable of inhibiting the 
proteolytic enzyme activity of Der D I, the agent optionally including one 
or more of a pharmaceutically acceptable carrier, adjuvant or excipient. 
In a ninth aspect the invention provides an agent for attenuating or 
inactivating the allergenicity of Der p I which includes as active 
ingredient an effective amount of a compound having Der p I inhibitor 
activity, the agent optionally including one or more of a carrier, 
adjuvant, excipient. 
In a tenth aspect the invention provides an agent for reducing or 
destroying the viability of house dust mites which includes as active 
ingredient an effective amount of a compound having Der p I inhibitor 
activity, the agent optionally including one or more of a pharmaceutically 
acceptable carrier, adjuvant, excipient. 
In an eleventh aspect the invention provides a process for producing a 
compound or ligand of the invention which comprises synthesising a 
cysteinyl protease inhibitor compound or ligand and optionally conjugating 
said compound or ligand to a carrier 
Therefore, in summary, the present invention is based upon our appreciation 
that the major allergen of house dust mite faeces (Der p I), is capable of 
cleaving CD23 (the low affinity receptor for IgE) from the cell-surface of 
B-lymphocytes and presumably from other cell types. We demonstrate that 
this activity is stimulated by cysteine and can be abolished by the 
well-known cysteinyl protease inhibitor E64. 
The invention relates particularly to compounds capable of inhibiting the 
proteolytic cleavage of CD23 from the plasma membrane of cells by 
exogenous proteases (such as Der p I) bromelain and certain proteases and 
parasites) and to compounds capable of inhibiting endogenous proteases 
which cleave CD23 from the cell. 
The compounds may also have a prophylactic effect against allergic 
disease--by preventing allergic sensitisation to environmental and 
occupational antigens when administered to at-risk individuals (e.g. those 
at genetic risk of asthma, and those exposed to occupational allergens). 
The compounds may also be used for the inactivation of the proteolytic 
activity of environmental allergens in situ (e.g. house dust mite faecal 
allergen Der p I in beds, carpets and vacuum cleaners). Inactivation of 
the proteolytic activity of these allergens may attenuate their 
allergenicity (i.e. their capability to provoke allergies and asthma) 
which is due to their capability to cleave CD23 from the cell-surface. 
The compounds may also kill house dust mites by nutritional deprivation.

EXAMPLES 
Here we demonstrate that Der p I, a major allergen of house dust mite 
(Dermatophagoides pteronyssinus), cleaves CD23 from the surface of 
cultured human B cells (RPMI 8866 B cell line). The cleavage of the 
receptor from the B cell surface was associated with a parallel increase 
in sCD23 in the culture supernatant. Labelled antibody experiments and 
protease inhibition assays clearly demonstrate that Der p I is a cysteine 
protease that directly cleaves a 25K fragment of CD23. The proteolytic 
affect of Der p I has specificity for CD23, since none of the other B cell 
markers tested (CD20, HLA-DR, CD71 and CD49d) were affected. These data 
suggest that Der p I elicits IgE antibody responses in 80% of patients 
suffering from dust mite allergy, by its ability to proteolytically 
release sCD23, and thereby upregulate IgE synthesis. 
We have affinity purified Der p I from dust mite extract and tested its 
ability to proteolytically cleave CD23 expressed on cultured RPMI 8866 B 
cells, using FITC labelled monoclonal anti-human CD23 (Bu38). The data 
show that, in the presence of cysteine (5 mM), Der p I cleaves in a 
dose-dependent manner membrane CD23, thereby releasing sCD23 into the 
culture supernatant (FIG. 1a and 1b). The proteolytic activity of Der p I 
was inhibited by E64 (a cysteine protease inhibitor), but not by 
alpha-1-antitrypsin (a serine protease inhibitor), thereby confirming the 
cysteine protease nature of Der p I (FIG. 1c). We have in fact 
demonstrated that Der p I completely cleaves alpha-1-antitrypsin (1:10 
molar ratio) to yield a degradation pattern (FIG. 1d) similar to that 
generated by papain, a well characterised cysteine protease. A more 
detailed description of FIG. 1 is as follows: 
Cells were analysed on a FACScan (Becton Dickinson, Oxford, UK) with a 
linear fluorescence setting of 660 volts. The fluorescence (FL1) profile 
versus forward scatter (FSC) was used to monitor the cells, the 
amplification scale was altered according to the level of fluorescence. 
For each sample 4000 events were collected and then analysed using the 
flowMATE programme (DAKO, High Wycombe, UK). Data presented are 
representative of 3 replicate experiments, each point in a to c represents 
the mean of duplicate determinations, FIG. 1(a). Dose and cysteine 
dependency of CD23 cleavage by Der p I (for method of purification see 
FIG. 1(d) below). Der p I was pre-incubated (15 min at 37.degree. C.) with 
or without 5 mM cysteine and added to 2-3.times.10.sup.5 RPMI 8866 cells 
in a total volume of 200 ml RPMI 1640+10 mM HEPES. The mixture was then 
incubated (1 h at 37.degree. C.) and the cells, collected by 
centrifugation, were washed in RPMI 1640+10 mM HEPES and incubated (30 min 
at room temperature) with FITC conjugated anti-CD23 monoclonal antibody 
(Bu38, The Binding Site, Birmingham, UK). FIG. 1(b). Cleavage of membrane 
CD23 was associated with a parallel dose dependent release of sCD23 in the 
culture supernatant. The supernatant was collected from cultured RPMI 8866 
cells treated with Der p I (as described above) and diluted 1/5 for sCD23 
determination by ELISA (open circles) (The Binding Site, Birmingham, UK). 
In this ELISA there was no cross-reactivity between Der p I and sCD23. 
FIG. 1(c). Class specific inhibitor of cysteine proteases prevent membrane 
CD23 cleavage by Der p I. E64 (L-trans-epoxysuccinylleucylamido 
(4-guanidino) butane) (Sigma, Poole, UK) completely inhibits cleavage of 
CD23 by Der p I, whereas no such inhibitory effect was demonstrable with 
alpha-1-antitrypsin, a naturally occurring human serine protease 
inhibitor. One hundred ml of 5 g/ml Der p I was pre-incubated (30 min at 
37.degree. C.) with 10 ml of either E64 or alpha-1-antitrypsin and then 
added to the RPMI 8866 cells (as described above). The arrows indicate 
level of CD23 expression in the absence (upper arrow) and presence (lower 
arrow) of Der p I. FIG. 1(d). Silver stain SDS-PAGE (12% gel) analysis of 
the Der p I preparation, human alpha-1-antitrypsin and the effect of Der p 
I on alpha-1-antitrypsin. Der p I was purified by affinity chromatography 
using anti-Der p I antibody (4Cl, Indoor Biotechnologies, Clwyd, UK). The 
purity of the preparation was confirmed by N-terminal sequencing carried 
out on an automatic amino acid sequencer (Applied Biosystems, Foster City, 
Calif., USA). The sequence obtained 
(Thr-Asn-Ala-Cys-Ser-Ile-Asn-Gly-Asn-Ala, or TNACSINGNA SEQ ID No. 1) 
matches the published sequence of Der p I. The activity of the 
alpha-1-antitrypsin preparation was ascertained by active site titration 
against bovine chymotrypsin (Dr. David Lomas, personal communication). The 
gel shows single bands for Der p I (lane 1) and alpha-1-antitrypsin (lane 
2). Incubation (2 h at 37.degree. C.) of Der p I (0.25 mg) with 
alpha-1-antitrypsin (5 mg), in a total volume of 10 ml, results in the 
cleavage of a large fragment (arrow) from alpha-1-antitrypsin (lane 3). 
This pattern is in agreement with that generated by papain. The mass 
standards are indicated on the left. To investigate the enzymatic 
specificity of Der p I for CD23, we monitored the expression of other B 
cell markers following treatment with 2.5 mg/ml (final concentration) of 
Der p I. At this Der p I concentration, which has been shown to give 
maximum cleavage of CD23 (FIG. 1a), there was no significant loss of CD23, 
HLA-DR, CD71 and CD49d expressions (FIG. 2). A more detailed description 
of FIG. 2 is as follows: 
RPMI 8866 cells were treated with 100 ml of 5 mg/ml Der p I and the 
expression of membrane CD23 was monitored in parallel with other B cell 
surface markers (CD20, HLA-DR, CD71 and CD49d). These markers were 
detected using anti-CD20 (L27), anti-HLA-DR (L243) (Becton Dickinson, 
Oxford, UK), anti-CD71 (Ber-T9) (Dako, Buckinghamshire, UK) and anti-CD49d 
(HP2.1) (Immunotech, Westbrook, Me., USA) antibodies respectively. Paired 
results represent the expression of markers in the absence (open bars) and 
presence (solid bars) of Der p I. Data presented are representative of 3 
replicate experiments, each point represents the mean of duplicate 
determinations. 
To gain insight as to the Der p I cleavage site on CD23, we onitored the 
proteolytic cleavage process using Bu38 and EBVCSI monoclonal anti-CD23 
antibodies, which are directed against the lectin domain and the stalk 
region respectively. Thus, Bu38 detects all fragments down to 25 kDa, 
whereas EBVCSI recognises only fragments larger than 25 kDa (J. Gordon, 
personal communication). The results show that Der p I cleaves CD23 at a 
site close to the lectin domain, since EBVCS1 antibody was still capable 
of binding to the residual membrane bound portion of the receptor (FIG. 
3). However, at a Der p I concentration of greater than 1 mg/ml there also 
appeared to be some cleavage of CD23 fragments larger than 25 kDa. Since 
the highest concentration of Der p I (2.5 g/ml) resulted in complete loss 
of Bu38 binding and only partial loss of EBVCS1 binding, the preferred 
site of initial cleavage of CD23 by Der p I appears to be close to the 
lectin domain. A more detailed description of FIG. 3 is as follows: 
RPMI 8866 cells were treated with Der p I, as described above, and the 
expression of membrane CD23 was monitored using two monoclonal anti-CD23 
antibodies: Bu38 (recognises the lectin domain) and EBVCS1 (recognises the 
stalk region between 25 kDa fragments). Thus, Bu38 recognises the intact 
molecule and all soluble fragments, while EBVCS1 recognises the intact 
molecule and the residual membrane bound portion after cleavage of the 25 
kDa fragment (sCD23) (J. Gordon, personal communication). The experiment 
demonstrates that Der p I, at concentrations of up to 1 mg/ml, 
preferentially releases a 25 kDa fragment of CD23. Data presented are 
representatuve of 3 replicate experiments, each point represents the mean 
of duplicate determinations. 
The preferred cleavage site of CD23 giving rise to the 25 kD fragment has 
been identified by us as detailed above. 
Soluble CD23 is one of the signals known to induce IgE producing B cells to 
become plasma cells which are required for IgE production. Therefore the 
nature of the proteases that cleave CD23 in vivo is of considerable 
interest. Although it has been suggested that CD23 has autoproteolytic 
activity, we were previously unaware of what proteases cleave membrane 
CD23. We have demonstrated that Der p I, an exogenous cysteine protease, 
fulfils this function. Der p I elicits IgE antibody responses in 80% of 
patients suffering from dust mite allergy, and there is in vivo evidence 
that such patients have high circulating levels of sCD23. This ubiquitous 
inhaled allergen is clearly highly immunogenic, and we believe its 
immunogenicity may be due in part to its enzymatic activity. It has indeed 
been demonstrated that the allergenicity of papain, a cysteine protease 
showing sequence homology with Der pI, is highly related to its enzymatic 
activity. 
The demonstration that Der p I proteolytically cleaves membrane CD23 raises 
the question of the role of IgE in the allergic process. Firstly, IgE 
specific to Der p I could target Der p I to B lymphocytes and other CD23 
bearing cells (e.g. eosinophils), thereby helping to build a high 
concentration of this allergen on the cell surface. Secondly, the binding 
of IgE to CD23 may protect the receptor from proteolytic attack by Der p 
I. 
Purification of Der p I Protein 
Crude mite extract (.about.100 mg, SmithKline-Beecham) was dissolved in 5 
ml Phosphate Buffered Saline (PBS; 50 mM potassium phosphate; pH 7.4 
containing 150 mM NaCl). Der p I was purified by affinity column 
chromatography using 4 C1 antibody (Indoor Biotechnology, Deeside, U.K.) 
immobilised onto CNBr activated Sepharose 4B (Pharmacia, Milton Keynes, 
U.K.). The crude preparation was mixed with -2 ml of the affinity resin 
for 2 h at 4.degree. C. and then washed with 2-3 volumes of PBS. Elution 
of bound protein was carried out using 5 mM glycine containing 50% (v/v) 
ethylene glycol. Fractions (1-2 ml) were collected and neutralised with 
0.8 ml of 0.2 M sodium phosphate buffer, pH 7.0. The fractions were pooled 
and dialysed overnight against 4 L PBS followed by a second dialysis 
against 2 L PBS for 2-3 h. The total protein was concentrated as required 
by ultrafiltration (MacroSep; Flowgen, U.K.). 
This yielded protein of greater than 95% purity as judged by denaturing 
polyacrylamide gel electrophoresis in the presence of sodium dodecyl 
sulphate, C4 reverse phase high performance liquid chromatography 
(RP-HPLC) and high pressure size exclusion chromatography (HP-SEC) and no 
other contaiminating protease activity could be detected. 
Inhibitors of Der p I 
Using the purified Der p I it was surprisingly found that inhibitors to the 
enzyme could be made. These inhibitors are of the general formula 
##STR2## 
where X, Y, and Z may be N or CH. 
R.sub.1 is a blocking group for the N-terminal amino acid nitrogen (T. 
Greene. Protective Groups In Organic Synthesis). R.sub.2, R.sub.3, and 
R.sub.4 are side-chains on X, Y, and Z. 
W is a group that reactions irreversibly with an active cysteine thiol of 
Der p I. 
Where X and Y are CH, stereochemistry is exclusively of the "S" 
configuration, providing L -alpha-amino acid residues. Where Z is CH, the 
configuration may be "R" or "S" dependent upon W, but the chiral centre is 
derived stereospecifically with retention of configuration from the L 
-alpha-amino acid precursor. Where X, Y or Z are nitrogen, the residue is 
a peptidomimetic, an "azapeptide". 
Preferably, R.sub.1 represents an optionally substituted hydrophobic aryl 
or heteroaryl group optionally connected through a heteroatom (O, S, N, P) 
to the carbonyl. When connected through N or P the heteroatom may be mono 
or diaryl or mono or diheteroaryl substituted. 
Alternatively, R.sub.1 represents a hydrophobic aliphatic group of 3 
carbons or more, linear or branched optionally connected through a 
heteroatom (O, S, N, P) to the carbonyl. When connected through N or P, 
the heteroatom may be mono or di-substituted. 
These compounds can also be optionally substituted aryl for example 
optionally substituted phenyl, naphthyl or unsubstituted 2-naphthyl or 
9-anthracyl. Additionally, optionally substituted phenyl may be 
uonsubstituted phenyl or phenyl having 1 to 5 fluoro substituents or 
phenyl having 1 to 3 substituents where the substituents are independently 
selected from the group which comprises lower alkyl, lower alkoxy, nitro, 
halo, acetyl, benzoyl, hydroxyl, amino, methylamino, --COOH, --CONH.sub.2, 
--COOR.sup.2, and NHCOR.sup.2 wherein R.sup.2 is lower alkyl. 
Optionally substituted 1-naphthyl includes unsubstituted 1-naphthyl and 
1-naphthyl substituted at the 2-position with lower alkyl, lower alkoxy or 
trifluoromethyl. 
Optionally substituted heteroaryl includes optionally substituted, 5 or 6 
membered aromatic group containing 1 to 4 heteroatoms chosen from O, S, N, 
a 1 or 2-naphthyl or a 9-anthracyl group which may contain 1 to 4 
heteroatoms chosen from O, S, and N. 
Most preferably R.sub.1 represents pkhenyl, diphenyl amino radical, 
9-xanthenyl, piperonyl, phenyl amino radical, tert-butoxy, CF.sub.3 
-phenyl, a mono or disubstituted phenyl where the substituent is a lower 
alkyl C1-3, lower alkoxy C1-3, mono 2 or 3 amino or carboxy substituted 
phenyl, These criteria will also apply for diphenylamino radical and 
9-xanthenyl. In addition, straight chain or branched aliphatics such as 
pivolyl, n-butyl and variants thereof upto C8. 
Preferably R.sub.2 represents a hydrophobic side-chain as found bonded to 
the C-alpha of commercially available amino acids. Hydrophobic refers to 
straight or branched chain alkyl (Methyl such as Ala); cyclohexylmethyl; 
2-methylpropyl i.e. Leu; n-butyl i.e. Norleucine; 1-methylethyl i.e. Val; 
1-methylpropyl i.e. Ile; 3-methylbutyl, i.e. homoleucine; ethyl i.e. Abu. 
Alternatively, the hydrophobic chain may contain a heteroatom such as N, O, 
S such as 2-methylthioethyl (methionine), 4-aminobutyl i.e. Lys; or 
ethyl-2-carboamide i.e. Gln. 
Alternatively, the hydrophobic chain may be a phenylmethyl radical 
optionally containing a nitrogen atom or be substituted on the phenyl ring 
with --OH, alkoxy, phenyl, or alkyl at C1-3. 
Most preferably R.sub.2 represents biphenylmethyl, 1-methylethyl i.e. 
valine; methyl i.e. alanine; or cyclohexylmethyl i.e. cyclohexylalanine. 
Preferably R.sub.3 represents a C1 alkyl group optionally substituted with 
a heteroatom, O, or F. Alternatively, R.sub.3 may be 4-aminobutyl i.e. 
Lys; ethyl-2-carboxamide i.e. Gln; 2-(methylthiooxy) ethyl i.e. Met(O). 
Most preferably, R.sub.3 represents methyl i.e. alanine. 
Preferably, R.sub.4 represents a hydrophobic side-chain defined and with 
residues as described for R.sub.2. In addition, 2-hydroxyethyl, i.e. Thr; 
or 2-fluoroethyl. 
Most preferably R.sub.4 represents 3-methylbutyl i.e. homoleu; 
cyclohexylmethyl i.e. cha; 2-methylpropyl i.e. leucine; or n-butyl i.e. 
norleucine. 
Preferably W is selected from the group which comprises: 
##STR3## 
Preferably E is selected from the group which comprises: 
i) OAr or SAr 
##STR4## 
iii) heteroaryl iv) halogen 
##STR5## 
Preferably R is selected from the group which comprises alkyl and Ar. 
Preferably Ar is selected from the group which comprises optionally 
substituted aryl of heteroaryl. 
Preferably Y is selected from the group which comprises esters, sulphones, 
carboxylates, amides, phosphonates, ketones, sulfonates, nitriles, 
sulphonamides and nitro compounds. 
DEFINITIONS 
Optionally substituted aryl is preferably optionally substituted phenyl, 
benzyl or naphthyl. Optionally substituted phenyl is preferably 
unsubstituted phenyl or phenyl having 1 to 5 fluoro substituents or phenyl 
having 1 to 3 substituents where the substituents are independently 
selected form the group comprises lower alkyl, lower alkoxy, nitro, halo, 
acetyl, benzoyl, hydroxy, amino, methylamino, dimethylamino, diethylamino, 
methylthio, cyano, trifluoromethyl, phenylsulfonamidecarbonyl 
(--CONHSO.sub.2 C.sub.6 H.sub.5), --COOH, --CONH, --COOR, NHCOR wherein 
.sub.2 R is lower alkyl and 2,3,5,6,-tetramethyl-4-carboxy-phenyl 
(--C.sub.6 H.sub.5 (CH.sub.3).sub.4 --COOH). 
Optionally substituted 1-naphthyl includes unsubstituted 1-naphthyl and 
1-naphthyl substituted at the 2-position with lower alkyl, lower alkoxy, 
or trifluoromethyl. 
Halogen is preferably bromo, chloro or fluoro. 
Alkyl is preferably a branched or unbranched, saturated aliphatic 
hydrocarbon radical, having the number of carbon atoms specified, or if no 
number is specified, having up to 8 carbon atoms. The prefix "alk--" is 
also indicative of a radical having up to 8 carbon atoms in the alkyl 
portion of that radical, unless otherwise specified. Examples of alkyl 
radicals include methyl, ethyl, n-propyl, iospropyl, n-butyl, tert-butyl, 
n-pentyl, n-hexyl, and the like. The terms "lower alkyl" and "alkyl of 1 
to 4 carbon atoms" are, within the context of this specification, 
synonymous and used interchangeably. 
Optional or optionally indicates that the subsequently described event or 
circumstance may or may not occur, and that the description includes 
instances where said event or circumstance occurs and instances in which 
it does not. For example, "optionally substituted phenyl" means that the 
phenyl radical may or may not be substituted and that the description 
includes both unsubstituted phenyl radicals and phenyl radicals wherein 
there is substitution. 
These inhibitors were exemplified by the following examples which were 
produced as detailed. 
Synthesis of Der p I Inhibitors 
Potential inhibitors for Der p I were synthesised according to the general 
methods described below. Following synthesis the compounds were subjected 
to electrospray or MALDI-TOF mass spectrometry (MS) and the results are 
indicated. 
Compound 1: N-Benzoyl-L-valyl-L-alanyl-L-norleucine 
Solid phase benzoylated peptide synthesis. 
Resin Loading (Step 1) 
2-Chlorotritylchloride resin (4.9 g, 1.05 mmol/g, Novabiochem) was swelled 
in dichloromethane (40 ml) and a suspension of Fmoc-L-norleucine added and 
stirred for 5 minutes. A solution of diisopropylethylamine in DCM (10 ml, 
57 mmol in 30 ml) was added over 5 minutes and the resulting mixture 
stirred at room temperature for 2 hours. Methanol (5 ml) added and 
reaction mixture stirred for a further 10 minutes before resin filtered 
and washed with 3.times.DCM, 2.times.DMF, 2.times.2-propanol, 2.times.DMF, 
2.times.2-propanol, methanol, 2.times.ether and dried under vacuum for 24 
hours. 
Amino Acid Deprotection (Step 2) 
Fmoc-L-norleucine loaded resin was deprotected by treatment with 20% 
piperidine in DMF over 4 hours. The swollen resin was filtered, washed 
with 5.times.DMF, 2.times.ether and dried under vacuum for 24 hours. 
Peptide Chain Extension (Step 3) 
L-Norleucine loaded resin (5 mmol) was added to a solution of 
Fmoc-L-alanine (6.23 g, 20 mmol), hydroxybenzotriazole (3.0 g, 20 mmol). 
2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate 
(7.59 g, 20 mmol) and diisopropylethylamine (6.97 ml, 40 mmol) in DMF (20 
ml) and allowed to swell over 4 hours with mild agitation. Resin was 
filtered and washed with 4.times.DMF, 2.times.ether and dried under vacuum 
overnight. Steps (2) and (3) were carried out repetitively with 
Fmoc-L-alanine and Fmoc-L-valine to afford resin bound tripeptide 
H-L-valyl-L-alanyl-L-norleucine. 
Peptide Chain Benzoylation (Step 4) 
L-Valyl-L-alanyl-L-norleucine loaded resin (1 g, approx. 1 mmol) was added 
to a solution of benzoic acid (0.488 g, 4 mmol), hydroxybenzotriazole (0.6 
g, 4 mmol), 2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium 
hexafluorophosphate (1.52 g, 4 mmol) and diisopropylethylamine (1.40 ml, 8 
mmol) in DMF (5 ml) and allowed to sell over 6 hours with mild agitation. 
Resin was filtered and washed with 4.times.DMF, 2.times.ether and dried 
under vacuum overnight. 
Resin Cleavage (Step 5) 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine loaded resin (1.0 g, appr. 1 mmol) 
was treated with a 1% solution of trifluoroacetic acid in dichloromethane 
(20 ml) containing triethylsilane (320 .mu.l, 2 mmol) for 1 hour. Resin 
was removed by filtration and washed with dichloromethane (3.times.10 ml). 
Organic layer was collected, evaporated and triturated with ether to 
afford N-benzoyl-L-valyl-L-alanyl-L-norleucine (285 mg). 
Electrospray MS m/z 407 [MH.sup.+ ]. 
Compound 2 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine (140 mg, 0.34 mmol) was suspended 
in dry THF (3 ml) and dry DMF was added dropwise to afford homogeneity. 
The reaction mixture as cooled to -10.degree. C. and isobutylchloroformate 
(129 .mu.l, 1.0 mmol) and N-methylmorpholine (109 .mu.l, 1.0 mmol) added 
with stirring under Argon. The mixture was stirred for 30 minutes before a 
solution of diazomethane in ether (5 ml, approx. 2 mmol) was added. The 
reaction mixture was allowed to warm to room temperature over 1 hour 
before a 1:1 solution of acetic acid and 50% HBr (1 ml, 3.0 mmol HBr) was 
added dropwise and stirred for 15 minutes. The organic phase was diluted 
with ethylacetate (40 ml), washed with water (10 ml), brine (10 ml) and 
sat. bicarbonate (2.times.10 ml), dried over MgSO.sub.4 solvent removed 
under vacuum. This afforded an off white solid (152 mg) which could be 
further purified as required by preparative HPLC. Electrospray MS m/z 482 
[MH.sup.+ ] and 484 [MH.sup.+ ]. 
Compound 3 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-bis (trifluoromethyl)benzoyloxy 
methyl ketone 
A mixture of potassium fluoride (0.1 mmol, 6 mg) and 
2,6-bis(trifluoromethyl)benzoic acid (0.066 mmol, 17 mg) in dry DMF (500 
.mu.l) was stirred over molecular sieves at room temperature for 5 
minutes. A solution of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl 
ketone (0.033 mmol), 16 mg) in dry DMF (500 .mu.l) was added and the 
reaction mixture stirred for 1 hour. The reaction mixture was passed 
through a short silica plug and washed with 5% methanol in 
dichloromethane. Solvent was removed under vacuum and the residue purified 
using prep. HPLC. Freeze drying afforded (6.4 mg) as a white lyophilisate. 
Electrospray MS m/z 660 [MH.sup.+ ]. 
##STR6## 
Similarly the following compounds were prepared. 
Compound 4 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-dimethyl benzoyloxy methyl 
ketone 
(Electrospray MS m/z 552 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
2,6-dimethylbenzoic acid. 
Compound 5 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-hydroxybenzoyloxy methyl ketone 
(Electrospray MS m/z 540 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
2-hydroxybenzoic acid. 
Compound 6 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-dichlorobenzoyloxymethyl ketone 
(Electrospray MS m/z 592 [MH.sup.+ ] and 594 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
2,6-dimethylbenzoic acid. 
Compound 7 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine benzoyloxymethyl ketone 
(Electrospray MS m/z 524 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and benzoic 
acid. 
Compound 8 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,3,4,5,6-pentafluoro benzoyloxy 
methyl ketone 
(Electrospray MS m/z 614 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
2,3,4,5,6-pentafluorobenzoic acid. 
Compound 9 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 1,1-dimethylpropyloxymethyl ketone 
(Electrospray MS m/z 504 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
1,1-dimethylpropanoicacid. 
Compound 10 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 
N-(-benzyloxycarbonyl)-D-serinyl-(O-tert-butyl)oxymethyl ketone 
(Electrospray MS m/z 697 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
N-benzyloxycarbonyl-D-serinyl-O-tert-butylether. 
Compound 11 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine N(-benzyloxycarbonyl)-D-serinyloxy 
methyl ketone 
(Electrospray MS m/z 641 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethly ketone and 
N-benzyloxycarbonyl-D-serine. 
Compound 12 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-furanoxy methyl ketone 
(Electrospray MS m/z 514 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 2-furan 
carboxylic acid. 
Compound 13 
N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-dichlorophenylacyloxy methyl 
ketone 
(Electrospray MS m/z 606 [MH.sup.+ ], 608 [MH.sup.+ ]) from of 
N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 
2,6-dichlorophenylacetic acid. 
Standard preparative HPLC conditions were used to analyse these compounds 
thus C4 preparative HPLC system (Vydac, 22.times.250 mm) eluting at 10 ml 
per minute a gradient of 5-95% (90% acetonitrile (0.1% TFA)) over 30 
minutes. 
Compound 14 
N-Benzoyl amino-L-valyl-L-alanyl-L-norleucyl-hydroxamic acid 
To a suspension of Bz-Val-Ala-norLeu-OH (50 mg, 0.12 mmol) in THF (5 ml) in 
a plastic reaction vessel was added diazomethane (0.3 mmol) in ether 1.5 
ml. Gas evolution was observed and to the resulting clear solution was 
added acetic acid (0.05 ml) and the solution was evaporated to dryness. 
The residue was dissolved in methanol (2 ml) and hydroxylamine (2 mmol) in 
methanol (2 ml) was added and the solution stirred for 5 hours at room 
temperature. The solution was concentrated water added (2 ml), and the 
resulting solid was filtered and dried to yield 33 mg, 65%. 
Compound 15 
N-(Benzoyl amino-L-valyl-L-alanyl-L-norleucyl)-O-benzoyl hydroxamate 
To a solution of Bz-Val-Ala-norLeu-NHOH (10 mg, 0.022 mmol) in dry pyridine 
at -10.degree. C. was added benzoyl chloride (0.004 ml, 0.03 mmol) and 
stirred for 2 hours. The solution was evaporated and purified according to 
the method described in the preparation of Ethyl-(S)-(E)-3-((N-benzoyl 
valyl alanyl)amino-6-methyl-hept-2-enoate, collecting the peak elution at 
25-27 min. and lyophilised to yield 0.5 mg, 5%. 
Electrospray MS m/z 525 [MH.sup.+ ]. 
Compound 16 
N-(N-benzoyl-L-valyl-L-alanyl-L-norleucyl)-O-2,6-dimethyl-benzoyl 
hydroxamate 
To a solution of 2,6-dimethylbenzoic acid (4 mg, 0.024 mmol) in dry DMF (1 
ml) cooled to 0.degree. C. was added 1-hydroxy-7-azabenzotriazole (3.2 mg, 
0.023 mmol), O-7-azabenzotriazole-1-yl-1,1,3,3-tetramethyl uronium 
hexafluorophosphate (9 mg, 0.023 mmol) and N-methylmorpholine (0.008 ml, 
0.07 mmol) and the solution was stirred for 5 minutes. The hydroxamic acid 
Bz-Val-Ala-norLeu-NHOH (10 mg, 0.02 mmol) was added and the reaction 
stirred overnight. The solution was evaporated and purified according to 
the method described in the preparation Ethyl-(S)-(E)-3-((N-benzoyl valyl 
alanyl) amino-6-methyl-hept-2-enoate collecting the peak eluting at 26-28 
min. and lyophilised to yield 0.9 mg, 6%. 
Electrospray MS m/z 553 [M.sup.+ +H], 575 [M.sup.+ Na] 
Compound 17 
Preparation of N,O-dimethyl(tert-butoxycarbonyl 
amino-L-leucyl)hydroxylamine 
A solution of Boc-Leu-OH.H.sub.2 O (80.3 mmol) and N-methyl morpholine (88 
mmol) in THF (35 ml) was added to a pre cooled solution of isobutyl 
chloroformate (88 mmol) in THF (65 ml) under nitrogen at between -10 and 
-15.degree. C. over 40 minutes. The reaction was stirred at -10.degree. C. 
for 1 hour after which time N-methyl morpholine (88 mmol) was added 
followed by N,O-Dimethylhydroxylamine hydrochloride (88 mmol) portion wise 
between -10 and 0.degree. C. The reaction was then stirred at -10.degree. 
C. for 1 hour and then allowed to warm up to room temperature over night. 
The THF was then removed under vacuum and water (50 ml) and ethylacetate 
(200 ml) added. The organic layer was then washed with 0.1 M citric acid 
solution (4.times.50 ml), then saturated sodium bicarbonate (4.times.50 
ml), dried over magnesium sulphate and then concentrated under vacuum to 
give the product. 
Compound 18 
Preparation of N,O-dimethyl(amino-L-leucyl) hydroxylamine 
Hydrogen chloride in dioxane (4M, 75 mL) was added to Boc-Leu-N(OMe)Me (33 
mmol) with cooling and then stirred at room temperature for 1 hour. The 
solution was then concentrated under vacuum. Diethyl ether (100 ml) added 
and concentrated down to dryness three times to give the product. 
Compound 19 
Preparation of N,O-dimethyl (tert-butoxycarbonyl amino-L-alanyl-L-leucyl) 
hydroxylamine 
A solution of Boc-Ala-OH (46 mmol) and N-methyl morpholine (46 mmol) in THF 
(20 ml) was added to a pre cooled solution of isobutyl chloroformate (46 
mmol) in THF (30 ml) under nitrogen at between -10 and -15.degree. C. over 
30 minutes. The reaction was stirred at -10.degree. C. for 1 hour after 
which time a solution of N-methyl morpholine (46 mmol) and 
HCl.H2N-Leu-N(OMe)Me (41.8 mmol) in 1,4-dioxane (20 ml) was added drop 
wise slowly. The reaction was left for 1 hour at -10.degree. C. and then 
allowed to warm up to room temperature. After concentrating the solution 
under high vacuum, water (50 ml) and ethylacetate (200 ml) was added. The 
organic layer was then washed with 0.1 M citric acid solution (4.times.50 
ml), then saturated sodium bicarbonate (4.times.50 ml), dried over 
magnesium sulphate and then concentrated under vacuum to give the product. 
Compound 20 
Preparation of N,O-dimethyl(amino-L-alanyl-L-leucyl)hydroxylamine 
Hydrogen chloride in dioxane (4M, 80 mL) was added to Boc-Ala-Leu-N(OMe)Me 
(33 mmol) with cooling and then stirred at room temperature for 1.5 hour. 
The solution was then concentrated under vacuum. Diethyl ether (100 ml) 
was then added and concentrated down to dryness three times to give the 
product. 
Compound 21 
Preparation of N,O-dimethyl(tert-butoxycarbonyl 
amino-L-valyl-L-alanyl-L-leucyl)hydroxylamine 
A solution of Boc-Val-OH (46 mmol) and N-methyl morpholine (46 mmol) in THF 
(20 ml) was added to a pre cooled solution of isobutyl chloroformate (46 
mmol) in THF (30 ml) under nitrogen at between -10 and -15.degree. C. over 
30 minutes. The reaction was stirred at -10.degree. C. for 1 hour after 
which time a solution of N-methyl morpholine (46 mmol) and 
HCl.H2N-Ala-Leu-N(OMe)Me (41.8 mmol) in 1,4-dioxane (30 ml) was added drop 
wise slowly. The reaction was left for 1 hour at -10.degree. C. and then 
allowed to warm up to room temperature. After concentrating the solution 
under high vacuum, water (50 ml) and ethylacetate (200 ml) was added. The 
organic layer was then washed with 0.1 M citric acid solution (3.times.50 
ml), then saturated sodium bicarbonate (3.times.50 ml), dried over 
magnesium sulphate and then concentrated under vacuum to give the product. 
Electrospray MS m/z 445 [MH.sup.+ ] 
Compound 22 
Preparation of tert-butoxycarbonyl amino-L-valyl-L-alanyl-L-leucyl aldehyde 
A solution of lithium aluminium hydride (4.5 mmol)in THF (24.5 mL) was 
cooled to between -15 and -10.degree. C. Boc-Val-Ala-Leu-N(OMe)Me (2.2 
mmol) in THF (10 mL) was then added very slowly to maintain the low 
temperature. After 40 minutes ethyl acetate (10 mL) was added slowly at 
-15.degree. C. and then left for 10 minutes. Water (2 mL) was then added 
very slowly, again at -15.degree. C. and the reaction then allowed to warm 
up to room temperature. Citric acid solution (100 mL, 0.5M) was then added 
and the product extracted into ethyl acetate. The ethyl acetate layer was 
washed with 100 ml saturated sodium bicarbonate solution, followed by 100 
ml water and then dried over magnesium sulphate. The solution was then 
concentrated to give the product which was subsequently used crude. 
Electrospray MS m/z 386 [MH.sup.+ ] 
Compound 23 
Ethyl-(S)-(E)-3-((tert-butoxycarbonyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
To a suspension of sodium hydride (46 mg, 1.9 mmol) in anhydrous THF (4 ml) 
cooled to 0.degree. C. was added a solution of triethylphosphonoacetate 
(420 mg, 1.9 mmol) in THF (2 ml) dropwise over 5 minutes and the mixture 
stirred until gas evolution ceased. The solution was added dropwise to a 
solution of Boc-Val-Ala-Leucyl aldehyde (600 mg, 1.56 mmol) in dry THF 
cooled to -10.degree. C. The reaction mixture was stirred for 1 hour and 
saturated ammonium chloride (10 ml) was added. A white solid precipitated 
which was removed by filtration and the filtrate was partitioned between 
ethyl acetate and water. The organic layer was dried with magnesium 
sulphate and evaporated to give an oil which was crystallised from 
acetonitrile water to yield the title compound, 640 mg, 91%. 
Electrospray MS m/z 456 [M.sup.+ +H], 356 [(M.sup.+ -.sup.t BOC)+1] 
Compound 24 
(S)-(E)-3-((tert-butoxycarbonyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoic acid 
Ethyl-(S)-(E)-3-((tert-butoxy carbonyl amino valyl alanyl) 
amino-6-methyl-hept-2-enoate (455 mg, 1 mmol) was dissolved in dioxane (10 
ml) was water added followed by lithium hydroxide (126 mg, 3 mmol). The 
solution was stirred for 3 hours and 1M HCl aq was added until the pH 
reached neutrality. The dioxane was removed by rotary evaporation and the 
pH adjusted to 4 with 1M HCl aq. The title compound precipitated, filtered 
and washed with water to yield 420 mg, 98%. 
Electrospray MS m/z 428 [M.sup.+ +H] 
Compound 25 
1,1,1-Trifluoroethyl-(S)-(E)-3-((tert-butoxycarbonyl 
amino-L-valyl-L-alanyl) amino-6-methyl-hept-2-enoate 
The acid (Boc-Val-Ala-Leu-OH) (50 mg, 0.117 mmol) and dimethylaminopyridine 
(29 mg, 0.24 mmol) was dissolved in dry dichloromethane (1 ml) and cooled 
to 0.degree. C. Water soluble carbodiimide hydrochloride salt (26 mg, 0.13 
mmol) in 0.5 ml dichloromethane was added and the solution stirred for 5 
minutes, 1,1,1-Trifluoroethanol (0.017 ml, 0.23 mmol) in 0.5 ml 
dichloromethane was added and the reaction was allowed to warm to room 
temperature after 1 hour and the reaction mixture stirred overnight. The 
reaction mixture was washed 2.times.2 ml 0.5M citric acid solution, 
1.times.2 ml water, 1.times.2 ml saturated sodium bicarbonate solution, 
1.times.2 ml water, dried with magnesium sulphate and evaporated to 
dryness to give the title compound 
Electrospray MS m/z 510 [M.sup.+ +H], 410 [(M.sup.+ -.sup.t BOC)+1], 454 
[(M.sup.+ -.sup.t Bu)+1] 
Compound 26 
Ethyl-(S)-(E)-3-((N-benzoyl-L-valyl-L-alanyl) amino-6-methyl-hept-2-enoate 
The Ethyl-(S)-(E)-3-((tert-butoxycarbonyl amino valyl alanyl) 
amino-6-methyl-hept-2-enoate (16.6 mg, 0.036 mmol) was dissolved in 4.0M 
HCl in dioxane (2 ml) stirred at room temperature for 30 minutes and 
evaporated to dryness. The residue was dissolved in DMF (0.5 ml) and 
N-methylmorpholine (7.36 mg, 0.073 mmol) added followed by benzoyl 
chloride (5.4 mg, 0.038 mmol) in DMF 0.5 ml. The reaction stirred for 2 
hours, diluted with 0.1% trifluoroacetic acid solution (4 ml) and 
acetonitrile (2 ml) and injected onto a C4 preparative HPLC system 
(22.times.250 mm) eluting at 10 ml per minute, monitoring at 215 nm and a 
gradient of 10-90% system B over 25 minutes and holding at 90% for 15 
minutes. System A=0.1% TFA in water, system B=90% acetonitrile, 10% system 
A. The peak eluting at 26-28 minutes was collected and lyophilised to a 
white solid, yield 4.5 mg, 27%. 
Electrospray MS m/z 460 [M.sup.+ +H] 
In an identical manner to the above, the following compounds were prepared: 
Compound 27 
Ethyl-(S)-(E)-3-((2-trifluoromethyl-N-benzoyl-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
A yield of 3.7 mg, at 22% was obtained. Electrospray MS m/z 528 [M.sup.+ 
+H]. 
Compound 28 
Ethyl-(S)-(E)-3-((Piperonyloyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
yield 3.8 mg, 23%. 
Electrospray MS m/z 504 [M.sup.+ +H]. 
Compound 29 
Ethyl-(S)-(E)-3-((Phenyl carbamoyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
As per method above, except that phenyl isocyanate was used in place of an 
acid chloride, yield 1.5 mg, 10%. 
Electrospray MS m/z 475 [M.sup.+ +H]. 
Compound 30 
Ethyl-(S)-(E)-3-((Diphenyl carbamoyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
yield 2.3 mg, 13%. 
Electrospray MS m/z 551 [M.sup.+ +H]. 
Compound 31 
Ethyl-(S)-(E)-3-((Naphthoyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
yield 1 mg, 6%. 
Electrospray MS m/z 510 [M.sup.+ +H]. 
Compound 32 
Ethyl-(S)-(E)-3-((Quinazoloyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
yield 1.5 mg, 9%. 
Electrospray MS m/z 512 [M.sup.+ +H]. 
Compound 33 
Ethyl-(S)-(E)-3-((Morpholinoyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
yield 2.9 mg, 19%. 
Electrospray MS m/z 469 [M.sup.+ +H]. 
Compound 34 
Ethyl-(S)-(E)-3-((Xanthene-9-oyl amino-L-valyl-L-alanyl) 
amino-6-methyl-hept-2-enoate 
As per method above, except that xanthane-9-carboxylic acid (8.1 mg, 0.036 
mmol) was used in place of the acid chloride. Coupling of this acid was 
effected using 2-(1H-benzotriazole-1-yl)-1,1,3,3-teramethyluronium 
hexafluorophosphate (13.6 mg, 0.036 mmol), as activator and 
1-hydroxybenzotriazole (5.5 mg, 0.036 mmol) as catalyst in the presence of 
N-methylmorpholine (10.8 mg, 0.108 mmol). 
Yield 1.7 mg, 9%. 
Electrospray MS m/z 564 [M.sup.+ +H]. 
Compound 35 
Diethyl Phenylsulfonylmethylphosphonate 
(Adapted from I. Shahak, J. Almog, Synthesis 145 (1970).) The commercially 
available diethyl phenylthiomethylphosphonate (1.0 ml, 4.1 mmol) was 
dissolved in dichloromethane (10 ml). Sulphuric acid (10 ml, 25%) was 
added and the mixture cooled in ice. Solid Potassium permanganate was then 
added portionwise (3.times.0.5 g) with stirring after which time the 
reaction appeared to be complete. Solid sodium metabisulfite was added 
slowly until the mixture turned colourless. This was then extracted with 
ethyl acetate (.times.3) and the combined organic washings washed with 
saturated sodium bicarbonate solution followed by brine before drying over 
sodium sulphate. The volatiles were then removed in vacuo. The residue was 
purified by flash chromatography on silica eluting initially with ethyl 
acetate/hexane 8/2 followed by pure ethyl acetate. In this way the desired 
product, diethyl phenylsulfonylmethylphosphonate (1.0 g, quant) was 
obtained as a colourless solid. 
MS (MALDI-TOF): required (M.sup.+ (C.sub.11 H.sub.17 O.sub.5 PS)+1)=292; 
obtained (M.sup.+ +1)=292 
##STR7## 
Compound 36 
(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl)-L-alanyl)amino-1-phenylsulfon 
yl-5-methyl-1-hexene 
Diethyl phenylsulfonylmethylphosphonate (38 mg, 129 mmol) was dissolved in 
dry THF (10 ml) and then cooled to 0.degree. C. under an atmosphere of 
nitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 mmol) was 
added and the mixture stirred for 15 mins (effervescence). The aldehyde 
.sup.t Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was then added to the 
resulting solution and the mixture was stirred for 60 mins. The reaction 
was quenched by addition of dilute hydrochloric acid (0.1 M), followed by 
extraction with ethyl acetate (.times.3). The separated organic phase was 
sequentially washed with saturated sodium bicarbonate solution and brine 
before drying over sodium sulphate. The volatiles were removed in vacuo. 
The residue was purified by flash chromatography on silica eluting with 
ethyl acetate/hexane 4/6. An unidentified by-product was eluted first (12 
mg) followed by the desired product 
(S)-(E)-3-(tert-butoxycarbonyl-amino-L-valyl-L-alanyl)amino-phenylsulfonyl 
-5-methyl-1-hexene (22 mg, 32%) as a solid. 
Electrospray MS m/z 546 [M.sup.+ +Na], 424 [(M-.sup.t Boc)+1] 
##STR8## 
Compound 37 
Diethyl Methylsulfonylmethylphosphonate 
The commercially available Diethyl methylthiomethylphosphonate was 
converted to the title compound using the method of I. Shahak and J. 
Almog, Synthesis 171 (1969). 
##STR9## 
Compound 38 
(S)-(E)-3-((tert-butoxycarbonylamino-L-valyl)-L-alanyl)amino-1-methylsulfon 
yl-5-methyl-1-hexene 
Diethyl methylsulfonylmethylphosphonate (30 mg, 130 mmol) was dissolved in 
dry THF (5 ml) and then cooled to 0.degree. C. under an atmosphere of 
nitrogen. Sodium hydride (7 mg of 60% dispersion in oil, 175 mmol) was 
added and the mixture stirred for 15 mins (effervescence). The aldehyde 
.sup.t Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was then added to the 
resulting solution and the mixture then stirred for 60 mins. The reaction 
was quenched by addition of dilute hydrochloric acid (0.1 M), followed by 
extraction with ethyl acetate(.times.3). The separated organic phase was 
sequentially washed with saturated sodium bicarbonate solution and brine 
before drying over sodium sulphate. The volatiles were then removed in 
vacuo. The residue was purified by flash chromatography on silica eluting 
with ethyl acetate/hexane 8/2. An unidentified by-product was eluted first 
(4 mg) followed by the desired product 
(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-methylsulfonyl-5-m 
ethyl-1-hexane (24 mg, 40%) as a solid. 
Electrospray MS m/z 484 [M.sup.+ +Na], 362 [(M-.sup.t Boc)+1] 
##STR10## 
Compound 39 
Ethyl Diethylphosphorylmethylsulfonate 
Prepared in accordance with procedure B in L. Ghosez et. al. Tetrahedron 43 
5125 (1987). 
Electrospray MS m/z 261 [M.sup.+ +H], 283 [M.sup.+ +Na]. 
##STR11## 
Compound 40 
Ethyl(S)-(E)-3-((tert-butoxycarbonyl 
amino-L-valyl)-L-alanyl)amino-5-methylhexenylsulfonate. 
Ethyl diethylphosphorylmethanesulfonate (36 ml, .about.138 mmol) was 
dissolved in dry THF (5 ml) and then cooled to 0.degree. C. under an 
atmosphere of nitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 
mmol) was added and the mixture stirred for 15 mins (effervescence). The 
aldehyde .sup.t Boc-Val-Ala-Leucyl aldehyde (50 mg, 129 mmol) was added to 
the resulting solution and the mixture stirred for 30 mins. The reaction 
was quenched by addition of dilute hydrochloric acid (0.1 M), followed by 
extraction with ethyl acetate (.times.3). The separated organic phase was 
sequentially washed with sodium bicarbonate solution and brine before 
drying over sodium sulphate. The volatiles were then removed in vacuo. The 
residue was purified by flash chromatography on silica eluting with ethyl 
acetate/hexane 1/1. The desired product, 
Diethyl(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-5-methylhex 
enyl sulfonate, (22 mg, 35%) was obtained as a solid. 
electrospray MS m/z 492 [M.sup.+ +1], 392 [(M.sup.+ -.sup.t Boc)+1] 
##STR12## 
Determination of Kinetic Constant For Der-p I Substrates 
All Der-p I enzyme assays were routinely carried out in 50 mM potassium 
phosphate; pH 8.25 containing 1 mM ethylenediaminetetraaceticacid (EDTA) 
and 1 mM dithiothreitol (DTT). Product formation was monitored with 
respect to time by measuring the increase in fluorescence emission at 420 
nm and exciting at 320 nm. All assays were carried out at 25.degree. C. 
Stock solutions of the various substrates and/or inhibitors were made up 
in 100% dimethylsulphoxide (DMSO). 
The kinetic constants (K.sub.M and k.sub.cat) were calculated from the 
initial velocities of the enzymatic reaction at various substrate 
concentrations. These data were fitted, by regression analysis, to the 
Michaelis-Menten equation and the kinetic constants obtained. 
Inactivation Kinetics 
##STR13## 
The reaction of enzyme and inhibitor is comprised of two steps. The first 
is binding of enzyme and inhibitor to produce the enzyme inhibitor complex 
(E.I). This step is assumed to be rapid and reversible, relative to the 
other steps, and no chemical reaction occurs. In this case k.sub.1 is the 
second order rate constant for the formation of the E.I complex and 
k.sub.-1 is the first order rate constant for the breakdown of the E.I. 
The second step in the process, occurring at a rate k.sub.2, is the 
formation of the enzyme-inhibitor covalent complex (EI) resulting in 
irreversible inactivation of the enzyme. 
The practice of inactivation kinetics of enzymes have been described by two 
standard accepted methods (Schemes 1 and 2). The first (Scheme 1) is the 
dilution method described by Kitz, C. G. and Wilson, I. B., (1962), J. 
Biol. Chem., 237, 3245-3249. In this case enzyme and inhibitor are 
pre-incubated for a set period of time prior to quenching of this reaction 
by the addition of an excess of substrate. The second method (Scheme 2), 
is monitoring enzyme inactivation in the presence of substrate and 
irreversible inhibitors. This method has been described previously (Tian, 
W. -X. & Tsou, C. -L, (1982), Biochemistry, 21, 1028-1032; Morrison, J. F. 
& Walsh, C. T., (1988), Adv. Enzymol. Relat. Areas Mol. Biol., 61, 
201-301) and the equations describing the kinetics are shown below (Eq. 1, 
2 and 3). In both cases the inhibitor concentration employed is at least 
10 times greater than the enzyme concentration in order to maintain 
pseudo-first order conditions. 
EQU k.sub.app =k.sub.2 [I]/1+[S]/K.sub.M [I]+K.sub.1) Eq. 1 
EQU [Product]=v.sub.s t+(v.sub.0 -v.sub.5)[1-exp(-k.sub.app t)]/k.sub.app +dEq. 
2 
EQU second order rate constant=(k.sub.app /[I])(1+[S]/K.sub.M) Eq. 3 
The apparent inactivation rate constant (k.sub.app) was calculated using 
Eq. 2; where v.sub.o is the initial velocity of the reaction, v.sub.s is 
asymptotic steady-state velocity of the reaction, d is the intercept at 
time zero. The second order rate was calculated using Eq. 3. 
Inhibition Kinetics of Der-p I 
Assays were routinely carried out in 50 mM potassium phosphate; pH 8.25 
containing 1 mM ethylenediaminetetraaceticacid (EDTA) and 1 mM 
dithiothreitol (DTT). The fluorogenic substrate was 
2-aminobenzoylvalylalanylnorleucylseryl-(3-nitro)tyrosinyl aspartylamide. 
Product formation was monitored with respect to time by measuring the 
increase in fluorescence emission at 420 nm and exciting at 320 nm. Assays 
were carried out at 25.degree. C. Stock solutions of the various 
inhibitors were made up in 100% dimethylsulphoxide. 
Inactivation kinetics for various inhibitors were carried out using the 
techniques already described. In the dilution method, generally 100 nM Der 
p I was mixed and incubated with 0.5-10 uM of the inhibitor and aliquots 
were taken out at given time points (sampling time) and the residual 
enzyme activity determined by a ten-fold dilution into assay buffer 
containing saturating amounts of substrate. The residual activity was 
related to the sampling time and the curve fitted by computational 
non-linear least square regression analysis. In cases where the second 
order rate constants were greater than 10.sup.5 M.sup.-1 s.sup.-1, second 
order conditions were employed (i.e. equimolar amounts of enzyme and 
inhibitor). Generally stoichiometric amounts of enzyme and inhibitor were 
incubated for given time intervals (sampling time) and the reaction 
stopped by a ten-fold dilution of this mixture by saturating amounts of 
substrate in assay buffer. A plot of reciprocal enzyme concentration 
versus sampling time was fitted by linear least square regression analysis 
to obtain the second order inactivation rate constant. 
In cases where inactivation kinetics were calculated in the presence of 
enzyme, inhibitor and substrate the following conditions were employed. 
Generally a solution containing 12.5 mM substrate and 0.1-10 mM inhibitor 
was incubated at 25.degree. C. for 5 min. prior to addition of enzyme (10 
nM) to initiate the reaction. In the absence of inhibitor, product 
formation was linear with time. Inactivation of enzyme was exhibited by 
the downward curvature in the increase in fluorescence. The apparent 
inactivation rate constant (k.sub.app) was determined by fitting these 
curves to Eq. 2, using least square regression analysis, and the second 
order rate constant determined using Eq. 3. 
Assay Results 
______________________________________ 
Compound 
k.sub.obs /[I] 
number (M.sup.- s.sup.-1) 
______________________________________ 
3 &gt;10.sup.7 
4 1.6 .times. 10.sup.7 
6 6.8 .times. 10.sup.7 
7 3.7 .times. 10.sup.5 
8 &gt;10.sup.7 
9 2.3 .times. 10.sup.4 
10 1.9 .times. 10.sup.5 
11 1.2 .times. 10.sup.6 
12 1.9 .times. 10.sup.5 
13 6.6 .times. 10.sup.5 
15 1.5 .times. 10.sup.5 
16 1.6 .times. 10.sup.4 
23 1.7 .times. 10.sup.3 
25 3.1 .times. 10.sup.3 
26 4.1 .times. 10.sup.3 
27 6.3 .times. 10.sup.3 
28 6.8 .times. 10.sup.3 
29 4.6 .times. 10.sup.3 
30 7.5 .times. 10.sup.3 
34 1.1 .times. 10.sup.4 
36 6.4 .times. 10.sup.3 
38 1.1 .times. 10.sup.3 
40 6.9 .times. 10.sup.4 
______________________________________ 
Compounds for which no inhibition data is shown were key intermediates in 
the formation of further compounds or were too unstable to be tested and 
hence were intermediates to more stable compounds. 
Pharmacophore Definition and Specification 
A collection of compounds with biological activity for Der p I was provided 
as a training set. Each compound in the training set was subjected to full 
conformational analysis (J. Comp. Chem., 1995, 16, 171-187). A 
representative number of conformers were generated over a given energy 
range above the lowest energy conformation (J. Chem. Inf. Comp. Sci., 
1995, 35, 285-294 and J. Chem. Inf. Comp. Sci., 1995, 35, 295-304). 
This information was used to derive a pharmacophore (based on seven 
chemical feature type rules) (J. Chem. Inf. Comp. Sci., 1994, 34, 
1297-1308) that correlates to the observed biological activity. It was 
assumed that the molecules in the training set all act at the same target 
in the same manner of action. 
A pharmacophore consisting of at least the following chemical features 
defines the chemical motif of potential inhibitors of Der p I: 
A Hydrogen bond acceptor feature, three Hydrophobe (J. Comp. Chem., 1986, 
7, 565-577) features and a Hydrogen bond donor feature. 
A Hydrogen bond acceptor feature matches the following atom types or groups 
of atoms which are surface accessible. 
sp or sp.sup.2 nitrogens that have a lone pair of electrons and a charge 
less than or equal to zero 
sp.sup.3 oxygens or sulphurs that have a lone pair of electrons and charge 
less than or equal to zero 
non-basic amines that have a lone pair of electrons. 
A Hydrogen bond donor feature has the same chemical rules, i.e. it matches 
the same atoms or groups of atoms, as the Hydrogen bond acceptor except 
that it also includes basic nitrogen. There is no exclusion of 
electron-deficient pyridines and imidazoles. This feature matches the 
following atom types or groups of atoms. 
non-acidic hydroxyls 
thiols 
acetylenic hydrogens 
NH moieties (except tetrazoles and trifluoromethyl sulfonamide hydrogens). 
A Hydrophobe feature is defined as 
a contiguous set of atoms that are not adjacent to a concentration of 
charge (charged atoms or electronegative atoms), in a conformation such 
that the atoms have surface accessibility, including phenyl, cycloalkyl, 
isopropyl and methyl. This may also include residues which have a partial 
hydrophobic character such as Lysyl or Glutaminyl amino acid side-chains. 
The term "pharmacophore" used herein is not meant to imply any 
pharmacological activity. The term refers to chemical features and their 
distribution in three-dimensional space which constitute and epitomise the 
preferred requirements for molecular interaction with a receptor. For 
example the receptor may be the catalytic active site of the cysteine 
protease Der p I. 
FIG. 4 graphically shows the pharmacophore of Der p I. In the figure the 
Hydrogen bond acceptor is represented by a vector function consisting of 
two spheres. The smaller sphere (at least 1.6 Angstroms radius up to 2.6 
Angstroms) defines the centroid of the hydrogen bond acceptor on the 
ligand while the large sphere (at least 2.2 Angstroms radius up to 2.6 
Angstroms) defines the projected point of the hydrogen bond acceptor from 
the receptor. These two spheres are at least 3.0 Angstroms apart. 
Similarly the Hydrogen bond donor is represented by a two sphere vector 
function defined in the same way as above for the Hydrogen bond acceptor. 
The Hydrophobe features are represented by spheres of at least 1.6 
Angstroms radius up to 2.6 Angstroms. 
The absolute sphere centroid positions of each feature are defined in three 
dimensions as follows: 
Hydrophobe 1 has Cartesian XYZ co-ordinates of -6.272, 3.372, -1.200 
Hydrophobe 2 has co-ordinates of -3.320, -2.305, 0.906 
Hydrophobe 3 has co-ordinates of -0.612, -4.088, -1.740 
Hydrogen Bond Donor origin co-ordinates of 0.007, 0.926, 4.168 
Hydrogen Bond Donor projected point co-ordinates of -0.743, 0.926, 4.168 
Hydrogen bond acceptor origin co-ordinates of 5.155, -0.25, -2.528 
Hydrogen bond acceptor projected point co-ordinates of 7.413, 0.349, -4.426 
The distance constraints are shown in FIGS. 5 and 10 to 19. The angle 
constraints are shown in FIGS. 6 and 10 to 19. 
The tolerances on all distances between the chemical features is +/-0.5 
Angstroms and the geometric angles +/-20 Degrees. 
REFERENCES 
1. Sutton, B. J. & Gould, H. J. Nature 366, 421-428 (1993). 
2. Flores-Romo, L. et al. Science 261, 1038-1041 (1993). 
3. Yu, P. et al. Nature 369, 753-756 (1994). 
4. Stief, A. et al. J. Immunol. 152, 3378-3390 (1994). 
5. Fujiwara, H. et al. Proc. Natl. Acad. Sci. USA 91, 6835-6839 (1994). 
6. Chapman, M. D. et al., J. Allergy Clin. Immunol. 72,27-33 (1983). 
7. Krillis, S. et al. J. Allergy Clin. Immunol. 74,132-141 (1984). 
8. Barrett, A. J. et al. Biochem. J. 201, 189-198 (1982). 
9. Mast, A. E. et al. Biochemistry 31, 2720-2728 (1992). 
10. Knapp, W. et al. eds. Leucocyte typing IV, Oxford University Press. 
142-154 (1989). 
11. Liu Y. J. et al. Eur. J. Immunol. 21, 1107-1114 (1991). 
12. Gordon, J. et al. Immunol. Today 10, 153-157 (1989). 
13. Letellier M. et al. J. Exp. Med. 172, 693-700 (1990). 
14. Kim, K -M. et al. Pediatric Res. 26, 49-53 (1989). 
15. Yanagihara, Y. et al. Clin. Exp. Allergy 20, 395-401 (1990). 
16. Chua, K. Y. et al. J. Exp. Med. 167, 175-182 (1988). 
17. Finkelman, F. D. & Urban J. F. Parasitol. Today 8, 311-314 (1992). 
18. Lombardero, M. et. al. J. Immunol. 144, 1353-1360 (1990). 
19. Ghadieri, A. A. et al. Immunol. Lett. 27, 113, (1991). 
20. Ghose, A. et al. J. Comp. Chem., 1986, 7, 565-577 
21. Smellie, A. et al. J. Comp. Chem., 1995, 16, 171-187 
22. Smellie, A. et al. J. Chem. Inf. Comp. Sci., 1995, 35, 285-294 
23. Smellie, A. et al. J. Chem. Inf. Comp. Sci., 1995, 35, 295-304 
24. Greene, J. et al. J. Chem. Inf. Comp. Sci., 1994, 34, 1297-1308 
25. Maeji, N. J. Bray, A. M. Valerio, R. M. and Wang, W., Peptide Research, 
8(1), 33-38, 1995. 
26. Valerio, R. M. Bray, A. M. and Maeji, N. J. Int. J. Pept. Prot. Res, 
44, 158-165, 1994. 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 1 
- - - - (2) INFORMATION FOR SEQ ID NO: 1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 10 amino - #acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: peptide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1: 
- - Thr Asn Ala Cys Ser Ile Asn Gly Asn Ala 
1 5 - # 10 
__________________________________________________________________________