Note: Descriptions are shown in the official language in which they were submitted.
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CANDlDA ALB~CANS TATA-BINDING PROTEIN. NUCLETC ACTD AND ASSAYS
ABSTRACT OF THE DISCLOSURE
The invention ellco.,.l,a~ses a novel Lldl~seliption factor from Candida
albicans, TBP, a nucleic acid seq~lenre encoding TBP, and methods of sc~ ,nillg for
inhibitors of Candida albicans growth by targeting TBP.
The invention relates in general to lr~nsc~ ion factors and to methods for
screening for allLirul.gal agents.
The invention was made in part using gove,.. ~-t funds, NIH grant no.
GM46498, and therefore the U.S. go~ lllent has certain rights in the invention.
BACKGROUND OF THE INVENTION
The yeast Candida albicans ~C. albicans) is one of the most pervasive
25 funga} pathogens in hllmqn.c. It has the capacily to o~o,~ irqlly infect a diverse
~yecLnlnl of colllpiolllised hosts, and to invade many diverse tissues in the human body.
It can in many ill~c~ es evade antibiotic tl~ and the immlm~ system. Although
Candida albicans is a member of the normal flora of the mucous membranes in the
respiratory, gastroint~stinql~ and female genital tracts, in such locations, it may gain
30 dominqn~e and be associated with pathologic conditions. Som~timps it producesprogressive systemic disease in debilitated or immlmosuppressed patients, particularly if
cell-m~iq-ted i~"~ ily is impaired. Sepsis may occur in patients with colll~lolllised
cellular i------~ y, e.g., those undergoing cancer chemotherapy or those with lymphoma,
AIDS, or other conditions. Candida may produce bloodstream invasion,
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thrombophlebitis, endocarditis, or infection of the eyes and virtually any organ or tissue
when introduced intravenously, e.g., via tubing, needles, narcotics abuse, etc.
Candida albicans has been shown to be diploid with b~l~nre~ lethals, and
therefore probably does not go through a sexual phase or meiotic cycle. This yeast
S appears to be able to syollL~lleously and reversibly switch at high frequency between at
least seven general phenotypes. Switching has been shown to occur not only in standard
laboratory strains, but also in strains isolated from the mouths of healthy individuals.
Nystatin, ketoconazole, and amphotericin B are drugs which have been used
to treat oral and systemic Candida infections. However, orally ~(lminictered nystatin is
10 limited to tre~tm~nt within the gut and is not applicable to systemic tre~tm~-nt. Some
systemic infections are susceptible to tre~tment with ketoconazole or amphotericin B, but
these drugs may not be effective in such L,~ enl unless combined with additional drugs.
AmphoL~lic,ll B has a relatively narrow the,ay~uLic index and ~lu~lerous undesireable side
effects and toxicities occur even at thcla~ulic col~ce"L~aLions. While ketoconazole and
other azole a~irungals exhibit .si~nifir~ntly lower toxicity, their m~c~nicm of action,
inactivation of cyLochroll~e P4soyluSll.r~;r group in certain el~y~es, some of which are
found in h-lm~n.c, precludes use in p~ti~ntc that are sim~ usly receiving other drugs
that are metabolized by the body's cyloch.ullle P4so el~y~lles. In addition, les;~ re to
these compounds is emerging and may pose a serious problem in the future.
There is a need in the art for an effective tre~m~ont of opportunistic
infections caused by Candida albicans. Therefore, one object of the invention is to
provide scleellillg assays for idellliryillg potential inhibitors of Candida albicans growth.
Another object of the invention is to provide scle~nil,g assays and to identify potential
inhibitors of Candida albicans growth that are based on inhibition of ~ seliytion in this
organism.
Synthesis of mRNA in eukaryotes requires RNA polymerase II and
accessory Llansc,iytion factors, some of which are general and act at most, if not all,
promoters, and others of which confer ~ecificily and control. Five general factors, a,
b, d, e, and g, have been purified to homogeneity from the yeast S. cerevisiae, and have
30 been identified as c~ulll~lparts of human or rat factors, TFIIE, TFIIH, TFIID, TFIIB and
TFIIF, respectively. These factors assemble at a promoter in a complex with RNA
polymerase II to initiate l~a~lsc~ Lion. Binding studies have shown that the order of
assembly of the initiation complex on promoter DNA begins with factor d (TFIID), is
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followed by factor e (TFIIB), and then by polymerase and the rem-q-ining factors. Factors
b (TFIIH), e (TFIIB) and g (TFIIF), however, bind directly to polymerase II, and as
many as four of the five factors may assemble with the polymerase in a holoenzyme
before promoter binding. The functional ~igni~l( Anre of interactions revealed by binding
5 studies is not clear in that only a few percent of initiation complexes may give rise to
ansclipls.
Many aspects of llansc~ ion by RNA polymerase II are conserved between
yeast and higher eukaryotes. For example, there is extens}ve amino acid sequencesimilarity among the largest subunits of the yeast, Drosphila and mqmmqli~n polymerases.
10 Other components of the transcription apparatus, such as TATA-binding and enh~nrer
binding factors, are in some i~xlAnres i~rcl-~nge~kle between yeast and mqmmAliqn in
vitro binding or ll~sclil.tion systems. There are, nonPth~l~s~, signific,qnt dirre,c,~ces
be~ the two ~y~llls. TATA el~rntontc are located from 40 to 120 or more base pairs
u~Ll~alll of the inithAti-~n site of an S. cerevisiae promoter, and where these el~PrnPnt~
15 occur, they are required for gene exl,lession. The fact that C. albicans genes function
in S. cerevisiae suggests that it also uses the 40 to 120 base pair spacing bcl~een the
TATA elPnnpnt and initiation site. In cOllLla~ ..s.,,,"qliqn (as well as S. pombe)TATA
elernPntc and ~ , ;plion start sites are only 25 to 30 bp apart, and deletion of a TATA
elem~nt does not always reduce the frequency of ~1~ scli~lion initiqti~m, although it may
20 alter the jnititZltiOn site. There are also varying degrees of homology bcl~,e.l
llAi-!i' - ;l-lion factor sequenres from yeast and ~ qli~n sources. Some of the
mllltic~kunit factors, such as RNA polymerase II, TFIIF, and TFIID, contain different
l~ul~lb.,.~ of subunits in hl-mAn~ and yeast. The molecular weights of corresponding
polypeptides differ b~lwcell hllmq-n~ and yeast, with seql)e~rçs being found in a given
25 yeast factor not being found in its human coullte.~ll and vice versa.
TATA-binding protein (TBP) is the central initiation factor for lldllscli~lion
by all three nuclear RNA polymerases, and is highly conserved throughout the eukaryotic
kingdoln The 180 amino acid carboxy-~ninql core domain is s~1fflri~nt for TATA
element binding, for all e~e~ l fimrtion~ in S. cerevisiae. and is 80% i-1Pntirql belwee
30 S. cerevisiae and hllmqn~. In vitro, yeast and human TBPs can functionally replace one
another in terms of basal RNA polymerase II llal}sc~iption, and they display nearly
i(ientirql DNA seq~lenre requirements for TATA elements. However, TBP exhibits
species-specific behavior in vivo. For example, human and yeast TBP's are not species
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hlLercl1angeable in supporting cell growth (Gill and Tjian, Cell 65:333-340, (1991);
Cormack et al., Cell 65:341-348 (1991)). Human and S. cerevisiae TFIIB's have 50-60%
amino acid seql~nre identity, and also are not species interchangeable in supporting cell
growth.
S Operative s~lbsti~ltion of the same l.ansc~ tion factor in transcription
systems of dirr~,~el~l yeast species is not predictable. This is true despite a high degree
of amino acid sequence identity among some llanscli~tion factors from different yeast
species. For example, the ability of a given t,anscliplion factor to support efficient and
accurate llansc,iption in a heterologous yeast species is not predictable. Li et al. (1994,
Science 263:805) tested the i~llc~ g~bility of S. cerevisiae and S. pombe llallScliption
factors in vitro, and report that many S. cerevisiae components cannot substitute
individually for S. pombe RNA l~lsc~ ion factors a, e, or polymerase II, but some
combinations of these col"pol~e"~s were effective. In one in~t~nre, active lla,-sc,illlion
could not be l.,co~-c~ cl when S. cerevisiae-derived TFIIB was the sole substitution into
a TFIIB-~lepleted set of factors from S. pombe. A TFIIB-RNA polymerase II combination
from S. cerevisiae was able to ~lilule, inrli~ ~ting that the r~mcliollal interaction of these
two co,l,l)o,l~ s is not only unlJo~ but also that the activity may be dependent on
species-s~ecir,c d~ that cannot be complemPntPd by either component derived
from a different org~nicm The unpredictability in making ~sLilulions of a given factor
among dirrel~,.,l yeast species is also evident in that such s~lbstit ltions are not reciprocal;
that is, substitutions of S. pombe fractions into an S. cerevisiae ll~.;,il,lion system are
less effective than the reverse s~bstitltions (Li et al., supra).
The yeast Candida albicans differs from most yeast strains in that it does
not use the same genetic code that most Ol~g~ , wllelher .. ~.. ~li~n or yeast, utilize.
25 Santos et al. (1995, Nucleic Acids Research, 23:1481) report that the codon CUG, which
in the universal code is read as a leucine, is ~l~co~ as a serine in Candida. Therefore,
any CUG codon .that is Secoded in Candida albicans as a serine, would be decoded as a
leucine in the transformed S. cerevisiae. Any gene cont~ining a CUG codon would
thtlcrole be tr~n~l~te~l as different amino acid sequences in Candida albicans and S.
30 cerevisiae. Such mistranslation may produce an inactive protein, since the amino acids
serine and leucine have m~rkrrlly different cl~mir~l l,rop~ ies and serine is known to be
an essenti~l residue in the active site of some el~y,lles. Repl~re~-.c..l of leucine by serine
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s
at CUG encoded residues is a serious problem in the use of many l~oller systems (e.g.
~-g~lqrtosi~-q-ce, Chlorqmrhrnirol acetyllral~r,,l~se, Flux) in Candida albicans. Previous
experiments have shown that tranclation by Candida of CUG as serine instead of leucine
often resulted in the production of inactive l~,pOlL,~ proteins.
Another object of the invention is to provide an assay for screening for
selective inhibition of Candida albicans growth and/or viability.
Yet another object of the invention is to provide a molecular target for
inhibition of Candida albicans llanSCli~liOn or l.dl~scli~lion initiation.
SUMMARY OF THE INVENTION
The invention el~co~ ac.sPs a recombinant nucleic acid cou~l"isillg a nucleic
acid seql.enre enr.otling Candida albicans TBP.
The invention also encomracses a vector colllplisillg a nucleic acid seql~enre
çnr.o-ling Candida albicans TBP, and a transformed host cell contAining a nucleic acid
sequence encoding Candida albicans TBP.
The invention also ~ o~nl~Ac~s~s a method for producing recombinant
Candida albicans TBP, COll~li~illg cultunng a host cell transformed with a nucleic acid
encoding Candida albicans TBP under conditions ~llrr~ .t to permit e~lession of the
nucleic acid enrotling Candida albicans TBP, and isolating Candida albicans TBP.The "l~ulion also e~o~ qcses a scl~,n~llg method for idenlirying an
inhibitor of Candida albicans growth, COlllp~i~illg dçtecting inhibition of mRNAllansclilllion in an in vitro llallSClillliOn assay col"~lising a DNA template, RNA
polymerase II, l~,cOlllbillallt Candida albicans TBP, and a c~n~ qt~ inhibitor, wherein
production of an mRNA transcript compl~prnpntqry to the DNA template occurs in the
q~bsenre if the c-qn.li~l-qte inhibitor.
The invention also enromrqc.ces a seleel~illg method for identifying an
inhibitor of Candida albicans growth, comprising d~PIeC~ g in the l,r~sellce of a cqn~ qte
inhibitor inhibition of formation of a complex COllll~liS~l~g a DNA template andrecombinant Candida albicans TBP, wll~ciu in the absence of the cqnf~ qte inhibitor,
~ 30 formation of the complex occurs. The method also may be pelro~ ed in the ple3e.1ce of
additional factors, such as TFIIB, RNA polyln~ se II and TFIIF.
The invention also encomraCses a S~;lccllmg method for identifying an
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inhibitor of Candida albicans growth, co~ lisil~g ~letecting in the pl~sellce of a c~n-lid~te
inhibitor, irlhibition of formation of a complex comprising Candida albicans TFIIB and
Candida albicans TBP, whereill in the absence of the c~n~ te inhibitor formation of the
complex occurs. Preferably, the complex will include a DNA template.
S The invention also e.~ro~ csP~s a scll,~nillg mPthnd for identifying an
inhibitor of Candida albicans growth, colll~)lisillg ~leL~cl;l~g in the ~lesence of a c~nt~ te
inhibitor inhibition of formation of a complex com~,lisillg RNA polymerase II, Candida
albicans TBP, and Candida albicans TFIIB, wherein in the absence of the c~n~ t~
inhibitor formation of the complex occurs. Preferably, the complex will include a DNA
template and the RNA polymerase II from C. albicans.
In the above-described s~ el~lg methods, detection may be performed in
the plesellce of a plurality of c~n~ te inhibitors. In screening methods of the invention
which involve SCl~.Cllillg of a plurality of c~n~ te inhibitors, the plurality of inhibitors
may be screened together in a single assay or individually using multiple .Cim~llt~nPous
individual ~letPcting steps.
The invention also enco-~ cs~s a method of ~ ell~hlg Candida albicans
growth in culture, comprising cont~rting the culture with an inhibitor that selectively
inhibits the biological activity of Candida albicans TBP.
The invention also el~co~r~cses a method of pl~,~ellling Candida albicans
growth in a m~mm~l, Co~ ;sillg ~mini~tpring to a ~---------~1 a thel~cl"ir~lly effective
amount of an inhibitor that inhibits the biological activity of Candida albicans TBP.
As used herein, "inhibition" refers to a reduction in the parameter being
measured, whether it be Candida albicans growth or viability, Candida albicans TBP-
mP~ tP(~ ,nscli~lion, or formation of a Candida albicans TBP lldnscli~Lion complex.
The amount of such reduction is measured relative to a sL~ndar~ (control). Rec~nse of the
multiple interactions of Candida albicans TBP in tl~nsc;li~tion initiation, the target
product for ~etection varies with respect to the particular SCl~ illg assay employed.
Three llrer~.led detection products p~scllled in this disclosure are; a) newly lldnsclibed
mRNA, b) a DNA-TBP complex, and c) a TBP-TFIIB-RNA polylllc.~se II complex.
"Reduction" is ~e~lnPfl herein as a decrease of at least 25% relative to a control,
preferably of at least 50%, and most preferably of at least 75%.
As used herein, "growth" refers to the no~nal growth pattern of Candida
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albicans, i.e., to a cell doubling time of 60 - 90 mimlt~. "Viability" refers to the ability
of Candida albicans to survive in culture for 48 hours.
"Biological activity" refers to the ability of TBP to form a L~nsc~ ion
complex with a DNA template or other ynoteil~- of the ~lansc-il lion complex, or to
S interact with other llans~ lion compollcllls so as to permit initiation of L-~nscliylion.
"DNA template~ refers to double stranded DNA and, where inrlic~tçd by
the particular binding assay to single stranded DNA, at least 10 nucleotides in length, that
may be lleg~Livcly supercoiled if double-stran-lP~l, possesses a promoter region, and
contains a yeast TATA conce~ C region. DNA templates useful herein preferably will
10 contain a TATA sequenre that is located from 40 to 120 or more base pairs upstream of
the inhit~tion site (~ict~nre measured from the first T of the TATA element to the 5'-most
initiation site). An especially efficient DNA template for use in methods of the invention
involving ~lanscliy~ion is devoid of ~ O~ f residues, and thelefo~ a "G-minus" or "G-
less" cassette is yrer~lcd~
"mRNA l~ refers to a full-length ~ s~;liyl as well as to truncated
,al scliyts, oligonucleotide lla~lSCliyLS and ~inllrlçotide RNAs.
"Formation of a complex~ refers to the binding of TBP to other
ll~nscli~lion factors (i.e., protein-protein binding) as well as to binding of TBP to a DNA
template; such binding will, of course, be a non-covalent ~Ccoci~tion
Other fe,dlul~es and advantages of the invention will be aypalclll from the
description, ylcfc~l~,d embo~ thereof, the dl~wing~, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 pl~se.lls the nucleotide and amino acid sequences of the Candida
2~ albicans k~scliylion factor TBP.
Fig. 2 yl~se~ nucleotide and amino acid sequenre of the Candida albicans
transcription factor TFIIB.
DESCRIPTION
The invention is based on the discovery of a novel protein, Candida
albicans TBP, and on the isolation of recon.bin~..l DNA encoding Candida albicans
transcription factor TBP. Rec~llse TBP is esse~ti~l for viability of the cell, a compound
that blocks the biological activity of the protein is expecte~ to have fimgiri~l yioy~ies~
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Therefore, the invention is also based on the development of assays for screening from
inhibitors of TBP.
Isolation and Chala~;Lcl;G~Iion of the Candida albicans TBP Gene
Given the unpredictability with respect to operative substitutions of a given
S transcription factor among dirr~,lel~l yeast strains, one cannot assume that strategies for
cloning of the gene encoding a given transcription factor which are based on factor
function, such as genetic compleme~t~tion, will work Other cloning strategies, which
do not require f~nrtion~l comple~ ion, such as those based on homology at the
nucleic acid level, may be utilized in an attempt to cil~;unlvent a requirement for factor
10 filnrtion For example, Southern hybridization of specific sequPn~ec to a library carried
in E coli and PCR amplification of potentially highly homologous regions of a gene are
two strategies that have been s~cc~r~llly used to clone homologous genes from dirÇer~
Ol~a~ ."~,
The approach used to clone the Candida albicans homolog of TBP
15 involved genetic colll~ on of mutant S. cerevisiae strains. A library of Candida
albicans genomic seq.~Pnres was introduced into a strain of S cerevisiae that contained
a m-lt~ted TBP gene (sptlS) This mutant strain was capable of growth at 30~ C, but was
non-viable at 37~ C, due to a le~pel~lu,e se.~i~ive mllt~tion in the TBP gene Following
transform~tio~ of the library into the strain, the cells were grown at 37~ C, and the
20 colonies which grew at this non-p~ ;.ve le~ alu~ were further studied as potentially
carrying a Candida albicans homolog of the d~f~;live gene. This a~ oach will only
work if a Candida albicans homolog is able to substi~l1te functionally in vivo for the
d~Çe~;Li~e gene.
After c~n~ clones were i~ol~ted by growth at the lonpell,lissive
25 t~llpelalul." the library plasmid DNA was recovered from the cell and retested to confirm
that the C. albicans sequen~ es on the pl~mirl were sub~ for the S. cerivisiae gene.
Subclones of the C. albicans seql~ent~çs were constructed by ~ndard cloning methods,
and the minim~l Candida DNA sequen~es that substituted were sequenced using standard
methods.
The nucleotide sequenre encoding Candida albicans TBP and the predicted
amino acid sequence of the encoded protein are p.~l,se~ed in Fig 1 (SEQ ID NOS: 1 and
2). I~e nucleotide seqllenre encoding Candida albicans TFIIB and the predicted amino
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acid sequPnce of the çnco~ed protein are l)lesell~ed in Fig. 2 (SEQ ID NOS: 3 and 4).
Methods For Screening Potential Inhibitors of Candida albicans Growth and/or Viability
Because TBP initiation factor is es~Pnti~l for lldnscliL)lion initiation, the
S recombinant Candida albicans TBP gene and recombinant protein e~oded by this gene
may be utilized in scl.,el~ing assays for inhibitors of Candida albicans growth and
viability. The screening assays of this invention detect inhibition of the Candida albicans
TBP-mPdi~e~l component of Iranscli~lion initiation, either by llleas~ g inhibition of
lldncli~lion, ll~s~ lion initation, or initiation complex formation, or by assaying
10 formation of a protein/DNA or a protein/protein complex.
EXAMPLE 1
Scleening for Inhibitors of TldnscliyLion
a) Tldnsclil,lion Assay Components.
An in vitro ~dnscli~lion assay con.~istin~ of the minim~l COlll~Ol~lltS
~IPcess~ y to synthesi7P an mRNA Lldns~;LipL from a DNA template can be used to screen
for inhibition of mRNA production. The cl~ ; of such an assay consist of; a) a DNA
template, b) RNA polyl,lc.~.sc II, c) 1cccjlllbilldnL Candida albicans TBP, and d) a TFIIB
which is preferably Candida albicans TFIIB. In order to increase the efficiency of
20 lldnsc~ ioll, additional components of the lldnscliplion complex may be included, as
desired; e.g., TFIIE, TFIIF, TFIIH, etc.
Parvin and Sharp (Cell 73, 533-540, 1993) have reco~ d gene
lldl~cli~tion in vitro with a minim~l reaction cont~ining a DNA template, RNA
polymerase II, TFIIB, and TBP. For efficient Ll~scli~lion under minim~l conditions, the
25 DNA template (a) is supercoiled, and (b) possesses a promoter region cont~ining a TATA
consel~us region. Additionally, Lue et al. (Science 246, 661-664, 1989) have detellllil~ed
that ll~nsclil~tion may be ~etected most efficiently with a DNA template devoid of
gl.~nosin~ residues (a G-minus or G-less cassette ). Promoter depe~Pnre is demonstrated
by the loss of signal when a plasmid lacking promoter sequences is utilized as a template.
30 Correct initiation is demonstrated by the production of a band with a mobility consistent
with the size of the expected product on del~luring polyacrylamide electrophoresis gels.
As stated above, Candida albicans TBP forms a ll~ns~ lion initiation
complex with RNA polymerase II. Therefore, it is desired that an in vitro transcription
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assay according to the invention contain RNA polymerase II. Although it is possible to
perform an inhibitor ~cl~,e~ ,g assay using RNA polymerase II from a yeast strain other
than Candida albicans, e.g., S. cerevisiae, it is most desirable to use a homologous assay
in which the transcription complex components are from Candida albicans .
S A method for S. ccr~isiae RNA polII purification is described in Edwards
et al. (Proc. Natl. Acad. Sci. USA 87: 2122-2126 (1990)). Alternatively, highly purified
RNA polymerase Il from Candida albicans was provided as follows.
RNA polymerase II activity was measured in reactions cont~ining 50 mM
Tris-C1, pH 7.9 (4~ C), 50 mM (NH4)2 SO4, 2.5 mM MnC12, 0.1 mM EDTA, 5 mM
DTT, 100 ~g/ml BSA, 0.6 mM ATP, CTP and GTP, 25 ~M UTP (2.5 ~Ci) ~a32P~ UTP
and 100 ~g/ml heat-denaLured calf thymus DNA in a final volume of 50 ~1. Reactions
were inrl-b~t~ri for 60 min. at 30~ C and le~ d by addition of 50 ~l 15% (w/v)
trichloroacetic acid. Acid-insoluble radioactivity was collected by filtration through glass
fiber filters and ql-~ntifiP~ by liquid scintillation ~c.,L,ophotometry. One unit of RNA
polymerase activity catalyzes the ulco~ tion of 1 pmol of UTP into acid-insoluble
material in 60 min. under the conditions described above.
Candida albicans was obtained from the American Type Culture Collection
(ATCC 10231) and cultured in YPD ~ (Current Protocols in Molecular Biology,
Vol. 2, 13, Suppl. 19 (1989)) at 30~ C with vigorous agitation and aeration. Allprocedures were carried out at 4~ C using 18 liter cultures. Cells were harvested by
centrifugation (5000 rpm, 10 min., Sorvall H6000 rotor), washed once with--11 ice-cold
deionized water and repelleted as above. The cell pellet (200-300 g wet weight) was
thoroughly resuspended in a volume of Buffer A (50 mM Tris-HCl, pH 7.9, 4~ C, 10%
glycerol, 1 mM EDTA, S mM MgCI2, and pfotease inhibitor) co-~ 300 mM (NH4)2
S04 equivalent to the packed volume of cells (~letermin~d by weight ~c.$~1ming a density
of 1 g/ml cells). RP-sll~pended cells were either processed im m~ tely as described
below or frozen by pipetting into liquid N2 and stored at -80 C. Frozen cells were thawed
on ice prior to proceeding. Following the addition of NP-40 to a final concentration of
0.1%, cells were disrupted by glh~diulg with 1 ml acid-washed glass beads/ml cell
suspension (Sigma, 400-625 ~M) using 12 bursts of 30 sec. each in a Bead Beater
(BioSpec). Glass beads were allowed to settle out and the :iU~Je~llal~lnt was centrifuged at
30,000 x g for 40 min. Solid (NH4)2 S04 was slowly added to a final collcellLlation of 0.4
glml sup~ and the resl~ltin~ cipl~te was pelleted by centrifugation at 100,000
CA 02250129 1998-09-30
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11
x g for 30 min. The pe}let was resuspended with a volume of Buffer A sufficient to yield
a conductivity equivalent to Buffer A cont~inin~ 75 mM (NH4)2 SO4.
Following centrifugation of the res~lsp~ ion at 10,000 x g for 10 min, this
~ .e...~ 1. 5 mg protein/ml) was loaded onto a 300 ml DE-52 DEAE-cellulose5 column equilibrated with Buffer A cont~ining 75 mM(NH4)2 SO4. After washing with S
column volumes Buffer A cont~ining 75 rnM (NH4)2 SO4, and 5 column volumes Buffer
A cont~ining 0.15 M (NH4)2 SO4, RNA polymerase II was eluted with 5 column volumes
Buffer A cont~ining 0.4 M (NH4)2 SO4. Fractions were collected cont~ining the peak of
protein, determin~d by absorbance at 280 nm and pooled. The pool was dialyzed against
10 Buffer A co..l~ 20% glycerol for 3 hr. at 4~ C.
The 0.4 M (NH4)2 SO4 eluate from DEAE-cellulose (261 mg protein, 290
ml) was diluted with sufficient Buffer A to lower the conductivity to the equivalent of
Buffer A co..l;.i..;l~ 0.15 M (NH4)2 SO4, centrifuged at 10,000 x g for 10 min. and the
~,Jl,c~ ..l was loaded at a flow rate of 30 ml/hr onto an 30 rnl DEAE-cell~llose column
15 equilibrated with Buffer A co~ ini~ 0.15 M (NH4)2 SO4. After ~Sl~lllg with 3 column
volumes of Buffer A COI~t~ 0.15 M (NH4)2 SO4, the column was developed with a 200
ml linear gradient of 0.15 - 0.4 M(NH4)2 SO4 in Buffer A at a flow rate of 45 ml/hr.
Fractions from the single peak of ~ -sensiLive RNA polymerase activity, eluting
around 0.22 M (NH4)2 SO4, were pooled (21.1 mg protein, 45 ml) and loaded directly
20 onto a 5 ml Heparin agarose column equilibrated with Buffer A co..~ g 0.2 M (NH4)2
SO4. The column was washed with 3 column volumes of Buffer A cont~inin~ 0.2 M
(NH4)2 SO4 and developed with an 80 ml linear gradient of 0.2 - 0.6 M (NH4)2 SO4 in
Buffer A. The active fractions, which eluted at ~ro~in~lely 0.42 M (NH4)2 SO4 were
pooled (2.0 mg protein, 15 ml), frozen in 300 ~l ~li a~lots in liquid N2, and stored at -80~
25 C where activity was stable for at least 6 months.
P- t;rr~ion of protein initiation factors used in the assay is accomplished
by standard meth~3s known in the art (e.g., phosphocellulose chromatography followed
by gel filtration), as described in (Nature 346, 387-390 (1990)).
To screen for Candida albicans TBP-mP~ ted l~ lion inhibition, a
30 ~ sclil,lion assay is l~,con~ l using recombinant Candida albicans TBP.
Supercoiled plasmid DNA cont~ining the CYC1 promoter linked to the G-less cassette
described by Lue et al. (Science 246, 661-664 (1989)), is purified by standard methods
for purification of supercoiled circular DNA (Current Protocols in Molecular Biology,
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Vol. 2, 13, Suppl. 19 (1989)). 10 - 100 ng of Candida albicans TFIIB, 10 - 100 ng of
Candida albicans TBP, 10 - 100 ng Candida albicans RNA polymerase II and 1 ,ug
plasmid DNA are added to 50 ~l reaction ~ ul~s cont~ining 50 rnM HEPES, pH 7.5,
10% glycerol, 90 mM pot~cci-lm ghlt~m~te, 0.75% polyethylene glycol (molecular weight
5 3350), 10 mM m~ s;l~ acetate, 5 rnM EGTA, 5 mM DTT, 0.4 mM ATP, 0.4 mM
CTP, 10 ~4M [~-32P]UTP, 0.2 mM 3'-O-methyl-GTP, and cont~ining or lacking a
ç~n-litl~t~. inhibitor molecule. Reactions are inr~lb~ted at 30~ C for 30 - 60 min. and RNA
synthesis is ~letecte~l as described below.
b) Detection of Tla~sclibed RNA.
The detection of newly transcribed RNA is achieved by standard methods
(Current Protocols in Molecular Biology, Vol. 1, 4.10, Suppl 24 (1989)). As one
example, RNA ~yll~lesis can be detectPd as h~col~olation of a radioactively or
fluorescelllly labeled nucleotide into higher molecular weight RNA products, de~ .il,Pd
by one of the following m~tho-ls: 1) acid-insoluble labeled material qll~ntit~tPd by the
15 approl,liate method (e.g. scintill~tion cu~ for ra~ioactive pre~,ul~ul~, fluorometry for
flUOl~SCell~ ,UI~jO1S); 2) labeled reaction product that llyblidi~es to oligonucleotides
compl~ml~nt~ y to the coll~,elly i~ lrd LlallSClipl (i.e., l~l~llclll blot analysis); 3) the
~sellce of a labeled band with the a~pr~lid~ mobility detcoct~d by autoradiography, on
delu~ulillg polyacrylamide elecL opholcsis gels: 4) any other method that discrimin~tes
20 mononucleotides from polynucleotides, where polynucleotides are the desired RNA
product. Such m~tho-1c may utilize one or more well known techniques of molecular
biology (Current Protocols in Molecular Biology, Vol. 2, 13, Suppl. 19 (1989)), for
example; UV analysis; affinity ~y~ s (e.g., affinity chromatography, nitrocellulose
filtration, biotin/s~ Lavidin systems, imm-lno~ffinity,) (Current Protocols in Molecular
25 Biology, Vol. 2, 13, Suppl. 19 (1989)); and high pe.rolllldnce liquid chromatography.
The inclusion of an inhibitor molecule that il~lclr l- s with Candida albicans
TBP biological activity inhibits lldnscli~>Lion. In this assay inhibition is measured as a
reduction in the amount of mRNA Llalls~lipt produced relative to the amount of mRNA
transcript produced in the absence of the inhibitor (the positive control). A decrease in
30 ~mollnt of mRNA Lransclipl is indicative of an inhibitor. The ~G~ ti-)n of effective
levels of mRNA Llanscli~L inhibition is described below.
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EXAMPLE 2
Screening for Inhibition of DNA-Protein Complex Formation
A DNA-protein binding assay consisting of the minim~l components
nPcessqry to permit DNA-Candida albicans TBP binding to occur can be used to screen
5 for inhibition of the formation of the DNA-Candida albicans TBP complex duringllans, li~Lion initiation. The ess~ntiql elçrnent~ of such an assay consist of; a) a DNA
template, b) recombinant Candida albicans TBP, and optionally c) a cq-n~ qte Candida
albicans TBP i~hibilor.
The inclusion of an inhibitor molecule that hlle~ ,s with the interaction
10 b~lweel~ the Candida albicans TBP and the DNA template inhibits Ll~nsclil,Lion initiation.
The inhibitor may interact directly with the Candida albicans TBP protein, and/or it may
interact with the DNA template at the DNA site of Candida albicans TBP binding. In this
assay inhibition is measured as a reduction in the amount of DNA- Candida albicans TBP
complex produced relative to the amount of DNA- Candida albicans TBP complex
15 produced in the absenre of the inhibitor (the positive control). A decrease in the amount
of DNA- Candida albicans TBP compleY is indicative of an inhibitor. Deterrninqtion of
erreclive levels of DNA- Candida albicansTBP inhibition is described below.
One DNA binding assay is co~ ,d as follows. 10 - 100 ng Candida
albicans TBP, e~lessed in and pulirled from E. Coli as desclibed above, is inrubatçd
20 with 0.5 ng labeled (e.g. rv lioactively or fluoresc.,l,~ly labeled) oligonucleotide contqining
a TATA el~mP~t such as the one ~scrihe(l by B~ldtow~ki et al. (Cell 56, 549-561 (1989))
in reactifm.c co~ g 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mM MgCI2,
12% glycerol, 10 mM dill"o~ e;lol (DTT), 100 ,ug/ml BSA, 5 - 20 ~g/rnl poly (dG-dC):(dG-dC) and a c-q-n~ qt~- inhibitor of complex formation. Reactions are in-~lbqte~l at
25 30~ C for 30-60 min.
Formation of a DNA-TBP complex may be detecte~l as retention of labeled
DNA (the label being cletect~d by an appl~pliate mPtho~ology such as scintillq-tion
counting for radiolabeled DNA or fluoro"Rtly for fluu,esce"lly labeled DNA) utili7.ing
known affinity m~thf (ls for protein immobilization (e.g., biotin/streptavidin, nitrocellulose
30 filtration, affinity chromatography, immllnnqfflnity). No~rl tention of labeled DNA due
to the failure of Candida albicans TBP-DNA complex formation is indicative of aneffective inhibitor.
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Complex formation may also be detected as retention of labeled Candida
albicans TBP (e.g. radioactively, fluo.~scelllly) ~1tili7ing known methods for immobilizing
DNA. Nonle~ellLion of labeled Candida albicans TBP due to the failure of Candidaalbicans TBP-DNA complex formation is indicative of an effective inhibitor. These
S mf th~-1c are suitable for high-throughput ch~mi~ ~l compound library screening
applications such as those commonly used in drug discovery.
A third example of detectin~ DNA/protein complex formation involves
detection of an electrophroretic mobility shift of labeled DNA on 4% polyacrylamide gels
cont~ining 5% (V/V) glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 mM MgCl2,
10 pH 8.3 in the presence of Candida albicans TBP. The position of the labeled
oligonucleotide is ~etected by approl)liate methods (e.g., autoradiography for radioactive
oligonucleotide). The ~hslo.nte or deviation of the eYpecte~ mobility shift due to DNA-
Candida albicans TBP complex formation is indicative of an effective inhibitor.
Finally, other methods for ~et~cting or separating DNA-protein complexes
15 may be used, inrllltlin~ W crosslinking analysis, high ~elrulll~a~ce liquid
chromatography, phage display technology (U.S. Patent No. 5,403,484. Viruses
E~res~ g Chimeric Binding PloLeins), floulesence polarization, and surface plasmon
.esona.~ce (Biacore, Pl.~ r;~ Biosensor, North America) as described below.
20 EXAMPLE 3
Screenin,~ for Inhibition of DNA-Protein Complex Formation
A DNA-protein binding assay consisting of the minim~l components
n~cess~ry to permit DNA-Candida albicans TBP a~soci~tion to occur can be used toscreen for inhibition of the formation of the DNA-TBP-Candida albicans TFIIB complex
25 during ll~nscli~tiorf initi~ion. The components of such an assay include: a) a DNA
temrl~te, b) recombinant Candida albicans TBP, c) TFIIB, preferably from C. albicans,
and optionally d) a canrlid~tP Candida albicans TBP inhibitor.
The inclusion of an inhibitor molecule that ill~.r~ with the interaction
between the Candida albicans TBP and the DNA template inhibits ll~nscli~Lion initiation.
30 The inhibitor may interact directly with the Candida albicans TBP protein, and/or it may
interact with TFIIB and/or with the DNA template at the site of TFIIB/TBP binding. In
this assay inhibition is measured as a reduction in the amount of DNA-TBP-TFIIB
complex produced relative to the amount of DNA-TBP-TFIIB complex produced in the
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absence of the inhibitor (the positive control). A decrease in the amount of DNA-TBP-
TFIIB complex is indicative of an inhibitor. De~ on of effective levels of DNA-
TBP-TFIIB inhibition is described below.
One DNA binding assay is constructed as follows. 10 - 100 ng Candida
5 albicans TBP, expressed in and purified from E. Coli as described above, is incubated
with 0.5 ng labeled (e.g. radioactively or fluorescently labeled) oligonucleotide cont~inin~
a TATA elem~nt such as the one described by Bu~tow~ki et al. (Cell 56, 549-561 (1989)
and 10 - 100 ng Candida albicans TFIIB in reactions cont~ining 10 - 20 mM HEPES (or
equivalent), pH 7.5 - B.0, 5 mM MgCI2, 12% glycerol, 10 mM dithiothreitol (DTT), 100
10 ~4g/ml BSA, 5 - 20 ~g/ml poly (dG-dC):(dG-dC) and a c~n~ te inhibitor of complex
formation. Reactions are inruhat~d at 30~ C for 30-60 min.
Formation of a DNA-TBP-TFIIB complex may be detecte(l as retention of
labeled DNA (the label being ~etected by an app.ul)lialc methodology such as scintill~tion
cc,ul.lhlg for radiolabeled DNA or fluorùnlc~ for fluorcsccl~lly labeled DNA) ntili~ing
15 known affinity m~th~l.c for protein immobilization (e.g., biotin/streptavidin, nitrocelllllose
filtration, affinity cl~oll~atography, immllno~fflnity). No-ll~,tel-lion of labeled DNA due
to the failure of Candida albicans TFIB-TBP-DNA complex form~tion is indicative of
an effective inhibitor.
Complex form~tion may also be ~etec.te~l as l.,t~ ion of labeled Candida
20 albicans TBP (e.g. r; ~ioactively~ fluo~cenLly) lltili7ing known m~thotl~ for immobilizing
DNA. Nolllcknlion of labeled Candida albicans TBP due to the failure of Candida
albicans TFIIB-TBP-DNA complex formation is indicative of an effective inhibitor. The
preceding two mPth-)tlc are suitable for high-throughput ch~miral compound library
sclccl~ing applications such as those commonly used in drug discovery.
A third example of detectin~ DNA/protein complex formation involves
~etection of an electrophoretic mobility shift of labeled DNA on 4% polyacrylamide gels
cont~ining 5% (v/v) glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 rnM MgCI2,pH 8.3 in the presence of Candida albicans TFIIB and TBP. The position of the labeled
oligonucleotide is ~letected by approl)l;ate methoflc (e.g., autoradiography for radioactive
oligonucleotide). The absence or deviation of the e~e~;led mobility shift due to DNA-
Candida albicans TBP complex formation is indicative of an effective inhibitor.
Finally, other m~th~-~e for clet~cting or s~:~a~ lg DNA-protein complexes
may be used, including UV cros~lin~ing analysis, high performance liquid
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16
chromatography, phage display technology (U.S. Patent No. 5,403,484. Viruses
E,~ essillg Chimeric Binding Proteins), and surface plasmon r~sondllce (Biacore,Pharmacia Biosensor, North America) as described below.
5 EXAMPLE 4
Screenin~ for Inhibition of Protein-Protein Complex Formation
A protein-protein binding assay CO.~ g of the minim~l components
n~ces.c~. ~ to permit Candida albicans TBP-Candida albicans TFIIB binding to occur can
be used to screen for inhibition of the formation of the Candida albicans TBP-Candida
albicans TFIIB complex during ll~lsclil"ion initiation. The elem~ontc of such an assay
consist of; a) recombinant Candida albicans TBP, b) TFIIB, preferably a recombmant
Candida albicans TFIIB, and optionally c) a c2n~ te inhibitor of binding.
The inrlll5ion of an inhibitor molçcl-le that hl~r~,.cs with the hlteld.;~ion
be~weell the Candida albicans TBP and Candida albicans TFIIB inhibits ~ sc~ ion
15 initiation. The inhibitor may interact with the Candida albicans TBP or TFIIB protein and
thus induce a conrol,national change which ~levell~ binding, or it may directly inhibit the
clion of Candida albicans TFIIB and TBP plote~s. In this assay, inhibition is
meas~ d as a reduction in the amount of Candida albicans TBP-TFIIB complex produced
relative to the ~m~ nt of Candida albicans TBP-TFIIB complex produced in the absence
20 of the inhibitor (the ~osili~e control). A decrease in the amount of TFIIB-TBP complex
is indicative of an inhibitor. Detc ...;..-'ion of effective levels of inhibition of Candida
albicans TBP-TFIIB binding is described below.
One assay for fo.~ tio., of Candida albicans TBP-TFIIB complex is
provided as follows. 10 - 100 ng Candida albicans TFIIB and 10 - 100 ng Candida
25 albicans TBP are t:Apl~ssed in and purified from E. coli as described above, and are
added to re~ctionc con~ g 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mMMgCl2, 12% glycerol, 10 mM dithiothreitol (DTT) 100 ~g/ml BSA, and a c~m
inhibitor. The Illi~lUlC iS then in~ b~d at 30~ C for 30 - 60 min.
Pormation of a complex comprising Candida albicans TBP and Candida
30 albicans TFIIB may be ~l~tected by an electrophoretic mobility shift of labeled (e.g.
radioactive or fluor~,scenl) TBP or TFIIB on 4% polyacrylamide gels cont~ining 5% (v/v)
glycerol, 25 mM Tris, 100 mM glycine, lmM EDTA, 5 mM MgCl2, pH 8.3 in the
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~,csence of the llnl~behpd partner. The position of the labeled partner is detPcte~ by
apploplidLe metho-lc (e.g., autoradiography for radioactive oligonucleotide). The ~hsen~e
or deviation of the eYpectPd mobility shift due to Candida albicans TFIIB-TBP complex
formation is indicative of an effective inhibitor.
S Formation of a complex col~ illg Candida albicans TBP and Candida
albicans TFIIB may be AetPctPd as retention of labeled TBP utili7.ing known affinity
metho-lc for irnmobilizing the Candida albicans TFIIB protein (e.g., biotin/streptavidin,
nitroce~ lose filtration, affinity chrol"atography, immlmoaf~lnity). The failure of
formation of the Candida albicans TFIIB-TBP complex is indicative of inhibition, and is
10 in(lic~tP,d by llomete~ on of labeled TBP. Alternatively, the immobilized elem~rt may
be Candida albicans TBP and the labeled partner Candida albicans TFIIB.
In the above example, a stronger signal may be co~ c;d in the presence
of both TBP and TFIIB and, in addition, a DNA template cont~ining a TATA ehP~nPnt
The complex is then .~ ed by lntor~iography~ Phos~holi~llager technology, or
15 scintill~tion CUUllLillg for ra~ y labeled factors, fluoro~ h~ for fluol~sc.,.llly
labeled factors, l.. ;.,(,.. rtry for factors labeled with ligands that are detected using
chPmil~ sc~l or phnsl~hoJ~ ce~ll pl~g mPth-dologies, or other similar ~lPtectionmPtho~s or materials labeled as described above that are s~lldard in the art.
Other mPth-~Ac for ~lPL~,ti"g or s~aratillg protein-protein complexes may
20 be used, inrh~rling W crosslinking analysis, high pe~ro~ nre liquid chromatography,
phage display technology, and surface plasmon lesonallce as described herein.
EXAMPLE 5
Assa~ for Formation of TBP-TFIIB-RNA Poly,llcl~se II-DNA Complex
Formation of a TBP, TFIIB, RNA polymerase II, DNA complex is known
to be n~rkP~lly stim~ te~ by the ad~lition of another factor, TFIIF. Previous data
inrlic~tto.s that TFIIF from S. cerevisiae can function in species as distantly related as
Schiznsaccharom~ces po~e and hl-m~n.c, sL~on~ly su~ge~Lmg that this factor can
functionally replace its C. albicans homolog. Accordingly, this factor is purified from
5. cerevisiae by published methods (Sayre, 1992,J. Biol. Chem. 267:23383) and used to
reconstitute formation of a complex cont~ining C. albicans TBP, TFIIB, RNA polymerase
II and promoter cont~ining DNA such as that described for reconstitution of the TFIIB-
TBP-DNA complex.
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18
Complex formation is carried out in reactions cont~ining~ for example, 10 -
100 ng Candida albicans TBP, 10 - 100 ng Candida albicans TFIIB, 10 - 100 ng
Candida albicans RNA polymerase II, 10 - 100 ng S. cerevisiae TFIIF, 0.5 ng double-
stranded TATA element cont~ining-oligonucleotide (same as that used for TFIIB-TBP-
5 DNA complex assay), 10 - 20 mM HEPES (or equivalent), pH 7.5 - 8.0, 5 mM MgCl2,
12% glycerol, 10 mM dithio~.~eilol (DTT), 100 ~ug/ml BSA, 5 - 20 ~g/ml poly (dG-dC);
(dG-dC) and compound(s) to be tested for inhibitory activity. Following inrllb~tinn at 30~
C for 30 - 60 min, complexes are detected by one of the metho~s described above for the
TBP-TFIIB-DNA complex. The TBP-TFIIB-RNA polymerase II-DNA complex has a
10 slower electrophoretic mobility than the TBP-TFIIB-DNA complex i~lentifi~d by using
the elecLIùphol~ic m~thod In addition, complex formation can be ~letected as TBP,
TFIIB-depen-lent retention of RNA polymerase II activity (lllea;,ul~,d by incoly ,l~Lion of
labeled nucleotide pl.,CUl~Ol~ into acid-insoluble product using the assay for RNA
polyll-e~ase activity desclil)ed in the RNA polymerase II purifi~tion protocol above) on
15 a matrix with bound TATA el~ ~..e .l co..~ g DNA. The IC50 of inl~ilo,~ colllyuui~ds
will be ~el~ ...i..~ by titration into reaclions recol.~ c~ as described above. The ICso
of these colllpoullds against re~~ on~ l~col.~ d with human TBP, TPIIB and RNA
polylllelase II will also be d~ ~.. in~d by the same mPth~ Human RNA pOlyl~c.asc II
and TFIIF are l,u,irled as described pl~ iuusly (Flores et al., 1990, J. Biol. Chem.
20 265:5629-5634; Reinberg et al., J. Biol. Chem 262:3310-3321). Those compounds whose
ICso against re~ction~ cont~inin~ C. albicans factors is < 1/5 of their ICso against
reactions l~co~ ed with human factors will be tested for their ability to inhibit C.
albicans growth as described below.
25 EXAMPLE 6
Phage Display Inhibitor Sc~ ,nillg
In addition to the above mentioned ~ dard tec~ni~ es of the art, other
technologies for molecular identifir~tion can be employed in the i~entifi-~tion of
inhibitor molecules. One of these technologies is phage display technology (U.S. Patent
30 No. 5,403,484. Viruses E~l,les~ing Chimeric Binding ~lcins). Phage display permits
identifir~tion of a binding protein against a chosen target. Phage display is a protocol
of molecular SCl~,e,ning which utilizes recolllbh~n~ bacteriophage. The technology involves
transforming bacteriophage with a gene that encodes an applopliate ligand (in this case,
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19
a c~n~ te inhibitor) capable of binding to the target molecule of interest. For the
purposes of this disclosure, the target molecule may be Candida albicans TBP, or a DNA-
protein or protein-protein complex formed using TBP and/or TFIIB, as described herein.
The transformed bacteriophage (which preferably is tethered to a solid support) express
S the c~n~ te inhibitor and display it on their phage coat. The cells or viruses bearing the
c~n~ t~ inhibitor which recogmze the target molecule are isolated and amplified. The
successful inhibitors are then characterized.
Phage display technology has advantages over standard affinity ligand
scl~e~ g technologies. The phage surface displays the microplot~ ligand in a three
10 r~imPn~ional conformation, more closely resembling its naturally occl~rring conro~ ation.
This allows for more specific and higher affinity binding for scl,,.,ni~lg purposes.
EXAMPLE 7
Biospecific Interaction Analvsis
A second relatively new sclee~ lg technology which may be applied to the
inhibitor scleenillg assays of this invention is biospecific interaction analysis (BIAcore,
ph~rm~ria Bios~n~or AB, Uppsala, Sweden). This technology is described in detail by
Jonsson et al. (Bioterhniq~les 11:5, 620-627 (1991)). Biospecific ~lt~,~dclion analysis
utilizes surface plasmon lesol~lce (SPR) to monitor t_e adsorption of biomolecular
20 complexes on a sensor chip. SPR measules the changes in l~;rlac~ e index of a polarized
light directed at the surface of the sensor chip.
Specific ligands (i.e., c~n~ late inhibitors) capable of binding to the target
molecule of interest (i.e., Candida albicans TBP or a protein-protein or protein-DNA
complex co~t~ining TBP) are immobilized to the sensor chip. In the ~les~"~ce of the
25 target molecule, specific binding to the immobilized ligand occurs. The nascent
immobilized ligand-target molecule complex causes a change in the refractive index of the
polarized light and is ~etected on a diode array. Biospecific ill~.aclion analysis provides
the advantages of; 1) allowing for label-free studies of molecular complex formation; 2)
studying molecular interactions in real time as the assay is passed over the sensor chip;
30 3) d~l~ti.-g surface concentrations down to 10 pg/mm2; d~tecting interactions between two
or more molecules; and 4) being fully aulolllated (Biotçcllniq~lPs 11:5, 620-627 (1991)).
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EXAMPLE 8
Hi~h Throu~hput Screenin~ of Potential Inhibitors
It is con~e~ lated according to the invention that the ~ ,.,.ling methods
disclosed herein encompass s.;leenillg of multiple sa-m--ples .simllltqnPously, also referred
5 to herein as 'high throughput' scret;ning. For example, in high throughput screel~ing,
from several h.~ ,d to several thousand c~ntli~qte inhibitors may be screened in a single
assay. Several examples of high throughput screening assays useful according to the
invention are as follows.
A protein A (pA)-C. albicans TBP fusion protein is ge~ dt~ by inserting
10 the coding sequen~e of TBP in fr~ne dow~ ~ll of the pA coding sequence of theplasmid pRIT2T (Phqrmqriq Biotech). The fusion construct is intl~ced, and the res~ltq-nt
recombinant protein is e~ aclcd and purified according to the m~nllfactllrer's
recommPn~lp~d con~ition~. This plocedurc can also be carried out for the pl~,~aldlion of
a pA-Candida albicans TFIIB fusion protein except that the dow,.sl,~a." coding sequence
15 is that of TFIIB protein; all other steps would remain the same.
A Dynatech Microlite 2 IlPicl. lil~,r plate or equivalent high protein-binding
capacity plate is coated with 1 ~g/well human IgG by inrllb~ing 300 ~l 3.33 ~4g/ml
human IgG (Sigma) in coating buffer (0.2 M sodium call,onate, pH 9.4) in the well for
4-12 hr at 4~C. The coating buffer is then ~l~c~ and the wells are washed five times
20 with 300 ~1 PBS. 300 ,ul blocking buffer (SuperBlock~ blocking buffer; Pierce)
cont~ining 3.33 ~g/ml pA-TBP or pA-TFIIB are added and the plate is inruhate~l for 4
or more hours at 4~C. The plates may be stored in this form at 4~C until ready for use.
When ready for use the plates are washed five times with 300 ,ul PBS. Test compound
at a final concellllation of 20-200 ~M, labeled TBP or TFIIB (i.e., nonfusion protein),
25 whichever is not added during the coating step, and 10 - 1000 fmol plulllotel-cont~ining
oligonucleotides are s~lspen~ in HEG buffer cont~ining 200 ~g/m~ BSA in a total
volurne of 150 ~l and are added and the reaction is inrubated at room telll~J.,lalulc with
gentle agitation for 60 min. The plate is then washed five times with PBS using a
Dynatech plate washer or equivalent. Bound labeled protein is qn~ntit~terl by adding 250
30 ~l Miclusci~lt (Packard) per well and is counted in a microtiter plate-compatible
seintill~tion ~pe~ ophotometer.
As an ~ltern~tive, the protein A fusion and the second, non-fusion protein
can be ine~lb~t~ in the presence of test compound in polypropylene microtiter plates
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21
under the same buffer and in~ub~tion conditions described above. The reaction mix is then
transferred to the wells of a microtiter plate coated with human IgG (which is prepared
as described above, and is stored in blocking buffer and is washed five times with 300 ~1
PBS immPfli~tely before use) and is inr'lb?~d for 60 min at room tc.n~eldture with gentle
S agitation. RPt~ntion of radioactive protein is q~l~nti~lP(~ as above.
Interaction of TBP and TFIIB, which is measured as retention of
rar~ioa~tivity on the plate, is d~pe ~-ient on human IgG coating the plate and wild-type
Candida albicans TBP or TFIIB, one of which must be fused to pA. (~n~ te
inhibitors or extracts that inhibit retention of r~ion~tivity by more than 30% are i-lPIltifi~d
10 and the inhibiloly activity is further purified if nrces~.y.
I~lhil,ilols i~PntifiP~l as deccri~ed above are then tested for their ability toinhibit Candida albicans TBP-dependent L-~ulscl;~lion in an in vitro ~ sclil,tion system
as described herein, and also may be tested for their ability to inhibit Candida albicans
growth.
~ther fusion or modified protein ~ S that are contemplated include, but
are not limited to, glutathione-S-l~ lase, maltose binding protein, infllnPn7~ virus
hPm~ tinin, FLAGn~ and hPY~ ti~linP fusions to Candida albicans TBP or Candida
albicans TFIIB which are pl~arcd, eA~.es~ed, and purified by published mPtho~c or
biotinylated Candida albicans TBP or TFIIB which are pl~aled using reactive biotin
20 precursors available co.. lfl,,ially. The ~ulirled fusion or ml tlifiPd protein is
immobilized on a microtiter plate cont~inin~ the a~,r~l,liale ligand for each fusion protein
(e.g. gll~t~thione~ amylose, CA157 antibody, etc., respectively) and the assay is carried
out and the results evaluated in esse .~;~lly the same ~ r as described above.
25 EXAMPLE 9
C~ndid~te Inhibitors
A "c~n~ te inhibitor," as used herein, is any compound with a potential
to inhibit Candida albicans TBP-m~ ted llal~scliplion initiation or complex formation.
A c~n~ tP inhibitor is tested in a concenlIdtion range that d~p~n~1s upon the molecular
30 weight of the molecule and the type of assay. For example, for inhibition of
protein/protein or protein/DNA complex formation or llai~s~;liplion initi~tion, small
molecules (as defined below) may be tested in a concelll àlion range of lpg - 100 ug/ml,
preferably at about 100 pg - 10 ng/ml; large molecules, e.g., peptides, may be tested in
CA 02250129 1998-09-30
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22
the range of 10 ng - 100 ug/ml, preferably 100 ng - 10 ug/ml.
Inhibitors of Candida albicans growth or viability may target the novel
transcription factor described herein, TBP, or it may target a protein or nucleic acid that
hlLe,ac~ with the novel transcription factor so as to prevent the natural biological
5 interaction that occurs in vivo and leads to tldnsclipLion initiation in Candida. Thus, an
inhibitor idPntifiPd as described herein will possess two ~r~clLies: 1) at some
concentration it will inhibit Candida albicans growth or viability; and 2) at the same
concellLlàtion, it will not signfir~ntly affect the growth of ~ n, particularly human,
cells.
~n~ te inhibitors will include peptide and polypeptide inhibitors having
an amino acid sequence based upon the novel TBP sequences described herein. For
PY~mplP, a fragment of TBP may act as a co...pe~;~ive inhibitor with respect to TBP
binding to other protems involved in Candida L~ scli~lion, e.g., RNA polymerase II,
TFIIB, or with respect to binding of the lldnscli~ion complex to the DNA template.
C~n~1id~tP inhibitor colllpoullds from large libraries of synthetic or natural
compounds can be scl~,ned. Numerous means are ~;u~ ly used for random and directed
synthesis of saccharide, peptide, and nucleic acid based compounds. Synthetic compound
libraries are commercially available from a llulll~l of co~.pAniPS inr!-ltlin~ Maybridge
ChPmir~l Co. (Trevillet, Cornwall, UK), Co~lge.le~ lc.,lon, NJ), Brandon Associates
20 (Me~im~r~ NH), and Miclùsoulce (New Milford, CT). A rare ch~mir~l library is
available from Aldrich (Milwaukee, WI). ColllbindL~l;al libraries are available and can
be prepared. ~Itc~ ely, libraries of natural culll~uunds in the form of bactPri~l1
fungal, plant and animal extracts are available from e.g., Pan Laboldtolies (Bothell, WA)
or MycoSearch (NC), or are readily produceable. Additionally, natural and synthPtir~lly
25 produced libraries and compounds are readily mo-lifi~d through conventional chr~llir~l,
physical, and bioch~.~.ir~l means.
Useful compounds may be found within lwlllelous çhPmical classes, though
typically they are organic compounds, and preferably small organic compounds. Small
organic compounds have a molecular weight of more than 50 yet less than about 2,500
30 daltons, preferably less than about 750, more preferably less than about 350 daltons.
Exemplary classes include heterocycles, peptides, saccharides, steroids, and the like. The
colllpuullds may be mo(lifiPd to e.~h~i~reefficacy, stability, ph~ celltir~l compatibility,
and the like. Structural id~ rlr~lion of an agent may be used to identify, generate, or
.. . . ....
CA 02250129 1998-09-30
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23
screen additional agents. For example, where peptide agents are i~lentifipd~ they may be
modified in a variety of ways to ~ernh~nre their stability, such as using an ululaluldl amino
acid, such as a D-amino acid, particularly D-alanine, by functionqli7ing the amino or
carboxylic termim~, e.g. for the amino group, acylation or alkylation, and for the
5 carboxyl group, e~ .cdtion or qmi-lifi-~tion, or the like. Other methods of stabilization
may include e~ ..lqtion, for example, in liposomes, etc.
EXAMPLE 10
Measurement for effective inhibition
The amount of inhibition by a cqndid-q-te inhibitor is qu-q-ntified using the
following formula, which describes reactions n~co~ çcl with a ra~lioartively labeled
moiety.
(CPMposijve Con~rol ~ CPMsamplc)
Percent Inhibition = - x 100
(CPMPosilive Conlrol)
where CPMPoilivc Conool iS the a~ gc of the cpm in complexes or RNA molecules formed
in reactions that lack the cqn~ qt~ inhibitor, and CPMs~ p~c is the cpm in complexes
20 formed in reactions co~tqinin~ the cqn~iid-qte inhibitor. C-q-n-lidq-te inhibitors for which the
percent inhibition is 50% are titrated into re~ction~ contqining either Candida albicans
TBP or hlunan TBP (eA~l~,s~d in and purified from E. coli using e~i.cting recombinant
clones (P~telsoll et al., Science 248, 1625-1630, 1990; Kao et al., Science 248, 1646-
1650, 1990; Hoffman, et al., Nature 346, 387-390, 1990, and assayed as described above)
25 and their IC50 with respect to human and Candida albicans TBP de~e~ninPd from graphs
of compound colr~ ion vs. % inhibition. The IC50 is defined as the conce~ dLion
that results in 50% inhibition. C~n~1id~te inhibitors for which the IC50 against Candida
albicans TBP-co~ g reactions is less than or equal to 1/5 the IC50 against human TBP-
cont~ining reactions are further tested for their ability to inhibit the growth of Candida
30 albicans in culture as described below.
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24
EXAMPLE 11
Measurement for inhibition of Candida albicans ~rowth in culture
Once an inhibitor is identified in one or more of the binding or transcription
assays described herein, it may be desirable to Cletf~ f' the effect of the inhibitor on the
S growth and/or viability of Candida albicans in culture. A cq-n~il1qtp~ i.~il,i~or is tested for
its ability to inhibit growth of Candida albicans cells in culture as follows. Methods for
pelro~ ing tests on growth inhibition in culture are well-known in the art. Once such
yruce lul~ is based on the NCCLS M27P method (The National Committ~Ae for Clinical
Laboratoly Standards, Refe.~,~ce Method for Broth Dilution ~..lir~ gdl Su~ce~libility
10 Testing of Yeasts; proposed ~ndd~-l, 1992), as follows. Serial dilutions (two- or three-
fold steps starting from a mqxim--m concellll~tion of 100 - 200 ~g/ml) of cqn~ qte
inhibitor are ç,l~,all d using RPMI-1640 .~ as diluent and an aliquot of 100 ,ul of
each dilution is added to the wells of a 96-well poly~ ne lllicrotil, . plate. Five
Candida albicans colonies, picked from a Sabo~laud De~ use Agar plate in~Clllqtpd 14-20
15 hr previously with the test Candida albicans strain (Cat,alog ..w~e. 10231 from the
ican Type Culture CollPction Yeast Catalog), are resuspended in RPMI-1640
m~dillm such that the density of cells is 10,000 - 30,000 cells/ml. 100 ~1 of the cell
SUSpen~iQn is added to each of the wells of the 96-well lnicluli~. plate co..~i..;..g diluted
cqn~ A inhibitor and mP~Ihlm control. Cultures are mixed by agitation and inruba~e~ at
20 35~ C for 48 hr. without agita~iQn~ after which cell growth is monitored by visual
eclion for the formqtion of turbidity and/or myce!ial colonies. The minimllm
concentration of cqndi~lqt~ inhibitor at which no cell growth is det~Fcte~l by this method is
defined as the minimllm inhibitory co.~F .~.~(ion (MIC) for that co~ uul~d. Examples of
MICs for known anlirul~l colll~uui~ds obtained using this techniq~le are 0.125 - 0.5
25 ~g/ml for flllronq7ole and 0.25 - 1.0 ~g/ml for ~n~~h~ icin B (The National Co~ Pe
for Clinical Laboratory Standards, ReÇt~ c~ Method for Broth Dilution Anlir~ al
Susceptibility Testing of Yeasts; l)loposesd s~ldard, 1992). An inhibitor identified by
the mP~hnfl~ described herein, will have MIC which is equivalent to or less than the MICs
for fluconq7Ole or a nphotericin B.
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EXAMPLE 12
Tlanscllplion Inhibition Coul~lers~;.cen Using Human TBP
A compound identified as an inhibitor of Candida albicans according to one
or more of ehe assays described herein may be tested further in order to determine its
5 effect on the host organism. In the development of useful antifungal compounds for
human thc.dpcuLics, it is desirable that such compounds act as effective agents in
inhibiting the viability of the fungal pathogen while not .signifir~ntly inhibiting human cell
systems. Specifically, inhibitors of Candida albicans ide~tifip(l in any one of the above
described assays may be c~ u~ ened for inhibition of human TBP.
Recombinant human TBP can be obtained from existing sources and
purified by published methods (for example, see Peterson et al., Kao et al., and Hoffman
et al., supra) and contacted with the c~n-lid~te inhibitor in assays such as those described
above but using a human system. The effectiveness of a Candida albicans TBP inhibitor
as a human th,l~y~uLic is determinP~ as one which exhibits a low level of inhibition
15 against human TBP relative to the level of inhibition with respect to Candida albicans
TBP. For example, it is yl~çelled that the amount of inhibition by a given inhibitor of
human TBP in a human system be no more than 20% with respect to the amount of
inhibition of Candida albicans TBP/TF~B in a Candida system when tested in any of the
assays described above.
Dosage and Pharrn~re~tir~l Formulations
For thel~yculic uses, inhibitors idPntifiPd as described herein may be
mini.stered in a ph~ e.~lic~lly acceptable/biologically colllpalible formulation, for
example, in the form of a cream, ointm~nt, lotion or spray for topical use, or in a
25 physiological solution, such as a salt solution, for intPrn~l ~flmini~tration. The amount
of inhibitor ~mini~tered will be determined according to the degree of pathogenic
infection and whether the infection is systemic or loc~li7ed, and will typcially be in the
range of about lug - 100 mg/kg body weight. Where the inhibitor is a peptide or
polypeptide, it will be a~mini.~tered in the range of about 100 - 500 ug/ml per dose. A
30 single dose of inhibitor or multiple doses, daily, weekly, or intermittently, is contemplated
according to the invention.
The route of a~mini~tration will be chosen by the physician, and may be
topical, oral, transdermal, nasal, rectal, intravenous, intr~mnsc~ r~ or subcutaneous.
CA 022~0l29 l998-09-30
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26
Budapest Treaty Deposit
E. coli transformed with a plasmid cont~ining the gene encoding Candida
albicans TBP has been deposited in an international depository, the A.T.C.C., Roclcville,
MD, under the accession number 69900, on September 15, 1995. E. coli transformedS with a plasmid cu"~ g the gene encoding Candida albicans TFIIB has been deposited
in an international depository, the A.T.C.C., Rockville, MD, under the accession number
69899, on September 15, 1995. A.T.C.C. Nos. 69900 and 69899 will be available to the
public upon the grant of a patent which discloses the accession l~ulllbtl~ in conjull.;lion
with the invention described herein. The deposits were made under the Budapest Treaty,
10 will be available beyond the enrolceable life of the patent for which the deposit is made,
and will be m~int~in~d for a period of at least 30 years from the time of deposit and at
least 5 years after the most recent request for the r"".;~ of a sample of the deposit is
received by the A. T. C. C. It is to be understood that the availability of these deposits does
not COl~lilul~ a license to practice the subject invention in derogation of patent rights
15 granted for the subject invention by gO~"l...llr..l;~l action.
OTHER EMBODIMENTS
The foregoing examples demona~lale experiments pelro~ ed and
co..lP~-,pl~t~d by the present hlvt~ ls in making and call~illg out the invention. It is
believed that these examples include a disclosure of tt~chniqlles which sene to both apprise
20 the art of the practice of the invention and to demol~llate its usefulness. It will be
appreciated by those of skill in the art that the techniques and embo~im~ntc disclosed
herein are preferred embo~imPnt~ only that in general llulllel~us equivlaent methods and
terhniquec may be employed to achieve the same result.
All of the references identifi-od hereinabove, are hereby expressly
25 hlcoll,olaled herein by reference to the extent that they describe, set forth, provide a
basis for or enable compositions and/or methods which may be hll~ol~nl to the practice
of one or more embo~im~onts of the present inventions.
~ . . . ..
CA 022~0129 1998-09-30
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SEQUENCE LISTING
(i) GENERAL INFORMATION
(i) APPLICANT: SCRIPTGEN PHARMACEUTICALS, INC.
(ii) TITLE OF THE INVENTION: NOVEL TATA-BINDING PROTEIN FROM CANDIDA
ALBICANS, NUCLEIC ACID SEQUENCE CODING THEREFORE, AND METHODS OF SCREENING
FOR INHIBITORS OF CAMDIDA ALBICANS GROWTH
(iii) NUMBER OF ~QU~N~S: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: DARBY & DARBY P.C.
(B) STREET: 805 Third Avenue
(C) CITY: New York
(D) STATE: New York
(E) COUN1~Y: United States of America
(F) ZIP: 10022-7513
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ Version 1.5
(vi) CURRENT APPLICATION DATA:
~A) APPLICATION NUMBER:
(B) FILING DATE:
~C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/626,309
(B) FILING DATE: 01-APR-1996
4û
(viii) AllORN~Y/AGENT INFORMATION:
(A) NAME: S. PETER LUDWIG, ESQ.
(B) REGISTRATION NUMBER: 25,351
(C) REFERENCE/DOCKET NUMBER: 0342/2C488-WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212)527-7700
5û (B) TELEFAX: (212)753-6237
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO:l:
(i) S~YU~N~ CHARACTERISTICS:
(A) LENGTH: 219 amino acids
(B) TYPE: amino acid
(C) STR~Nn~nN~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
~ (iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CA 022~0l29 l998-09-30
W O 97l36925 PCTrUS97/06170
~8
Met Lys Ser Ile Glu Glu Asp Glu Lys Asn Lys Ala Glu Asp Leu Asp
5 10 15
Ile Ile Lys ~ys Glu Asp Ile Asp Glu Pro Lys Gln Glu Asp Thr Thr
20 25 30
Asp Ser Asn Gly Gly Gly Gly Ile Gly Ile Val Pro Thr Leu Gln Asn
35 40 45
Ile Val Ala Thr Val Asn Leu Asp Cys Arg Leu Asp Leu Lys Thr Ile
50 55 60
Ala Leu His Ala Arg Asn Ala Glu Tyr Asn Pro Lys Arg Phe Ala Ala
Val Ile Met Arg Ile Arg Asp Pro Lys Thr Thr Ala Leu Ile Phe Ala
Ser Gly Lys Met Val Val Thr Gly Ala Lys Ser Glu Asp Asp Ser Lys
100 105 110
Leu Ala Ser Arg Lys Tyr Ala Arg Ile Ile Gln Lys Leu Gly Phe Asn
115 120 125
Ala Lys Phe Cys Asp Phe Lys Ile Gln Asn Ile Val Gly Ser Thr Asp
130 135 140
Val Lys Phe Ala Ile Arg Leu Glu Gly Leu Ala Phe Ala His Gly Thr
145 150 155 160
Phe Ser Ser Tyr Glu Pro Glu Leu Pro Pro Gly Leu Ile Tyr Arg Met
165 170 175
Val Lys Pro Lys Ile Val Leu Leu Ile Phe Val Ser Gly Lys Ile Val
180 185 190
Leu Thr Gly Ala Lys Lys Arg Glu Glu Ile Tyr Asp Ala Phe Glu Ser
195 200 205
Ile Tyr Pro Val Leu Asn Glu Phe Arg Lys Asn
210 215
(2) INFORMATION FOR SEQ ID NO:2:
( i ) S~UU~N~h' CHARACTERISTICS:
(A) LENGTH: 344 amino acids
(B) TYPE: amino acid
tC) STRA~n~n~S: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) ~Y~Ol~-llCAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE: internal
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Ser Pro Ser Thr Ser Thr Ala Val Gln Glu Tyr Ile Gly Pro Asn
Leu Asn Val Thr Leu Thr Cys Pro Glu Cys Lys Ile Phe Pro Pro Asp
20 25 30
Leu Val Glu Arg Phe Ser Glu Gly Asp Ile Val Cys Gly Ser Cys Gly
35 40 45
Leu Val Leu Ser Asp Arg Val Val Asp Thr Arg Ser Glu Trp Arg Thr
50 55 60
Phe Ser Asn ASp Asp Gln Asn Gly Asp Asp Pro Ser Arg Val Gly Asp
65 70 75 80
Ala Gly Asn Pro Leu Leu Asp Thr Glu Asp Leu Ser Thr Met Ile Ser
85 90 95
Tyr Ala Pro Asp Ser Thr Lys Ala Gly Arg Glu Leu Ser Arg Ala Gln
100 105 110
Ser Lys Ser Leu Val Asp Lys Lys Asp Asn Ala Leu Ala Ala Ala Tyr
115 120 125
Ile Lys Ile Ser Gln Met Cys Asp Gly Tyr Gln Leu Pro Lys Ile Val
130 135 140
Ser Asp Gly Ala Lys Glu Val Tyr Lys Met Val Tyr Asp Glu Lys Pro
145 150 155 160
.. , . _ . . ..
CA 022~0129 1998-09-30
W O 97/36925 PCTrUS97/06170
29
Leu Arg Gly Lys Ser Gln Glu Ser Ile Met Ala Ala Ser Ile Phe Ile
165 170 175
Gly Cys Arg Lys Ala Asn Val Ala Arg Ser Phe Lys Glu Ile Trp Ala
180 185 190
Lys Thr Asn Val Pro Arg Lys Glu Ile Gly Lys Val Phe Lys Ile Met
195 200 205
Asp Lys Ile Ile Arg Glu Lys Asn Ala Ala Asn Pro Asn Ala Ala Tyr
210 215 220
Tyr Gly Gln Asp Ser Ile Gln Thr Thr Gln Thr Ser Ala Glu Asp Leu
225 230 235 240
Ile Arg Arg Phe Cys Ser His Leu Gly Val Asn Thr Gln Val Thr Asn
245 250 255
Gly Ala Glu Tyr Ile Ala Arg Arg Cys Lys Glu Val Gly Val Leu Ala
260 265 270
Gly Arg Ser Pro Thr Thr Ile Ala Ala Thr Val Ile Tyr Met Ala Ser
275 280 285
Leu Val Phe Gly Phe Asp Leu Pro Pro Ser Lys Ile Ser Asp Lys Thr
290 295 300
Gly Val Ser Asp Gly Thr Ile Lys Thr Ser Tyr Lys Tyr Met Tyr Glu
305 310 315 320
Glu Lys Glu Gln Leu Ile Asp Pro Ser Trp Ile Glu Ser Gly Lys Val
325 330 335
Lys Leu Glu Lys ~le Pro Lys Asn
340
~2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 657 base pairs
(B) TYPE: nucleic acid
(C) sTRA~n~s single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
ATGAAGTCAA TAGAGGAAGA TGAAAAAAAT AAAGCCGAAG ATTTGGATAT TATAAAAAAC 60
GAAGATATTG ATGAACCTAA ACAAGAAGAT ACCACTGATA GTAATGGTGG TGGAGGTATT 120
GGTATAGTGC CCACATTACA AAATATTCTT GCTACGGTGA ATCTTGATTG TCGACTTGAT 180
AAAACAATTG CTTTACATGC TAGAAATGCC GAATATAATC CAAAACGTTT TGCTGCGGTG 240
ATTATGAGAA TTAGAGATCC AAAAACTACG GCATTAATCT TTGCTTCGGG GAAAATGGTT 300
GTGACTGGGG CTAAATCCGA AGACGATTCC AAGTTGGCTT CAAGAAAGTA TGCTAGAATC 360
ATTCAAAAGT TGGGGTTCAA TGCTAAATTT TGTGATTTTA AAATTCAAAA TATAGTGGGG 420
TCAACAGATG TTAAGTTTGC TATTAGATTA GAAGGCTTAG CTTTTGCTCA TGGTACTTTT 480
TCTTCATATG AACCAGAATT AlllCLlGGG TTAATTTATA GAATGGTGAA ACCAAAAATT 540
GTTTTACTTA TAll~L-lllC TGGGAAAATT GTTTTGACGG GTGCCAAAAA GACAGAAGAA 600
ATTTATGATG CATTTGAACT GATTTATCCG GTTTTAAATG AATTTCGTAA AAATTGA 657
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQu~NL~ CHARACTERISTICS:
(A) LENGTH: 1095 base pairs
(B) TYPE: nucleic acid
(C) sTR~Nn~n~Fss
(D) TOPOLOGY:
CA 022~0l29 l998-09-30
WO 97/36925 PCT/US97/06170
(1i) MOLECULE TYPE:
(iii~ HYPOTHETICAL: NO
(iv) ANTISENSE: NO
(v) ERAGMENT TYPE:
(vi) ORIGINAL SOURCE:
(xi) S~uU~ DESCRIPTION: SEQ ID NO:4:
TAAGCTTGTA TTACTAAGCA TATTATGTCG CCATCAACAT CTACGGCAGT ACAGGAGTAT 60
ATTGGACCAA ACTTGAATGT TACATTAACA l~lC~lGAGT GTAAGATATT TCCACCTGAT 120
TTGGTAGAGA GGTTCAGCGA AGGTGACATT ~l~l~lGGCA GTTGTGGGCT AGTATTGAGT 180
GAlC~l~llG TGGATACGAG ATCAGAATGG AGAACTTTCA GTAACGATGA CCAAAATGGT 240
GATGATCCTT ClC~l~llGG TGATGCAGGT AAcc~lllAT TAGACACAGA GGACTTGTCC 300
ACAATGATTT CTTATGCTCC TGATACTACC AAAGCAGGAA GAGAGTTAAG CCGAGCCCAA 360
TCTAAATCTC TAGTCGATAA AAAAGAcAAT GCATTGGCTG CAGCATATAT CAAGATTTCT 420
CAAATGTGCG ATGGTTATCA ATTGCCTAAA ATAGTTCTGG ATGGGGCCAA GGAAGTCTAC 480
AAAATGGTTT ATGACGAGAA ACCATTGCGA GGAAAATCAC AAGAGAGTAT CATGGCAGCT 540
TCTATCTTTA TTGGTTGCAG AAAGGCCAAT GTTGCTCGTT CATTCAAAGA GATATGGGCA 600
AAGACTAATG TACCTCGTAA GGAAATTGGT AAA~l~ll~A AGATCATGGA CAAGATCATT 660
CGTGAAAAGA ATGCAGCCAA CCCTAATGCT GCATATTACG GTCAAGACAG CATTCAAACC 720
ACCCAAACTT CGGCCGAGGA TTTGATTAGA AGA1l~l~ll CTCACTTGGG TGTTAACACA 780
CAAGTTACAA ATGGTGCGGA ATACATAGCC AGAAGATGTA AGGAAGTCGG GGTTTTAGCA 840
GGTAGATCGC CAACTACAAT TGCTGCAACT GTAATTTACA TGGCTTCACT A~l~lllGGA 900
TTTGACTTAC CTCCATCCAA GATATCTGAT AAAACTGGTG TCAGTGATGG TACTATCAAA 960
ACTTCATACA AGTACATGTA CGAGGAGAAA GAACAATTGA TTGATCCATC TTGGATAGAA 1020
AGTGGTAAAG TAAAATTGGA AAAAATACCA AAAAACTAAT ACAGCGGAGT CGCCACTGTT 1080
AATCCTTTAC CCTCT 1095
