ENZYME
FIELD OF THE INVENTION
The present invention relates to a novel enzyme capable of catalyzing conversion of the monophosphorylated form of abacavir, a purine nucleoside analogue, to the monophosphorylated form of carbovir and cyclopropylamine.
BACKGROUND
The antiretroviral chemotherapeutic agent abacavir is converted in vivo to an HIV reverse transcriptase inhibitor by a multi-step process. Abacavir is first phosphorylated to its monophosphate form (abacavir-MP) by adenosine phosphotransf erase. Abacavir-MP is then converted by a cy tosolic enzyme to the 6- oxopurine (guanosine) analogue monophosphate (carbovir monophosphate, or CBV- MP), which is then further phosphorylated by cellular kinases to the anti- viral agent carbovir triphosphate (CBV-TP). (Figure 10).
Although the antiviral agents abacavir and carbovir are both metabolized in vivo to the active agent carbovir-triphosphate, they do not utilize the same pathways. The majority of abacavir anabolism occurs through the pathway described above; only a small percentage of intracellular abacavir is first converted to unphosphorylated carbovir prior to becoming carbovir-triphosphate. Faletto et al., Antimicrob. Agents Chemother. 41: 1099 (1997). The in vivo pharmaco inetic, distribution, and toxicological profiles of abacavir are improved over those of CBN. Daluge et al. Antimicrob Agents Chemother41:1082 (1991).
SUMMARY
An aspect of the present invention is an isolated and purified deaminase having a molecular weight of from about 38 kilodaltons to about 40 kilodaltons based on gel filtration. The deaminase has the ability to catalyze the removal of the cyclopropyl amino moiety from abacavir-MP, is inhibited by GMP and is not inhibited by potassium cyanide.
A further aspect of the present invention is an isolated nucleotide molecule comprising a sequence that is selected from among SEQ ID NO: l, SEQ ID NO: 3 and SEQ ID NO:5 provided herein; nucleotide sequences encoding an enzyme with an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; nucleotide sequences that have at least about 90% sequence identity to above and which encode a deaminase; and nucleotide sequences which differ from the sequences of (a) or (c) above due to the degeneracy of the genetic code; and a nucleotide sequence capable of hybridizing under stringent conditions with the sequence of (a), (b) or (c) and which encodes a deaminase.
A further aspect of the present invention is a deaminase comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO:4, or amino acid sequences which have at least about 90% sequence identity with these sequences and which are deaminases.
A further aspect of the present invention is a process for producing a deaminase by establishing a culture of a host cell transformed by an expression vector containing a polynucleotide of the present invention, and isolating expressed enzyme from the culture.
A further aspect of the present invention is an antibody that specifically binds to a deaminase as described herein.
A further aspect of the present invention is a method of screening a nucleoside analog containing an amino group for the ability to be deaminated by an abacavir-MP
deaminase. The method includes exposing a nucleoside analog containing an amino group to an abacavir-MP deaminase under conditions suitable for the enzyme's activity; and determining whether the nucleoside analog is deaminated.
A further aspect of the present invention is a method for identifying nucleoside analogs as potentially useful for the treatment of viral infections in humans, where the nucleoside analog includes an amine moiety, by determining whether the nucleoside analog or its monophosphate form is deaminated by abacavir-MP deaminase.
A further aspect of the present invention is a method of screening a subject as an aid in predicting the subject's suitability for treatment with a compound that is converted in vivo to its active form by an abacavir-MP deaminase, by assessing abacavir-MP deaminase activity levels in target cells of the subject and comparing these levels with predetermined values that have been associated with a favorable response to the compound. A subject with values outside the predetermined values would be considered less suitable for treatment with the compound than a subject exhibiting the predetermined values.
A further aspect of the present invention is a method of screening a subject as an aid in predicting the subject's suitability for treatment with a compound that is converted in vivo to its active form by an abacavir-MP deaminase, by assessing levels of GMP or AMP in target cells of the subject and comparing the values obtained with predetermined values that have been associated with a favorable response to the compound. A subject with values outside the predetermined values would be considered less suitable for treatment than a subject exhibiting the predetermined values.
A further aspect of the present invention is a method of studying a compound that is converted in vivo to its active form by an abacavir-MP deaminase to determine whether there is a correlation between the clinical response of a subject to the compound and in vivo activity of the enzyme, by determining abacavir-MP deaminase
activity levels in a plurality of subjects, and then administering the compound to each of the subjects, assessing the clinical response of each subject, and comparing the abacavir-MP deaminase levels to the clinical response to identify any correlation between response and enzyme activity level.
A further aspect of the present invention is a method of studying a compound that is converted in vivo to its active form by an abacavir-MP deaminase to determine whether there is a correlation between the clinical response of a subject to the compound and in vivo levels of AMP or GMP, by determining AMP or GMP levels in a plurality of subjects, administering the compound to each subject, assessing the clinical response of each subject to the compound, and comparing AMP or GMP levels to the clinical response to identify any correlation between response and AMP or GMP level.
A further aspect of the present invention is a method of treating a subject with a compound that is converted in vivo to its active form by an abacavir-MP deaminase, by assessing abacavir-MP deaminase activity levels in target cells of the subject, then comparing these levels with predetermined values that have been associated with a favorable response to the compound; and administering the compound to the subject when the subject's abacavir-MP deaminase levels are similar to the predetermined values.
A further aspect of the present invention is a method of treating a subject with a compound that is converted in vivo to its active form by an abacavir-MP deaminase, by assessing levels of GMP or AMP in target cells of the subject, comparing the values obtained with predetermined values that have been associated with a favorable response to the compound, and administering the compound to the subject when AMP or GMP levels are similar to the predetermined values.
BRIEF DESCRIPTIONS OF THE DRAWINGS
Figure 1. Time-course for the spectral changes that occurred during deamination of abacavir-MP to carbovir monophosphate.
Figure 2. A selected portion of the chromatogram for elution of the abacavir- MP deaminating protein from the Mono Q resin. Enzyme purified through Step 3 was absorbed onto a Mono Q resin in a HRlO/10 column. The enzyme was eluted from the resin with a linear gradient of KCI from 0 to 150 mM. The portion of the activity chromatogram between 60 and 75 mM KCI is presented for two independent preparations of the enzyme.
Figure 3. Chromatogram for the final purification step of the abacavir-MP deaminating protein in which activity and A2go are compared (Step 8). 8,600 units of protein were applied to the column.
Figure 4 Kinetic parameters for deamination of abacavir-MP (indicated as
2456U89 in the figure). The deamination of 1.7 μM abacavir-MP by 135 units of the deaminating protein was monitored at 290 nm. The time-course was analyzed by the integrated rate equation (Equation 1) to give values for Vm and Km of 0.0045 ± 0.0001 μM/s and 0.14 + 1 μM, respectively. The solid line is the time-course predicted for these values.
Figure 5 Inhibition of the abacavir-MP deaminating protein by GMP. The velocity of deamination of 1.7 μM abacavir-MP by 135 units of the deaminating protein was monitored at 290 nm. The initial velocity was determined for the indicated concentrations of GMP. These velocity values were normalized to the velocity in the absence of GMP. Equation 2 was fitted to the concentration dependence to give an IC50 value of 15 ± 1 μM. The solid line was predicted with this IC 50 value and equation 2.
From this value and the Km value, a value for Kj was calculated to be 1.1 μM by equation 3.
Figure 6 Time-course for enzymatic deamination of abacavir-MP to carbovir monophosphate. The reaction of 20μM abacavir-MP with 172 units of the deaminating protein was monitored at 290 nm. The reaction was initiated by addition of enzyme to 1 mL of the standard buffer containing the substrate equilibrated to 25 °C. The solid line was calculated for an enzymatic reaction with Nm = 0.195 μM/min and Km = 1.8 μM.
Figure 7 A. Reverse phase HPLC of AQC derivatives: chromatogram of abacavir-MP minus deaminating enzyme. Abbreviations: AMQ, aminoquinoline; ΝH3, AQC-derivatized ammonia.
Figure 7B. Reverse phase HPLC of AQC derivatives: chromatogram of deaminating enzyme minus abacavir-MP. Abbreviations: AMQ, aminoquinoline; NH3, AQC-derivatized ammonia.
Figure 7C. Reverse phase HPLC of AQC derivatives: chromatogram of reaction products of deaminating enzyme plus abacavir-MP. Abbreviations: AMQ, aminoquinoline; NH3, AQC-derivatized ammonia; CPA, cyclopropylamine.
Figure 7D. Reverse phase HPLC of AQC derivatives: chromatogram of authentic AQC-derivatized cyclopropylamine (CPA). Abbreviations: AMQ, aminoquinoline; NH3, AQC-derivatized ammonia; CPA, cyclopropylamine.
Figure 8A. Mass spectrometry of AQC-derivatized cyclopropylamine: nanoES- MS spectrum of authentic AQC-derivatized cyclopropylamine.
Figure 8B. Mass spectrometry of AQC-derivatized cyclopropylamine: product ion spectrum of authentic AQC-derivatized cyclopropylamine.
Figure 8C. Mass spectrometry of AQC-derivatized cyclopropylamine: precursor ion spectrum for precursors of m/z 171.0.
Figure 9A. Mass spectrometry of AQC-derivatized cyclopropylamine: precursors of m/z 171.0 from a control deaminating protein reaction containing no abacavir-MP following AQC derivatization.
Figure 9B. Mass spectrometry of AQC-derivatized cyclopropylamine: precursors of m/z 171.0 from a deaminating protein/abacavir-MP reaction following AQC derivatization.
Figure 9C. Mass spectrometry of AQC-derivatized cyclopropylamine: product ion spectrum of m/z 228.1 ion observed of an AQC-derivatized deaminating protein/abacavir-MP reaction mixture.
Figure 10. Anabolic pathway of abacavir to carbovir-TP in human cells. Abacavir is phosphorylated by adenosine phosphotransf erase to abacavir-MP, which is converted to CBN-MP by the deaminase disclosed herein. CBN-MP is then anabolized to CBN-TP. Only a small percentage of abacavir is converted to carbovir prior to phosphorylation.
DETAILED DESCRIPTION
Abacavir (formula I on Figure 10) is a synthetic purine nucleoside analogue containing an unsaturated cyclopentene ring in place of the 2'deoxyriboside of natural deoxynucleosides, and containing a cyclopropylamino group. Abacavir sulfate is commercially available as ZIAGEN® (Glaxo Wellcome), and is used in combination
with other antiretroviral agents to treat HIN-infected subjects. The chemical name of abacavir sulfate is (1 S,cis )-4-[2-amino-6-(cyclopropylamino)-9 H -purin-9-yl]-2- cyclopentene-1-methanol sulfate (salt) (2:1). Abacavir sulfate is the enantiomer with IS, 4R absolute configuration on the cyclopentene ring. It has a molecular formula of (C 14 H 18 Ν 6 O) 2 -H2 SO 4 and a molecular weight of 670.76 daltons. To act as an effective anti-retroviral agent, abacavir must be converted in vivo to carbovir triphosphate (CBN- TP).
Carbovir (CBN; formula VII of Figure 10)) is also an anti-retroviral nucleoside (guanine) analog that is metabolized in vivo to the CBV-TP active anti-retroviral agent. Vince et al. , Biochem. Biophys. Res. Commun. 156:1046-1053 (1988); Parker et al., Antimicrob Agents Chemother31(5): 1004-9 (1993). However, in vivo use of CBV as an anti-HIV agent is associated with toxicological effects, including renal and cardiac toxicity, poor oral bioavailability, and inefficient brain penetration. Daluge et al., Antimicrob Agents Chemother 41 : 1082 (1997) .
The majority of in vivo abacavir is metabolized to CBV-TP without first being converted to CBV. Faletto et al., Antimicrob. Agents Chemother. 41: 1099 (1997) report that, using cells in which radiolabelled species were monitored, most abacavir was first phosphorylated by an AMP phosphotransferase to the monophosphate form of abacavir (ABV-MP), which was then converted by a cytosolic enzyme to carbovir monophosphate (CBV-MP) by removal of abacavir 's 6-cyclopropyl amino moiety. The CBV-MP was then further phosphorylated by cellular GMP kinase and nucleotide diphosphate kinase to carbovir-triphosphate. Levels of guanine analogue were less than 2% the level of abacavir in plasma of animals and man. Faletto et al. (1997) state that it is this different activation pathway of abacavir (versus carbovir) that overcomes some deficiencies of in vivo carbovir while maintaining selective anti-HIV activity. Deamination of abacavir-MP is not catalyzed significantly by adenosine deaminase or AMP deaminase (Faletto et al., (1997) Antimicrobial Agents & Chemotherapy. 41:1099).
The present inventors have identified and purified a novel mammalian cytosolic enzyme, herein called abacavir-MP deaminase, that converts abacavir monophosphate to carbovir monophosphate. DNA encoding human abacavir-MP deaminase has been isolated and sequenced.
The present invention provides mammalian abacavir-MP deaminase in an isolated and homogeneously purified form. The enzyme may be isolated and purified from mammalian tissues (e.g., from bovine, porcine, ovine or rodent tissues), preferably from liver tissue. The abacavir-MP deaminase in its native state has a molecular weight of from approximately 38 kilodaltons to about 40 kilodaltons based on SDS-PAGE gel filtration. The enzyme catalyzes the removal of the 6-cyclopropyl amino moeity of abacavir-MP to convert abacavir monophosphate to carbovir monophosphate; the enzyme is inhibited by guanosine 5' monophosphate (GMP), adenosine 5' monophosphate (AMP) and 2,6-diaminopurine ribose 5 '-monophosphate but is not inhibited by depletion of O2 or H2O2, or by potassium cyanide (KCN).
The present inventors purified an abacavir-MP deaminating protein approximately 500,000-fold from mature rat liver. The catalytic efficiency of the enzyme was found to be high. The value of kcat/Km for the abacavir-MP deaminating
-1 -1 protein was calculated from kinetic data to be 9 μM s . For comparison, the value
of kcat/Km for triosephosphate isomerase, which is diffusion limited, has a value of 240
μM"1 s_1(Putman et al. (1972) Biochem. J. 129: 301).
The possibility existed that the "deamination reaction" of abacavir-MP involved oxidation of the cyclopropyl ring followed by nonenzymatic elimination of guanine. If
O2 or H2O2 were reactants, the products could be complex mixtures and the enzyme
would be classified as an oxidase or peroxidase. However, it was determined that the abacavir-MP deaminating protein was not a peroxidase or oxidase. The general reaction catalyzed by the protein is given by:
abacavir-MP 7 Carbovir Monophosphate + Products
This reaction was not inhibited by depletion of O2 or H2O2 from the assay, or by KCN.
Because the reaction was not dependent on peroxide or oxygen, the amine product of the reaction was predicted to be cyclopropylamine, placing the enzyme in the deaminase class; cyclopropylamine was subsequently identified as the product of the deamination. The enzymatically generated amine product was characterised by chromatographic and mass spectrometric techniques after formation of a fluorescent derivative.
The overall reaction catalyzed by the abacavir-MP deaminase isolated from rat liver, with abacavir as substrate, is described by: abacavir-MP + H2O -x carbovir monophosphate + cyclopropylamine
Additionally, it was found that the abacavir-MP deaminating activity was potently inhibited by GMP and AMP. While not wishing to be held to a single theory, the present inventors suggest that this finding has relevance for the differential efficiency of abacavir in some subjects. Individuals who respond less well may possess elevated levels of AMP or GMP, potentially inhibiting the deamination of abacavir-MP to the active metabolite.
The present inventors have identified a protein, herein termed abacavir-MP deaminase, that converts abacavir monophosphate to carbovir monophosphate by removal of the 6-cyclopropyl amino moiety. A human cDNA encoding the abacavir- MP deaminase of the present invention was isolated as described in the Examples,
below. The sequence of the coding portion of the isolated cDNA is provided as SEQ ID NO: 1:
ATGATAGAGG CAGAAGAGCA ACAGCCπGC AAGTCAGACT TCTATTCTGA AπGCCAAAA 60 GTGGAACπC ATGCCCACπ GAATGGATCC A AGTTCTC ATACCATGAA GAAATTAATA 120
GTCCAGAAGC CAGATCπAA AATCCACGAT CAGATGACTG TGAHGACAA GGGAAAGAAA 180
AGAACTTTGG AAGAATGTπ CCAGATGTTT CAAACTAπC ATCAGCTTAC TAGTAGCCCT 240
GAAGATATTC TAATGGTCAC AAAAGATGTC ATAAAAGAAT πGCAGATGA CGGCGTCAAG 300
TACCTGGAAC TAAGGAGCAC ACCCAGAAGA GAAAATGCTA CCGGAATGAC TAAAAAGACT 360 TATGTGGAAT CTATACπGA AGGTATAAAA CAGTCCAAAC AAGAAAACTT GGACATTGAT 420
GTTAGGTATT TGATAGCAGT TGACAGAAGA GGTGGCCCH TAGTAGCCAA GGAGACTGTA 480
AAACTTGCCG AGGAGTTCTT CCTTTCTACT GAGGGTACAG TTCTTGGCCT TGACCTCAGT 540
GGAGACCCTA CTGTAGGACA AGCAAAAGAC TTCTTGGAAC CTCTTTTAGA AGCTAAGAAA 600
GCAGGTCTGA AGTTAGCAπ GCATCTTTCA GAGATTCCAA ACCAAAAAAA AGAAACACAA 660 ATACTCCTGG ATCTGCTTCC TGACAGAATC GGGCATGGAA CATTTCTCAA CTCCGGTGAG 720
GGAGGATCCC TGGATCTGGT GGACTTTGTG AGGCAACATC GGATACCACT GGAACTCTGT 780 πGACCTCAA ACGTCAAAAG TCAGACAGTC CCATCπATG ACCAGCACCA TTTCGGAπC 840
TGGTACAGCA πGCACATCC CTGTGATC TGTACTGATG ATAAGGGTGT TTTTGCAACA 900
CACCTTTCTC AAGAGTACCA GCTGGCAGCT GAAACATTTA ATTTGACCCA GTCTCAGGTG 960 TGGGATCTGT CTTATGAATC CATCAACTAC ATCTTTGCπ CTGACAGCAC CAGATCTGAA 1020 CTGAGGAAGA AATGGAATCA CCTGAAGCCC AGAGTGTTAC ATAπTAA 1068 (SEQ ID N0:1)
The amino acid sequence encoded by a cDNA molecule having SEQ ID NO:l is provided as SEQ ID NO: 2:
1 MIEAEEQQPC KSDFYSELPK VELHAHLNGS ISSHTMKLI VQKPDLKIHD
51 QMTVIDKGKK RTLEECFQMF QTIHQLTSSP EDILMVTKDV IKEFADDGVK
101 YLELRSTPRR ENATGMTKT YVESILEGIK QSQENLDID VRYLIAVDRR 151 GGPLVAKETV KLAEEFFLST EGTVLGLDLS GDPTVGQAD FLEPLLEAKK
201 AGLLALHLS EIPNQKETQ ILLDLLPDRI GHGTFLNSGE GGSLDLVDFV
251 RQHRIPLELC LTSNVSQTV PSYDQHHFGF YSIAHPSVI CTDDGVFAT
301 HLSQEYQLAA ETFNLTQSQV WDLSYESINY IFASDSTRSE LRKKWNHLKP
351 RVLHI*
(SEQ ID NO:2)
The present inventors additionally prepared a DNA sequence in which a CAT (histidine) codon replaced the ATA (isoleucine) codon at positions 4-6 in the cDNA sequence (above), to enable expression in vectors having Nsil endonuclease cleavage sites 3' to the promoter. (SEQ ID NO:3 encoding SEQ ID NO:4).
A DNA sequence suitable for bacterial expression (having prokaryotic codons) was also prepared:
ATGCATGAGG CAGAAGAGCA ACAGCCTTGC AAGTCAGACT TCTATTCTGA ATTGCCAAAA 60 GTGGAACπC ATGCCCACπ GAATGGCTCC ATTAGTTCTC ATACCATGAA GAAATTAATA 120
GTCCAGAAGC CAGATCTTAA AATCCACGAT CAGATGACTG TGATTGACAA GGGAAAGAAA 180
CGCACTTTGG AAGAATGTTT CCAGATGTπ CAAACTAπC ATCAGCπAC TAGTAGCCCT 240
GAAGATATTC TAATGGTCAC AAAAGATGTC ATAAAAGAAT πGCAGATGA CGGCGTCAAG 300
TACCTGGAAC TACGTAGCAC ACCCCGTCGC GAAAATGCTA CCGGAATGAC TAAAAAGACT 360 TATGTGGAAT CTATACTTGA AGGTATAAAA CAGTCCAAAC AAGAAAACπ GGACATTGAT 420
GTTCGCTAπ TGATAGCAGT TGACCGTCGT GGTGGCCCH TAGTAGCCAA GGAGACTGTA 480
AAACTTGCCG AGGAGTTCTT CCTTTCTACT GAGGGTACAG πcπGGCCT TGACCTCAGT 540
GGAGACCCTA CTGTAGGACA AGCAAAAGAC πCπGGAAC CTCTTTTAGA AGCTAAGAAA 600
GCAGGTCTGA AGπAGCATT GCATCTTTCA GAGATTCCAA ACCAAAAAAA AGAAACACAA 660 ATACTCCTGG ATCTGCTTCC TGACCGTATC GGGCATGGAA CATTTCTCAA CTCCGGTGAG 720
GGAGGTTCCC TGGATCTGGT GGACTTTGTG AGGCAACATC GGATACCACT GGAACTCTGT 780
TTGACCTCAA ACGTCAAAAG TCAGACAGTC CCATCTTATG ACCAGCACCA TTTCGGATTC 840
TGGTACAGCA TTGCACATCC UCTGTGATC TGTACTGATG ATAAGGGTGT TTTTGCAACA 900
CACCTTTCTC AAGAGTACCA GCTGGCAGCT GAAACATTTA ATTTGACCCA GTCTCAGGTG 960 TGGGATCTGT CTTATGAATC CATCAACTAC ATCTTTGCTT CTGACAGCAC CCGCTCTGAA 1020 CTGCGCAAGA AATGGAATCA CCTGAAGCCC CGTGTGTTAC ATATTTAA (SEQ ID NO:5, encoding SEQ ID NO:4)
The gene encoding the abacavir-MP deaminase has been mapped to human chromosome 15, map position 15ql5. The DNA sequence encoding the present deaminase is contained within contigs #1 through #7 of GenBank Accession No. AC018924.
The present invention provides polynucleotide molecules, such as a DNA molecule consisting of or comprising SEQ ID NO:l, that encode an abacavir-MP deaminase.
The enzyme of the present invention is termed herein "Abacavir-MP deaminase" due to its ability to catalyze the removal of the 6-cyclopropyl amino moiety from abacavir, resulting in a 6-oxopurine (guanosine) analogue monophosphate (herein termed 'carbovir monophosphate') and cyclopropylamine. Abacavir MP deaminases of the present invention may be identified by function (ability to catalyze removal of the 6- cyclopropyl amino moiety of abacavir) and/or by structure (sequence similarity to sequences provided herein). As would be apparent to one skilled in the art, the
enzymes of the present invention may act on substrates in addition to abacavir, including non-nucleoside substrates. Further, the action of the present enzymes are not limited to deamination of a substrate, as is well known for other enzymes that are referred to as 'deaminases' (e.g., adenosine deaminase is capable of acting on 6-OMe and 6-Cl to give 6-oxopurine species). Compounds that may be substrates for the present enzymes, and thus are suitable candidates for the present screening methods, include but are not limited to nucleoside analogs containing NRJR2 , OR3, or SR4 moieties at the 6 position, where R1 - R4 are independently selected from alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aralkenyl. Further compounds suitable for use in the present screening methods include, but are not limited to, azidopurines, dideoxypurines, 3'-F-purine ribosides, 9-(2-phosphonylmethoxy ethyl) guanine (PMEG) analogs (Hatse et al., Biochem Pharmacol. 58:311 (1999); Naesens et al., Oncol Res.11: 195 (1999)), O6-propyl-nucleoside analogs and N6-propyl-nucleoside analogs.
Potential substrate compounds to be screened by the methods of the present invention include those having the following general formula:
Where the bracketed portion [X] is a carbohydrate or a carbohydrate mimic (e.g., dioxolane, oxathiolane, cyclopentene, cyclopentane, or acyclic chains) and where Y1 = NR!R2 , OR3, or SR4 moieties, where R1 - R2 are independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aralkenyl, provided that R1 and R2 are not both hydrogen; R3 - R4 are independently selected from alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aralkenyl and Y2 = H or NH2.
Species homologs of the disclosed polynucleotides and proteins are also provided by the present invention. Species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source from the desired species.
The invention also encompasses polynucleotide molecules having sequences that are allelic variants of the sequences provided herein, that is, naturally occurring polynucleotide sequences that also encode an abacavir MP deaminase. Naturally occurring mRNA allelic variants will provide alternative forms of the cDNA of SEQ ID NO:l.
The invention also includes polynucleotide molecules with sequences complementary to those of the polynucleotides disclosed herein.
The present invention also includes polynucleotides capable of hybridizing under reduced stringency conditions, more preferably stringent conditions, and most preferably highly stringent conditions, to the polynucleotide molecules described herein. The stringency of hybridization conditions can be defined by salt concentration, the concentration of organic solvent (e.g. , formamide), temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, and/or raising the hybridization temperature. The washing steps, which follow hybridization, can also vary in stringency. Examples of stringency conditions are shown in the table below: highly stringent conditions are those that are at least as stringent as, for example, conditions 1-6; stringent conditions are at least as stringent as, for example, conditions 7-12; and reduced stringency conditions are at least as stringent as, for example, conditions 13-18.
5§ The hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides. When hybridizing a polynucleotide to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing polynucleotide. When polynucleotides of known sequence are hybridized, the
hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity. § SSPE (lxSSPE is 0.15M NaCI, lOmM NaH2PO4, and 1.25mM EDTA, pH 7.4) can be substituted for SSC (IxSSC is 0.15M NaCI and 15mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete.
* Tb - Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10°C less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (°C) = 2(# of A + T bases) + 4(# of G + C bases). For hybrids between 18 and 49 base pairs in length, Tm (°C.) = 81.5 + 16.6(logl0 [Na+ ]) + 0.41(% G + C) - (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for IxSSC ***= 0.165M).
Additional examples of stringency conditions for polynucleotide hybridization are provided in Sambrook, J., E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11, and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference.
Preferably, each such hybridizing polynucleotide has a length that is at least 25% (more preferably at least 50%, and most preferably at least 60%, 75%, 80% or more) of the length of the polynucleotide of the present invention to which it hybridizes, and has at least 60% sequence identity (more preferably, at least 75%, 80% or 85% identity; most preferably at least 90% , 95% , 97% or 98% or more identity) with the polynucleotide of the present invention to which it hybridizes.
The phrases "percent sequence identity" or "percent sequence homology" refer to the percentage of sequence similarity found in a comparison of two or more amino acid or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MegAlign™ program (DNASTAR, Inc., Madison Wis.). The MegAlign™ program can create alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73: 237-244.) Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183: 626-645.)
The term "substantial sequence similarity" as used herein means that DNA, RNA or amino acid sequences which have slight and non-consequential sequence variations from those disclosed herein are considered to be equivalent. Molecules with substantial sequence similarity will be functionally equivalent, i.e., function in substantially the same manner to produce substantially the same compositions and results as the sequences specifically disclosed herein. Molecules with substantial sequence similarity typically have at least about 70% sequence identity, and preferably are at least 75% , 80% , 85%, 90% , 95%, 97% or even 98% or more similar in sequence.
Use of the phrase "isolated" in reference to nucleic acid, peptide, or protein molecules means that the molecules have been separated from their natural in vivo cellular components through the efforts of human beings. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated nucleic acid molecules further includes such molecules produced synthetically.
Thus the present invention also encompasses nucleic acid molecules that hybridize under stringent hybridization conditions (as defined herein) to all or a portion of the nucleotide sequence represented by SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, or a complement thereof, wherein the nucleic acid molecule encodes a functional abacavir MP deaminase. The hybridizing portion of the hybridizing nucleic acids is typically at least 15 (and preferably 20, 25, 30, or 50 or more) nucleotides in length.
The hybridizing portion of the hybridizing nucleic acid is at least 60%, 75%, 80%, 85%, 90% , 95% , 97% , or at least 98% or more identical to the sequence of a portion or all of a nucleic acid encoding an abacavir-MP deaminase, or its complement. Hybridizing nucleic acids of the type described herein can be used, for example, as a cloning probe, a primer (e.g., a PCR primer), or a diagnostic probe. Additional guidance is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, NY; and Ausubel et al. (eds.) 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, NY).
The invention further provides oligonucleotide fragments of nucleotide molecules encoding mammalian abacavir-MP deaminase. An oligonucleotide fragment refers to a continuous portion of the nucleotide molecule that is less than the entire molecule. Also provided are oligonucleotide molecules having a nucleotide sequence complementary to a fragment of a nucleotide molecule encoding abacavir-MP deaminase. Such an oligonucleotide is useful as a probe to detect and/or hybridize to DNA molecules encoding the enzyme. To this end, the invention further provides a method of detecting a nucleotide molecule encoding abacavir-MP deaminase in a sample by contacting the sample with the oligonucleotide under conditions suitable for hybridization of the oligonucleotide to a nucleotide molecule encoding abacavir-MP deaminase. The method further provides for detecting the resulting hybridized DNA, thereby detecting presence of the DNA molecule encoding the enzyme in a sample. Oligonucleotide fragments are preferably at least about 15, 25, 30, 50, 100, 250 or more nucleotides in length.
The present invention provides an isolated protein capable of removing the 6- cyclopropyl amino moiety of abacavir-MP. The protein may comprise or consist of SEQ ID NO:2 or SEQ ID NO: 4. The present invention further provides proteins or peptides having a high degree of sequence similarity to the sequences disclosed herein,
and having abacavir-MP deaminase function. Preferred abacavir-MP deaminases of the present invention are of mammalian origin; more preferred are of human origin.
The present invention also provides for variants of abacavir-MP deaminase as disclosed herein. Natural variants of the enzyme sequences provided herein may differ by conservative amino acid sequence differences or by minor non-conservative sequence differences. Variant abacavir-MP deaminase proteins or peptides preferably have at least 60%, 70%, 75%, 80% , 85%, 90%, 95% , 97%, 98% or greater sequence identity to the sequences provided herein. Conservative amino acid substitutions include substitutions within the following groups:
Glycine, alanine; Valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine;
Lysine, arginine; Phenylalanine, tyrosine
Proteins and protein fragments of the present invention include proteins with amino acid sequence lengths that are at least 50%, and most preferably at least 70%, 75%, 80% , 85%, 90% or 95% or more, of the length of an abacavir-MP deaminase disclosed herein; and which have at least 60% sequence identity (preferably, at least 70% , 75% or 80% identity; more preferably at least 85%, 90%, 95%, 97% or 98% or more identity) with that of the disclosed proteins, where sequence identity is determined by comparing the amino acid sequences of the proteins when aligned so as to maximize overlap and identity while minimizing sequence gaps.
As used herein, an isolated protein of the present invention can be a full-length protein or any homolog of such a protein, such as a protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitoylation, amidation and/or addition of glycerophosphatidyl inositol) such that the homolog comprises a protein having an
amino acid sequence that is sufficiently similar to a natural abacavir-MP deaminase that a nucleic acid sequence encoding the homolog is capable of hybridizing under stringent conditions to (i.e. , with) the complement of a nucleic acid sequence encoding the corresponding natural abacavir-MP deaminase amino acid sequence, and wherein the homolog has abacavir-MP deaminase activity.
It will be apparent to those skilled in the art that although the present deaminase is referred to as abacavir-MP deaminase, such proteins may additionally deaminate other compounds in addition to abacavir-MP. It will be apparent to those skilled in the art that the rate at which homologs of the enzyme disclosed herein remove the cyclopropyl amino moiety from abacavir-MP will vary. As used herein, a "functional abacavir-MP deaminase" need not display the same kinetics as an enzyme having SEQ ID NO:2 or SEQ ID NO:4.
According to the present invention, an isolated, or biologically pure, protein, is a protein that has been removed from its natural milieu. As such, "isolated" and "biologically pure" do not necessarily reflect the extent to which the protein has been purified. An isolated deaminase can be obtained from its natural source, or produced using recombinant DNA technology or chemical synthesis.
A protein homologue of the present invention can be the result of natural allelic variation of an endogenous gene encoding a deaminase. Deaminase homologues can also be produced using techniques known in the art including, but not limited to, direct modifications to a gene encoding a protein using, for example, classic or recombinant DNA techniques to effect random or targeted mutagenesis. Isolated proteins of the present invention, including homologues, can be identified by the proteins' ability to deaminate abacavir-MP.
Having provided nucleotide molecules encoding human abacavir-MP deaminase and oligonucleotide fragments thereof and complementary thereto, such nucleotide
molecules can be utilized to produce proteins with abacavir-MP deaminase activity. The nucleotide molecules are inserted into a suitable expression vector, and the vectors are then utilized to transfect suitable host cells.
The present invention includes a recombinant vector, which comprises a nucleic acid molecule encoding an abacavir-MP deaminase, inserted into any vector capable of delivering the nucleic acid molecule into a host cell. Such a vector contains heterologous nucleic acid sequences, that is, nucleic acid sequences that are not naturally found adjacent to the abacavir-MP deaminase nucleic acid molecules of the present invention. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. Recombinant vectors can be used in cloning, sequencing, and otherwise manipulating the abacavir-MP deaminase nucleic acid molecules of the present invention.
DNA molecules encoding an abacavir-MP deaminase may be inserted into expression vectors using standard cloning procedures readily known in the art. This generally involves the use of restriction enzymes and DNA ligases, as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Host cells may then be transfected with suitable expression vectors by known methods. For example, mammalian cells or insect cells are infected with the appropriate viral vectors.
It is also possible to directly insert a DNA molecule encoding an abacavir-MP deaminase into a host cell via techniques such as conjugation, transduction, microinjection, chemically-mediated transfection (such as calcium phosphate coprecipitation), electroporation, and liposome fusion. Transformed nucleic acid molecules of the present invention can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.
Once the nucleotide molecule encoding an abacavir-MP deaminase has been inserted into a host cell, with or without the use of an intermediate expression vector, the host cell can be used to produce deaminase by culturing the cell under conditions suitable for translation of the DNA molecule, thereby expressing the deaminase. The host cell is cultured using a suitable culture medium as would be apparent to one skilled in the art, i.e. , a medium that supports the cell culture and allows expression of the desired protein. The deaminase can then be recovered from the cell. Depending on the vector and host systems used for production, the resulting abacavir-MP deaminases may remain within the recombinant cell or be secreted into the fermentation medium.
Suitable host cells for transformation include any cell that can be transformed and that can express the introduced abacavir-MP deaminase. Such cells are, therefore, capable of producing proteins of the present invention after being transformed with at least one nucleic acid molecule of the present invention. Host cells can be either untransformed cells or cells that are already transformed with at least one nucleic acid molecule. Suitable host cells of the present invention can include bacterial, fungal (including yeast), insect, animal and plant cells. Preferred host cells include bacterial (e.g., E. colϊ), yeast, insect (e.g., Lepidoptera; Trichoplusia ni), animal and mammalian cells. Mammalian host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue or primary explants. Materials and methods for baculo virus/insect cell expression systems are commercially available in kit form, e.g., from Invitrogen, San Diego, Calif. USA and such methods are well known in the art.
A recombinant cell is preferably produced by transforming a host cell with one or more recombinant molecules, each comprising one or more nucleic acid molecules of the present invention operatively linked to an expression vector containing one or more transcription control sequences. The phrase operatively linked refers to insertion of a
nucleic acid molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified nucleic acid molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors of the present invention include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, yeast, insect, animal, and/or mammalian cells. As such, nucleic acid molecules of the present invention can be operatively linked to expression vectors containing regulatory sequences such as promoters, operators, repressors, enhancers, termination sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of nucleic acid molecules of the present invention. As used herein, a transcription control sequence includes a sequence which is capable of controlling the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, insect, animal and mammalian cells, such as, but not limited to, tac, lac, tip, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters, baculo virus, Heliothis zea insect virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40, retrovirus actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in. prokaryotic or eukaryotic cells. Additional suitable transcription control sequences include tissue-specific promoters and enhancers as well as inducible
promoters. Transcription control sequences of the present invention can also include naturally occurring transcription control sequences naturally associated with a DNA sequence encoding an abacavir-MP deaminase.
Expression vectors of the present invention may also contain secretory signals (i.e., signal segment nucleic acid sequences) to enable an expressed abacavir-MP deaminase to be secreted from the cell that produces the protein. Suitable signal segments include any heterologous signal segment capable of directing the secretion of an abacavir-MP deaminase, including fusion proteins, of the present invention.
Expression vectors of the present invention may also contain fusion sequences which lead to the expression of inserted nucleic acid molecules of the present invention as fusion proteins. Inclusion of a fusion sequence as part of an abacavir-MP deaminase nucleic acid molecule of the present invention can enhance the stability during production, storage and/or use of the protein encoded by the nucleic acid molecule. Furthermore, a fusion segment can function as a tool to simplify purification of a protein, such as to enable purification of the resultant fusion protein using affinity chromatography. A suitable fusion segment can be a domain of any size that has the desired function (e.g., increased stability and/or purification tool). It is within the scope of the present invention to use one or more fusion segments. Fusion segments can be joined to amino and/or carboxyl termini of an abacavir-MP deaminase. Linkages between fusion segments and abacavir-MP deaminases can be constructed to be susceptible to cleavage to enable straight-forward recovery of the abacavir-MP deaminase. Fusion proteins are preferably produced by culturing a recombinant cell transformed with a fusion nucleic acid sequence that encodes a protein including the fusion segment attached to either the carboxyl and/or amino terminal end of an abacavir-MP deaminase.
Antibodies
Antibodies useful in the present invention include those which bind specifically to a peptides and proteins of the present invention, including polypeptides of SEQ ID NO:2 or SEQ ID NO:4; and fragments of such antibodies, which fragments bind specifically to a polypeptide of the present invention. Such antibodies and antibody fragments may be produced by a variety of techniques as are known in the art. The term "antibodies" as used herein refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE. Of these, IgM and IgG are preferred. The antibodies may be monoclonal or polyclonal, and may be of any species of origin, including but not limited to human, mouse, rat, rabbit and horse, and may be chimeric antibodies. Antibodies of the present invention include functional equivalents such as antibody fragments and genetically-engineered antibodies, including single chain antibodies, that are capable of selectively binding to at least one of the epitopes of the protein used to obtain the antibodies. Antibodies of the present invention also include chimeric antibodies that can bind to more than one epitope. Preferred antibodies are raised in response to proteins that are encoded by an abacavir-MP deaminase nucleic acid molecule of the present invention.
Monoclonal antibodies used in the present methods may be produced in a hybridoma cell line according to the techniques of Kohler and Milstein, Nature, 265:495 (1975) and other techniques as are known in the art. Monoclonal antibodies specific for an enzyme allow the use of immunoradiometric, immunoenzymatic, or immunohistochemical techniques (as are known in the art) to detect the presence of the enzyme.
Antibodies to the abacavir-MP deaminase of the present invention are useful to measure the amount of enzyme present in a sample. Anti-abacavir-MP deaminase antibodies can be used as tools to screen expression libraries and/or to recover desired proteins of the present invention from a mixture of proteins and other contaminants.
Antibodies useful in the present methods may be conjugated to detectable groups to produce a detectable signal, as is known in the art. Such detectable groups, or labels, include radioactive labels (e.g., 35S, 125I, 131I), fluorescent labels (e.g., fluorescein) and enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase). Examples of suitable immunoassays include radioimmunoassays, immunofluorescent assays, enzyme-linked immunoassays, and the like.
Antibodies which selectively bind an abacavir MP deaminase (i.e., bind the enzyme while showing essentially no binding to other, unrelated, proteins under the same binding conditions) may be conjugated to a solid support (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
Isolated antibodies are antibodies that have been removed from their natural milieu. The term "isolated" does not refer to the state of purity of such antibodies. As such, isolated antibodies can include anti-sera containing such antibodies, or antibodies that have been purified to varying degrees. As used herein, the term "specifically binds to" refers to the ability of such antibodies to preferentially bind to the deaminase against which the antibody was raised (i.e., to be able to distinguish that deaminase from unrelated components in a mixture.). Binding affinities typically range from about 103 M"1 to about 1012 M"1. Binding can be measured using a variety of methods known to those skilled in the art including immunoblot assays, immunoprecipitation assays, radioimmunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescent antibody assays and immunoelectron microscopy; see, for example, Sambrook et al., ibid.
Antibody assays (immunoassays) may, in general, be homogeneous assays or heterogeneous assays. In a homogeneous assay the immunological reaction usually involves the specific antibody, a labeled analyte, and the sample of interest. The signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte. Both the immunological reaction and detection of the
extent thereof are carried out in a homogeneous solution. Immunochemical labels which may be employed include free radicals, radioisotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
In a heterogeneous assay approach, the reagents are usually the specimen, the antibody of the invention and a system or means for producing a detectable signal. Similar specimens as described above may be used. The antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the specimen suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal. The signal is related to the presence of the analyte in the specimen. Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, and so forth. For example, if the antigen to be detected contains a second binding site, an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step. The presence of the detectable group on the solid support indicates the presence of the antigen in the test sample. Examples of suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like. Those skilled in the art will be familiar with numerous specific immunoassay formats and variations thereof which may be useful for carrying out the method disclosed herein.
Methods of screening
The deaminase provided by the present invention removes the 6-cyclopropyl amino moiety from the monophosphorylated form of abacavir, a 6-substituted purine carbocyclic nucleoside. Given the desirable anti-HIV properties of abacavir, and the role of the intracellular abacavir-MP deaminase in converting abacavir to the active CBV-TP form, it is desirable to identify other compounds that are substrates for this enzyme.
Accordingly, an aspect of the present invention is a method of screening nucleoside analogs that contain an amino group (and their mono-, bi- and tri- phosphorylated forms) for the ability to act as a substrate for abacavir-MP deaminase (i.e. , the abacavir MP deaminase catalyzes the removal of the amino group from the nucleoside analog). Suitable nucleoside analogs include purine nucleoside analogs and carbocyclic nucleoside analogs (containing a carbocyclic ring in place of the sugar residue of naturally occurring deoxynucleosides, including unsaturated carbocyclic rings, such as an unsaturated cyclopentene ring). The amino group may be a 6-amino substitution, and may contain a branched, straight-chain, or cyclic carbon group. See, e.g., US Patent No. 5,034,394 (Daluge).
A futher aspect of the present invention is a method of screening compounds for their ability to act as a substrate for abacavir-MP deaminase (i.e., the abacavir MP deaminase catalyzes the removal of a group from the starting compound). The group may be a -NR R2 , -OR3, or -SR4 moiety, where R1 - R4 are independently selected from alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aralkenyl. Compounds suitable for use in the present screening methods include, without limitation, azidopurines, dideoxypurines, 3'-F-purine ribosides, 9-(2-phosphonylmethoxyethyl)guanine (PMEG) analogs (Hatse et al., Biochem Pharmacol. 58:311 (1999); Naesens et al., Oncol Res.11 : 195 (1999)), O6-propyl-nucleoside analogs and N6-propyl-nucleoside analogs.
Suitable compounds to be screened by the methods of the present invention include those having the following general formula, where the bracketed portion [X] is a carbohydrate or a carbohydrate mimic (e.g., dioxolane, oxathiolane, cyclopentene, cyclopentane, or acyclic chains) and where Y1 = NR R2 , OR3, or SR4 moieties, where R1 - R2 are independently selected from hydrogen, alkyl, cycloalkyl, alkenyl,
cycloalkenyl, or aralkenyl, provided that R
1 and R
2 are not both hydrogen; R
3 - R
4 are independently selected from alkyl, cycloalkyl, alkenyl, cycloalkenyl, or aralkenyl and Y
2 = H or NH
2.
The method comprises obtaining a sample of a compound to be tested, exposing the compound to abacavir-MP deaminase under conditions conducive to the enzyme's activity, and determining whether the enzyme catalyzes the removal of a chemical group, as discussed above, from the starting compound. It will be apparent to those skilled in the art that different substrates of an enzyme may react at different rates.
The majority of intracellular abacavir is converted to the active agent CBV-TP via the abacavir-MP deaminase pathway (see Fig. 11; see also Faletto et al, Antimicrobial Agents Chemother. 41:1099 (1997)). In subjects having decreased levels of abacavir-MP deaminase activity, deamination of abacavir-MP would be hindered. While not wishing to be held to a single theory, the present inventors surmise that reduced activity of the present deaminase leads to insufficient metabolism of the nucleoside analog substrate or, in the case of abacavir, to increased reliance on the metabolic pathway that produces carbovir as an intermediate.
An aspect of the present invention is a method of screening subjects to determine levels of abacavir-MP deaminase activity. Such methods may aid in predicting whether a subject will respond to treatment with a nucleoside analog that is a substrate of the enzyme. Additionally, such methods may assist in interpreting results from clinical trials of nucleoside analog compounds, where stratification of subjects by enzyme activity levels and correlation with clinical results would suggest whether clinical results were dependent on or affected by enzyme activity levels.
Any suitable method of measuring abacavir-MP deaminase activity may be used.
Such methods may include assessing the amount of deaminase present (e.g., indirectly by measuring mRNA levels or more directly by using labelled antibodies to detect
enzyme molecules); assessing the deaminase activity levels (e.g., by measuring the amount of product produced by a cell extract incubated with substrate under appropriate conditions); or screening for genotypes that are known to be associated with less active or inactive forms of the enzyme. It is preferred that the deaminase activity be measured in target cells. As used herein, a target cell is an identified cell type in which the active compound is desired for its therapeutic effect, or in which it is desirable to avoid or reduce metabolism ofthe compound.
Additionally, it was found that the abacavir-MP deaminating activity was potently inhibited by GMP and AMP. In subjects having increased intracellular levels of
GMP or AMP (and/or dGMP, dAMP, and other purine nucleoside monophosphates), abacavir-MP deaminase activity may be inhibited, contributing to the variation in response to abacavir seen in clinical subjects.
Accordingly, an aspect of the present invention is a method of screening subjects to determine levels of intracellular GMP and/or AMP. Such methods may aid in predicting whether a subject will respond to treatment with a nucleoside analog that is a substrate of the present enzyme. Additionally, such methods may assist in interpreting results from clinical trials of nucleoside analog compounds, where stratification of subjects by AMP and/or GMP levels and correlation with clinical results would suggest whether clinical results were dependent on or affected by such levels. Any suitable method of measuring intracellular AMP and/or GMP may be used, as would be apparent to one skilled in the art.
As used herein, a predetermined value may be one that has been established by previous studies using a study population that did not include the subject currently being tested. Comparison of test results to a predetermined value may be accomplished by referring to a printed sheet setting forth predetermined values or ranges of values, and the correlation between such values and response to treatment.
It will be apparent to one skilled in the art that even where a correlation exists between a subject's enzyme activity level (or the level of an enzyme inhibitor such as GMP) and the clinical response to a nucleoside analog, methods of measuring enzyme activity levels (or levels of enzyme inhibitors) as an aid in predicting response to drug therapy will not be 100% predictive. Factors such as concomitant disease, general health of the subject, and severity of disease will affect clinical response to a pharmaceutical compound. It is further known in the art that a predictive test need not be either 100% specific nor 100% selective to be useful in clinical practice. The selection of drug therapy rarely depends on the assessment of a single marker; additional diagnostic, laboratory and clinical observations typically are considered in determining therapies. Where enzyme activity levels (or levels of enzyme inhibitors) are used as an aid in predicting a subject's suitability for drug therapy, comparison should be made to levels (or a range of levels) that have been determined to be associated with a desired or favorable clinical response to the drug (e.g., lessening of pathologic signs or symptoms, or a decreased incidence of an unwanted side effect). A subject having levels similar to those determined to be associated with a desirable clinical response is more likely to similarly have such a desirable clinical response, compared to a subject with levels outside of the determined range. Such levels may vary among cell types, the disease condition being treated, and the drug being used. Where suitable levels are found (i.e., levels associated with a desirable clinical response), the subject may further be treated with a therapeutically effective amount of a compound that is a substrate of the enzyme, where the compound is known to be a suitable treatment for the subject's condition.
Methods in addition to those discussed in Example 3 herein may be used to assay for abacavir-MP deaminating activity. Thin layer chromatography may be used to separate the reaction products and substrates, followed by UV or visible light detection of the native compound, or UV or visible light detection of chemically derivatized compounds (e.g., where derivatization is done directly on the plate by applying the reagents as a spray). High pressure liquid chromatography (HPLC) may be used to separate the reaction products and substrates with an anion exchange column
packing, eluted with a gradient of increasing ionic strength, and monitored using a photodiode array detector. Alternatively, when using radiolabelled substrates, a radioactive flow cell may be used to detect the compounds eluting from the HPLC column. Reverse phase HPLC may also be used to separate the nucleosides and nucleoside monophosphate substrates and products. This method is able to resolve nucleosides as well as monophosphates. Detection of compounds can then be obtained by photodiode array detector or radioactive flow cell.
HPLC methods are more general compared to spectrophotometric assays and may be used to screen a diverse array of nucleoside monophosphates as potential substrates for abacavir-MP deaminase. HPLC may be used to detect the activity of abacavir monophosphate deaminase with any of a number of 6-substitutions other than amino groups, such as halogens or alkyloxy, or thioalkyl.
The above methods are suitable for screening compounds to see if they are substrates for abacavir monophosphate deaminase, as well as to assess the level of activity of an abacavir-MP deaminase with a known substrate, such as abacavir monophosphate. By HPLC, the products of the enzyme are identified as new peaks arising from a decrease in the amount of substrate. The new products can be characterized by their UV spectra to differentiate them from the substrates.
EXAMPLES
Example 1 Purification ofthe abacavir-MP Deaminating Protein from Rat Liver The enzyme was purified over 500, 000-fold from rat liver. All purification steps were performed at 4 °C.
Homogenisation (Step 1): 900 grams of rat frozen liver was thawed at approximately 40 °C for 20 minutes. The thawed livers were suspended in 1800 ml of buffer A (25 mM Hepes K+ at pH 7.5), and the suspension homogenised for 3 minutes
with a commercial Waring blender. This mixture was centrifuged at 23,000 x g for 10 minutes and the supernatant pooled for subsequent purification.
Heat and Ammonium Sulfate Precipitation (Step 2): The pooled supernatant was heated to 64-66 °C for 10 minutes. The mixture was cooled to 30 °C in an ice bath prior to centrifugation at 23,000 x g for 10 minutes. The supernatant was brought to 45 % saturation with ammonium sulfate (277g/l). The mixture was stirred at 4 °C for 20 minutes prior to centrifugation for 15 minutes at 23,000 x g. The supernatant was pooled and brought to 65 % saturation with ammonium sulfate (134 g/1). The mixture was stirred at 4 °C for 20 minutes prior to centrifugation for 15 minutes at 23,000 x g. The pellet was dissolved in a minimal volume of buffer A.
Orange A Column (Step 3): A 7 cm X 5.5 cm column was packed with 100 ml of DyeMatrix Orange A (Amicon) equilibrated in buffer A. The dialysate from Step 2 was absorbed onto the resin at a flow rate of 3 ml/min. The resin was washed with buffer A until the absorbance of the effluent had an A28o < 0.1. The deaminating activity was eluted from the column with 250 mL of 1.0 mM GMP. Fractions containing deaminating activity were concentrated to approximately 4 ml by ultrafiltration with a Centriprep 30 (Amicon). The retention was diluted to 20 ml with buffer B (10 mM Tricine K+ (Sigma Chemical Company, St. Louis, MO) at pH 8.1). The sample was exchanged into buffer B by repeating the ultrafiltration step three times.
Anion Exchange Chromatography (Step 4): The sample from Step 3 was diluted to approximately 9 mL with buffer B. The solution was filtered through a 0.22 mμ low protein binding Millex-GV filter (Millipore Corporation) prior to absorption onto Mono Q resin (Amersham Pharmacia Biotech AB) (HRlO/10 column) equilibrated in buffer B. The resin was washed with buffer B at 2 ml/min until the absorbance of the effluent was constant. The abacavir-MP deaminating activity was eluted from the column with a 180
ml gradient from 0 mM KCI to 150 mM KCI in buffer B. The activity was collected in approximately 10 ml centered about 60 mM KCI.
Hydroxyapatite Chromatography (Step 5): The sample from Step 4 was absorbed onto Hydroxyapatite Bio-Gel HTP Gel resin (Bio-Rad Laboratories) in a Pharmacia HR5/5 column that was equilibrated with buffer A. The resin was washed with buffer A until the absorbance at 280 nm was constant. The abacavir-MP deaminating activity was eluted from the resin with a 15 ml gradient from 0 mM to 500 mM potassium phosphate in buffer A. The activity was collected in 5 ml that was centered about 300 mM potassium phosphate.
Superdex 75 Chromatography (Steps 6-8): The sample from Step 5 was concentrated to approximately 1 ml by ultrafiltration through a Centricon 30 (Amicon).
The concentrated sample was applied onto Superdex 75 resin in a Pharmacia HR 10/30 column (Amersham Pharmacia Biotech AB) that was equilibrated with buffer C (12.5 mM Hepes K+ at pH 7.5). The chromatogram was developed with buffer C at a flow rate of 0.5 ml/min. The enzyme was eluted from the resin in 1.5 ml that was centered at 11 mis. The volume of this solution was reduced to 1 ml by ultrafiltration through a Centricon 30. Chromatography on the Superdex 75 resin was repeated in Steps 7 and 8. An alternative procedure that was able to be substituted for the three sequential Superdex 75 columns was to utilize two Superdex 75 columns run in tandem, using a flow rate of 0.25 ml/minute.
The above steps are summarized in Table 1.
Table 1: Purification of Abacavir-MP Deaminating Activity from Rat Liver
Example 2 Results: Purification ofthe abacavir-MP deaminating protein The abacavir-MP deaminating protein was purified from mature rat liver as outlined in Example 1. The deaminating activity in the initial ho ogenate survived heating at 65 °C for 10 minutes or 20 minutes with similar recoveries of activity. Consequently, this procedure was an initial step of the purification protocol. The matrix orange resin was an affinity resin from which the deaminating activity could be selectively eluted by the competitive inhibitor GMP. This step resulted in the largest fold purification with approximately 100-fold increase in specific activity. The anion exchange chromatographic step (Step 4) resolved the deaminating activity into at least two species (Figure 2). The asymmetric elution profile for activity from the column suggested heterogeneity. Only the early eluting fractions with activity were pooled for subsequent purification. Finally, the abacavir-MP deaminating activity and A28o absorbing material were eluted from the Superdex 75 resin (Step 8) in symmetrical peaks that coincided (Figure 3).
Because of the large background absorbance at 290 nm, the activity of the abacavir-MP deaminating protein was not monitored until Step 2 of the purification procedure. Consequently, deaminating activity could have been destroyed by the heat step. However, the yield of abacavir-MP deaminating activity did not vary significantly when the duration of heat step was changed or the fractionation range of ammonium sulfate was increased. Thus, it was assumed that the total activity in the homogenate
was the same as that after the heat step. In Table 1 this value has been placed in parenthesis to denote this assumption.
Polyacrylamide gel electrophoresis (SDS-PAGE) of samples eluting from the Superdex 75 resin (Step 8) suggested that in some fractions the preparation of abacavir- MP deaminating protein was approaching homogeneity. The major protein band had a molecular mass of about 38 to about 40 kDa. SDS-PAGE electrophoresis of samples from an alternative purification procedure in which two Superdex 75 columns in tandem were substituted for the three individual Superdex columns yield similar results with slightly different impurities. Comparison of these results strongly suggested that the 38- 40 kDa species was the abacavir-MP deaminating protein.
Example 3 Assay of abacavir-MP deaminating activity Deamination of abacavir-MP was previously assessed by monitoring the formation of radiolabelled carbovir monophosphate after separation from the substrate by thin layer chromatography (Daluge et al., (1997) Antimicrobial Agents & Chemotherapy 41: 1082; Faletto et al. (1997) Antimicrobial Agents & Chemotherapy 41:1099). This procedure was considered to be somewhat cumbersome for monitoring activity during a routine purification procedure. Consequently, a spectrophotometric assay based on absorbance changes of the substrate associated with the deamination reaction was considered. Assays based on analogous changes for monitoring the deamination of nucleoside analogues by adenosine deaminase have been developed (Mohamedali et al. (1996) Biochemistry 35: 1672).
The spectrophotometric assay, in analogy with spectrophotometric assays for other deaminases, monitored the absorbance changes associated with deamination of the substrate abacavir-MP (Δε29o= 10 mM~ cm- ). The concentration of abacavir-MP was
-determined spectrophotometrically at pH 7.5 with S284= 15 mM" cm . The standard buffer used for the assay was 0.025 M Hepes K+ at pH 7.5. In the standard assay,
0.490 mL of 10 μM abacavir-MP was equilibrated in a 0.5 ml quartz cuvette at 25 °C for 3 min in a UVIKON 860 Spectrophotometer. The reaction was initiated with 10 μL of enzyme and was monitored at 290 nm for 5 to 20 minutes. From the time-course of the reaction, the initial rate of absorbance change at 290 nm was determined. This rate was converted to pmol/min. One unit of enzymatic activity was defined as the amount of enzyme catalyzing the deamination of 1 pmol of abacavir-MP per minute under these conditions. Enzyme concentration in mg/ml was estimated from the absorbance of the enzyme solution at 280 nm and an extinction coefficient for an average protein of ε 80 = , 1 mg -1 cm -1.
O2 was depleted from assay solutions with an enzymatic scavenging system that consisted of glucose oxygen, glucose and catalase. In this system, glucose oxidase catalyzed the reduction of O2 to H2O2. Catalase, which catalyzes the breakdown of
H2O2 to O2 and H2O was present to prevent the build up of H2O2 that could slowly generate O2. The overall reaction is the consumption of one mol of O2 and two mol of glucose to give H2O and the gluconolactone. The concentrations of glucose oxidase, glucose and catalase used were 9.6 units/ml (Sigma units), 20 mM glucose, and 3000 units/ml (Sigma units), respectively. The efficiency of reduction of O2 by glucose oxidase in the absence of catalase can be measured spectrophotometrically at 225 nm. Under these conditions, the solution was depleted of O2 within 5 seconds.
Example 4
Results: Assay of abacavir-MP Deaminating activity
Using partially purified abacavir-MP deaminating protein, large spectral changes were observed during the conversion of abacavir-MP to carbovir monophosphate. An example of the time-course for these spectral changes associated with the conversion of abacavir-MP to product are presented in Figure 1. These data indicated that 290 nm was the wavelength that gave the largest absorbance change for the deamination reaction.
The deamination of 8.8 μM abacavir-MP in standard buffer was initiated by the addition of 100 units of the deaminating protein (specific activity = 56,000 units/ A280) to the sample cuvette. An equal volume of protein was added to the reference cuvette that did not contain abacavir-MP. The sample was scanned from 220 nm to 320 nm every 5 minutes for 60 minutes. The starting spectrum had high absorbance at 285 nm. The spectrum of the final product was consistent with that of carbovir monophosphate.
With 10 μM abacavir-MP, the rate of absorbance change at 290 nm was constant until over 90 % of the substrate was consumed, and the rate was linearly dependent on the amount of extract added to the assay. Consequently, extracts containing the abacavir-MP deaminating protein were assayed by monitoring the absorbance decrease with 10 μM abacavir-MP as substrate.
Example 5
Deaminating activity: Data analysis
Inhibition of abacavir-MP deaminating activity was determined at a fixed concentration of abacavir-MP (1.7 μM) with varying concentrations of inhibitor. The IC50 value for the inhibitor was calculated from these data as described below.
Vm and Km values of the abacavir-MP deaminating enzyme for substrate were calculated from the time-courses of the reaction using the integrated rate equation (equation 1) where [S]0 was the initial concentration of substrate, [S] was the concentration of substrate at time t, t was time, Vm was the maximal velocity and Km
2.3 [S]0 _ 1 ([S]0 -[S]) | Vm [S] Km t Km was the concentration of S that gives 50 % of the maximal velocity.
(1)
The IC50 values for inhibitors were calculated from normalized velocity data at a fixed concentration of abacavir-MP (v) and selected concentration of inhibitory ([I]) by
IC 50 v = ■
(IC50 + [I]) equation 2.
(2)
The IC50 values were related to the Kj values by equation 3, which assumed that the inhibition was competitive.
(3)
Example 6
Kinetic properties ofthe abacavir-MP deaminating protein The effect of abacavir-MP concentration on the initial velocity of enzymatic deamination of abacavir-MP suggested that the value of the Km was less than 1 μM.
Because initial velocity determinations with substrate concentration less than 1 μM were difficult to determine spectrophotometrically, the integrated rate equation method was used to determine the value for the Km. An example of this analysis for the deamination of 1.7 μM abacavir-MP is presented in Figure 4. These data were analyzed by equation 1 to give a Km value of 0.14 + 0.01 μM. Because product is accumulating during the course of the reaction, product inhibition can increase the apparent Km value determined by the integrated rate equation. To demonstrate that product inhibition was not a significant contributor to the apparent value determined for the Km, the apparent value of the Km was determined with a larger starting concentration of abacavir-MP. Under these conditions more product would have
accumulated by the end of the reaction, such that the contribution of product inhibition would be more significant. With an initial concentration of 17 μM abacavir-MP, the value for the apparent Km increased to 0.55 ± 0.02 μM. These results indicated product inhibition was occurring but that it did not significantly affect the value for K^ determined with a starting concentration of 1.7 μM abacavir-MP.
The above results were determined using abacavir-MP deaminating protein that eluted early from the Mono Q resin (Figure 2). An independent preparation of the abacavir-MP deaminating protein from the Mono Q resin gave Km values for the early and late eluting peaks of activity of 0.166 ± 0.006 μM and 0.12 ± 0.01 μM, respectively. Because these values were similar, the kinetic parameters of the early and late eluting abacavir-MP deaminating activity were probably very similar.
The value of kcat was calculated from the specific activity of abacavir-MP deaminating protein as follows. The following assumptions were used for this calculation: 1) an A280 of 1 corresponded to 1 mg/ml protein, 2) the molecular mass of the protein was approximately 35 kDa, and 3) the best preparation of the enzyme was approximately 50 % pure so that the specific activity of homogeneous enzyme (at saturating [abacavir-MP]) was approximately 2 μmol/min/A280 (Table 1). Based on these assumptions, kcat was calculated to have a value of 1.2 s"1.
Example 7 - Inhibitors ofthe abacavir-MP deaminating protein Abacavir-MP deaminating activity was potently inhibited by GMP (Figure 5). This effect was exploited in the purification procedure for the selective elution of abacavir-MP deaminating protein from the matrix orange resin (Table 1). Titration of abacavir-MP deaminating activity with GMP in the presence of 1.7 μM abacavir-MP yielded an IC50 value of 15 ± 1 μM, which corresponded to a Kj value of 1.1 μM calculated from the competitive model described by equation 3. Similar analyses were
made with other nucleosides and nucleotides (Table 2). Purine nucleoside monophosphates were more potent inhibitors of the enzyme than pyrimidine nucleoside monophosphates. Of particular interest was the observation that 2,6-diaminopurine ribose 5 '-monophosphate was a potent inhibitor of the enzyme but was not a substrate. These results suggested that the cyclopropyl moiety and/or the carbocyclic ribosyl analogue contributed significantly to catalysis.
Table 2 Inhibition of abacavir-MP Deaminating Activity by Selected Ligands
Abacavir-MP, acyclovir monophosphate and 2,6-diaminopurine riboside -5'- monophosphate were synthesized from the respective nucleoside analogues as described previously (Miller et al (1992) J. Biol. Chem. 267: 21220).
Example 8
Effect of H2O2 or O2 on abacavir-MP deaminating activity
To determine whether the abacavir-MP deaminating protein was either an oxidase or peroxidase that required O2 or H2O2 for deamination of abacavir-MP, the effects of O2 and H2O2 on the initial velocity of the deaminating reaction were
determined. The results summarized in Table 3 demonstrated that depletion of O2 and H2O2 in the assay mixture with glucose oxidase, catalase, and glucose had little effect on the velocity of the deamination reaction. These results demonstrated that O2 and
H2O2 were not required for deamination. Furthermore, the enzyme did not catalyze the reduction of phenazine ethosulfate, an electron acceptor, in the presence of abacavir- MP. These results suggested that the deamination reaction was not a redox reaction catalyzed by a peroxidase or oxidase.
Table 3 Effect of 02 and H202 on abacavir-MP Deaminating Activity
Example 9 Other inhibitors ofthe abacavir-MP deaminating activity Numerous deaminases can be inhibited by metal chelating agents such as EDTA,
1,10 o-phenanthroline, and 8-hydroxyquinoline-5-sulfonate (Mohamedali et al. (1996) Biochemistry 35: 1672; Zielke and Seulter (1971) /. Biol. Chem. 246: 2179; Wilson et al. (1991) Science 252: 1278; Porter and Austin (1993) /. Biol. Chem. 268: 24005- 24011). However, the abacavir-MP deaminating protein was not inhibited by these reagents (1 mM) after a 80 minute incubation at 25 °C (Table 4). Because these reagents do not inhibit all deaminases (Smith et al. (1994) Biochemistry 33: 6468- 6474), this result did not definitively eliminate the possibility that the abacavir-MP deaminating protein is a metallodeaminase.
The abacavir-MP deaminating protein was not inhibited by phenylmethylsulfonyl fluoride, suggesting that the protein was not a serine protease. Finally, the activity was not inhibited by KCN, which suggested that the activity was not an iron based peroxidase or oxidase.
Table 4 Potential Inhibitors of abacavir-MP Deaminating Activity
Example 10 Enzymatic Deamination of Abacavir-MP Abacavir-MP deaminating protein was purified through the Mono Q chromatographic step as described above, and had a specific activity of 10,100 units/ A28o_ml- Cyclopropylamine solutions were prepared volumetrically using a formula weight of 57.1 and a density of 0.824 g/ml. (Cyclopropylamine from Aldrich was used without further purification.)
Primary and secondary amines in the reaction were derivatized with 6-amino quinolyl-N-hydroxysuccinimidyl carbamate as described by Waters (Cohen SA and
Michaud DP (1993) Analytical Biochemistry 211, 279). The fluorescent derivatives were resolved on a reverse phase column as described. The concentration of
cyclopropylamine in the enzymatic reaction was calculated by comparison of the cyclopropylamine-associated peak area from the HPLC chromatogram with that for known concentrations of authentic cyclopropylamine.
The conversion of 20 μM abacavir-MP to carbovir monophosphate by 172 units of the deaminating protein at 25 °C was monitored spectrophotometrically at 290 nm (Δε290 = 10 mM^cm"1) as described above (Figure 6). Control reactions without enzyme and/or without abacavir-MP were prepared similarly. Standard samples containing 15 μM and 30 μM cyclopropylamine were likewise prepared.
The reaction was allowed to go to completion such that abacavir-MP was converted completely to carbovir monophosphate, and the sample was analyzed for cyclopropylamine. The time-course was analyzed with the integrated rate equation (equation 1 herein) to give apparent values for Vm and Km of 0.195 ± 0.006 μM s" and 1.8 + 0.4 μM, respectively. Because of product inhibition, the value of Km in this experiment was significantly larger than the value of 0.12 μM (reported above).
Example 11 Derivatization and chromatographic resolution of the products of deamination Products were derivatized according to the standard Waters 'protocol for AccQ-
Tag amino acid analysis (Cohen SA and Michaud DP (1993) Analytical Biochemistry 211, 279). In summary, a 20 μl sample was derivatized with 60 μl borate buffer and 20 μl AccQ-Fluor reagent. The AQC derivatized products (10 μl injection volume) were chromatographed on a Waters 2690 Alliance Separations Module with a 474 Fluorescence Detector (excitation, 250 nm; emission, 395 nm) using a Waters reverse phase C-18 AccQ-Tag column (3.9 x 150 mm, 4 μm). AQC derivatives were eluted with an increasing acetonitrile gradient at a 1.0 ml/min flow rate as shown in Table 5, where Eluant A was AccQ-Tag Eluant A (Waters (Milford, MA)), Eluant B was acetonitrile and Eluant C was H2O:
Table 5
Example 12 Results: Analysis of deamination products of abacavir-MP by derivatization and reverse phase chromatography A peak with retention time of 28.9 min. was observed with authentic AQC- derivatized cyclopropylamine (CPA) following reverse phase chromatography (Figure 7D). The peak at 28.9 minutes was also observed in chromatography of the derivatized products of the deaminating protein/abacavir-MP reaction mixture (Figure 7C). This peak was not present in control reactions abacavir-MP minus deaminating protein (Figure 7A) and deammating protein minus abacavir-MP (Figure 7B). The peak at 28.9 minutes was collected manually from chromatography of both the derivatized authentic cyclopropylamine and from the deaminating/abacavir-MP reaction mixture for analysis by mass spectrometry. The corresponding region from chromatography of the control reaction in Figure 7B was also collected for background analysis by mass spectrometry. The peak in all chromatograms at retention time of 11.8 minutes is aminoquinoline (AMQ) which is a by-product of the derivatization chemistry. Peaks eluting at 23.5, 24.3, and 25.0 minutes were not identified.
Example 13 Mass spectrometric analysis of the cyclopropylamine derivative Collected chromatographic peaks corresponding to authentic AQC-derivatized cyclopropylamine and the putative AQC-derivatized cyclopropylamine peak from the deaminating protein/abacavir-MP reaction mixture were analyzed by nanoelectrospray (nanoES) ionization mass spectrometry using a Perkin-Elmer Sciex API-Ill triple- quadrupole mass spectrometer. Eluant from each sample was initially concentrated onto a pipette tip reversed-phase micro-column containing approximately 300 nL of Poros R3 reverse phase material. Each sample was washed with 1 % formic acid to remove buffers and salts, and eluted into nanoES needles using 1 μL of a 70% methanol/5% formic acid solution.
Example 14 Mass spectrometric analysis of cyclopropylamine product
Figure 8A shows the nanoES-MS spectrum of authentic cyclopropylamine derivatized with the AQC reagent collected from the LC separation in Figure 7D. The protonated molecular ion (abbreviated [M+H] +) of AQC-derivatized cyclopropylamine was observed at m/z 228.0, consistent with the calculated molecular weight of 227.1. Subjecting the ion at m/z 228.0 to fragmentation by collision-induced decomposition resulted in a product ion spectrum (Figure 8B) containing two fragment ions (m/z 171.0 and m/z 145.2) which are diagnostic for the functional group added by AQC- derivatization. By acquiring a spectrum only of ions which fragment to form an ion at m/z 171.0 (called precursor ion scanning, Figure 8C), only the ion at m/z 228.1 was detected illustrating the selectivity of an m/z 171.0 precursor ion scan for AQC- derivatized cyclopropylamine. The m/z 171.0 precursor ion spectrum obtained from analysis of the eluant collected from a control reaction containing the deaminating protein but no abacavir-MP (chromatogram shown in Figure 8B) is shown in Figure 9A. Only background ions at m z 244.3 and m/z 262.9 were observed, presumably due to an amine contained in the reaction buffer mixture which is derivatized with the AQC
reagent. Similar analysis of the reaction mixture containing both the deaminating protein and abacavir-MP (Figure 9B) showed the mixture to contain an additional component at m/z 228.1 which corresponds to the expected mass of AQC-derivatized cyclopropylamine. Fragmentation of the ion at m z 228.1 (Figure 9C) yields a fragmentation pattern identical to that of authentic AQC-derivatized cyclopropylamine (Figure 8B), confirming the formation of cyclopropylamine in the deaminating protein/abacavir-MP reaction mixture.
Example 15 Sequence Identification
Intact purified rat enzyme was subjected to Edman degradation after electroblotting to polyvinyldene difluoride (PVDF) membrane from SDS-PAGE. No sequence was obtained indicating the N-terminus was blocked. The deaminase band was subjected to in-gel digestion with trypsin or endoproteinase Lys-C. For Edman sequencing, the resulting peptides were separated using a capillary reverse phase C18 column and each applied directly to a Hewlett-Packard (HP) reverse phase sequencing support. Automated Edman degradation was performed using a HP G1006A Protein Sequencing System with on-line PTH analysis. In-gel digests were also analyzed by nanoelectrospray ionization mass spectrometry using a Micromass quadrupole time-of- flight (Q-TOF) mass spectrometer. Peptides observed in this analysis were subjected to tandem mass spectrometry in order to derive primary sequence data.
The peptide fragments were used as templates for the design of oligonucleotide primers. Using various primer sets, DNA for the human enzyme was identified in cDNA libraries prepared from fetal brain, kidney and testis and in 5 '-RACE libraries derived from mRNA from human cerebellum. After identifying the complete DNA sequence from overlapping fragments, oligonucleotide primers that hybridize to the 5'- and 3 '-ends were employed to generate the full-length coding sequence. Oligonucleotide primers were prepared as described by Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
The 5' oligonucleotide introduces EcoRI, Ndel and Nsil sites at the 5' end of the sequence; the initiation codon (ATG) is contained in the Ndel recognition sequence:
5'-GCT GAA TTC GCA TAT GCA TGA GGC AGA AGA GCA ACA GCC TTG CAA GTC AGA CTT C-3' (SEQ ID NO:6)
The 3'-oligo introduces Hindlll and BamHI cleavage sites and two termination codons:
5'-GCA AGC TTG GAT CCT ATT AAA TAT GTA ACA CTC TGG GCT TCAGGTGATTCCATTTCTTCCTC-3' (SEQIDNO:7)
The complete coding sequence for the human enzyme was cloned from a 5'- RACE cerebellum library with PCR primers homologous to the 5' and 3 '-regions of the full-length cDNA sequence. These primers also added restriction endonuclease cleavage sites useful for subcloning the deaminase encoding DNA.
Example 16
Expression & Purification of the deaminase The deaminase was expressed in Trichoplusia ni insect cells using techniques as are known in the art. The molecular weight and amino acid content of the heterologously expressed protein matched that predicted by translation of the human gene.
The recombinant baculovirus was generated following Life Technologies Bac-
To-Bac Baculovirus Expression System Manual, which utilizes the method as described in Luckow et al. , J. Virol. 67:4566 (1993). Spodoptera frugiperda (Sf 9) cells grown at 27°C with shaking (135 rpm) in supplemented Grace's insect culture medium (Gibco BRL) with 10% fetal bovine serum (Hyclone), 1 % Pluronic F-68 (Gibco BRL), and 50 mg/ml gentamicin (Gibco BRL) were used for transfections, virus amplification and titering. Trichoplusia ni (T. ni) cells grown in Ex-cell 405 insect medium (JRH Biosciences) with 50 mg/ml gentamicin, also at 27°C with shaking, were used for recombinant protein generation. Using the initial 2 ml transfection mix harvested 48
hours post-infection, the recombinant virus was amplified from 2 ml to 150 ml in Sf 9 cells and titered. Five hundred (500) ml E. ni cells at 1.0 x 10 6 cells per ml in 2-liter shake flasks were infected at a multiplicity of infection (m.o.i.) of 1 plaque-forming unit (pfu) per cell and harvested 48 hours post-infection at 800 x g for 20 minutes. Cell pellets were washed once with cold phosphate-buffered saline, frozen in an ethanol-dry ice bath, and stored at -70°C.
The human enzyme expressed in E. ni cells was purified several hundred fold by
(NH4)2SO4 precipitation and chromatography on the orange column and the Superdex 75 column as described for the rat liver enzyme. SDS gel electrophoresis of this preparation demonstrated that over 50% of the protein had the desired molecular weight. Sequencing of the band confirmed that it was the protein of interest. The specific activity of this preparation with abacavir-MP as substrate was similar to that of the rat enzyme. The Km for abacavir-MP was 0.27 + 0.03 μM and the Kj for GMP was 1.3 + 0.01 μM. These values were similar to those determined for the rat enzyme.
Example 17 Antibody Production Polyvalent antibody to the abacavir-MP deaminase is prepared in rabbits using standard techniques as are known in the art, and utilizing recombinantly produced enzyme (such as enzyme expressed in E. coli or T. ni cells) as the immunogen. Antibodies are used to evaluate the level of deaminase expression in eukaryotic cells and organisms, using enzyme purified from bacteria or insect cells as the quantitation standard.
References
1. Daluge et al. , (1997) Antimicrobial Agents <& Chemotherapy. 41: 1082-1093.
2. Trisdale et al., (1991) Antimicrobial Agents & Chemotherapy. 41: 1094-1098. 3. Faletto et al. (1991) Antimicrobial Agents & Chemotherapy. 41:1099-1107.
4. Miller et al. (1992) J. Biol. Chem. 267: 21220-21224.
5. Spector et al. (1983) Biochem. Pharmacol. 32: 2505-2509.
6. Mohamedali et al. (1996) Biochemistry 35: 1672-1680.
7. Zielke and Seulter (1971) . Biol. Chem. 246: 2179-2186. 8. Wilson et al. (1991) Science 252: 1278-1284.
9. Porter and Austin (1993) . Biol. Chem. 268: 24005-24011.
10. Smith et al. (1994) Biochemistry 33: 6468-6474.
11. Putman et al. (1972) Biochem. J. 129: 301-310.
12. Cohen SA and Michaud DP (1993) Analytical Biochemistry 211, 279.
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<222> (1) .. (1068)
<400> 3 atg cat gag gca gaa gag caa cag cct tgc aag tea gac ttc tat tct 48 Met His Glu Ala Glu Glu Gin Gin Pro Cys Lys Ser Asp Phe Tyr Ser 1 5 10 15 gaa ttg cca aaa gtg gaa ctt cat gcc cac ttg aat gga tec att agt 96 Glu Leu Pro Lys Val Glu Leu His Ala His Leu Asn Gly Ser He Ser 20 25 30
tct cat ace atg aag aaa tta ata gtc cag aag cca gat ctt aaa ate 144 Ser His Thr Met Lys Lys Leu He Val Gin Lys Pro Asp Leu Lys He 35 40 45 cac gat cag atg act gtg att gac aag gga aag aaa aga act ttg gaa 192 His Asp Gin Met Thr Val He Asp Lys Gly Lys Lys Arg Thr Leu Glu 50 55 60 gaa tgt ttc cag atg ttt caa act att cat cag ctt act agt age cct 240 Glu Cys Phe Gin Met Phe Gin Thr He His Gin Leu Thr Ser Ser Pro 65 70 75 80 gaa gat att eta atg gtc aca aaa gat gtc ata aaa gaa ttt gca gat 288 Glu Asp He Leu Met Val Thr Lys Asp Val He Lys Glu Phe Ala Asp 85 90 95 gac ggc gtc aag tac ctg gaa eta agg age aca ccc aga aga gaa aat 336 Asp Gly Val Lys Tyr Leu Glu Leu Arg Ser Thr Pro Arg Arg Glu Asn 100 105 110 get ace gga atg act aaa aag act tat gtg gaa tct ata ctt gaa ggt 384 Ala Thr Gly Met Thr Lys Lys Thr Tyr Val Glu Ser He Leu Glu Gly 115 120 125 ata aaa cag tec aaa caa gaa aac ttg gac att gat gtt agg tat ttg 432 He Lys Gin Ser Lys Gin Glu Asn Leu Asp He Asp Val Arg Tyr Leu 130 135 140 ata gca gtt gac aga aga ggt ggc cct tta gta gcc aag gag act gta 480 He Ala Val Asp Arg Arg Gly Gly Pro Leu Val Ala Lys Glu Thr Val 145 150 155 160 aaa ctt gcc gag gag ttc ttc ctt tct act gag ggt aca gtt ctt ggc 528 Lys Leu Ala Glu Glu Phe Phe Leu Ser Thr Glu Gly Thr Val Leu Gly 165 170 175 ctt gac etc agt gga gac cct act gta gga caa gca aaa gac ttc ttg 576 Leu Asp Leu Ser Gly Asp Pro Thr Val Gly Gin Ala Lys Asp Phe Leu 180 185 190 gaa cct ctt tta gaa get aag aaa gca ggt ctg aag tta gca ttg cat 624 Glu Pro Leu Leu Glu Ala Lys Lys Ala Gly Leu Lys Leu Ala Leu His 195 200 205 ctt tea gag att cca aac caa aaa aaa gaa aca caa ata etc ctg gat 672 Leu Ser Glu He Pro Asn Gin Lys Lys Glu Thr Gin He Leu Leu Asp 210 215 220 ctg ctt cct gac aga ate ggg cat gga aca ttt etc aac tec ggt gag 720 Leu Leu Pro Asp Arg He Gly His Gly Thr Phe Leu Asn Ser Gly Glu 225 230 235 240 gga gga tec ctg gat ctg gtg gac ttt gtg agg caa cat egg ata cca 768 Gly Gly Ser Leu Asp Leu Val Asp Phe Val Arg Gin His Arg He Pro 245 250 255
ctg gaa etc tgt ttg ace tea aac gtc aaa agt cag aca gtc cca tct 816 Leu Glu Leu Cys Leu Thr Ser Asn Val Lys Ser Gin Thr Val Pro Ser 260 265 270 tat gac cag cac cat ttc gga ttc tgg tac age att gca cat cct tct 864 Tyr Asp Gin His His Phe Gly Phe Trp Tyr Ser He Ala His Pro Ser 275 280 285 gtg ate tgt act gat gat aag ggt gtt ttt gca aca cac ctt tct caa 912 Val He Cys Thr Asp Asp Lys Gly Val Phe Ala Thr His Leu Ser Gin 290 295 300 gag tac cag ctg gca get gaa aca ttt aat ttg ace cag tct cag gtg 960 Glu Tyr Gin Leu Ala Ala Glu Thr Phe Asn Leu Thr Gin Ser Gin Val 305 310 315 320 tgg gat ctg tct tat gaa tec ate aac tac ate ttt get tct gac age 1008 Trp Asp Leu Ser Tyr Glu Ser He Asn Tyr He Phe Ala Ser Asp Ser 325 330 335 ace aga tct gaa ctg agg aag aaa tgg aat cac ctg aag ccc aga gtg 1056 Thr Arg Ser Glu Leu Arg Lys Lys Trp Asn His Leu Lys Pro Arg Val 340 345 350 tta cat att taa 106E Leu His He 355
<210> 4
<211> 355
<212> PRT
<213> Modified human deaminase sequence
<400>
Met His Glu Ala Glu Glu Gin Gin Pro Cys Lys Ser Asp Phe Tyr Ser 1 5 10 15
Glu Leu Pro Lys Val Glu Leu His Ala His Leu Asn Gly Ser He Ser 20 25 30
Ser His Thr Met Lys Lys Leu He Val Gin Lys Pro Asp Leu Lys He 35 40 45
His Asp Gin Met Thr Val He Asp Lys Gly Lys Lys Arg Thr Leu Glu 50 55 60
Glu Cys Phe Gin Met Phe Gin Thr He His Gin Leu Thr Ser Ser Pro 65 70 75 80
Glu Asp He Leu Met Val Thr Lys Asp Val He Lys Glu Phe Ala Asp 85 90 95
Asp Gly Val Lys Tyr Leu Glu Leu Arg Ser Thr Pro Arg Arg Glu Asn 100 105 110
Ala Thr Gly Met Thr Lys Lys Thr Tyr Val Glu Ser He Leu Glu Gly 115 120 125
He Lys Gin Ser Lys Gin Glu Asn Leu Asp He Asp Val Arg Tyr Leu 130 135 140
He Ala Val Asp Arg Arg Gly Gly Pro Leu Val Ala Lys Glu Thr Val 145 150 155 160
Lys Leu Ala Glu Glu Phe Phe Leu Ser Thr Glu Gly Thr Val Leu Gly 165 170 175
Leu Asp Leu Ser Gly Asp Pro Thr Val Gly Gin Ala Lys Asp Phe Leu 180 185 190
Glu Pro Leu Leu Glu Ala Lys Lys Ala Gly Leu Lys Leu Ala Leu His 195 200 205
Leu Ser Glu He Pro Asn Gin Lys Lys Glu Thr Gin He Leu Leu Asp 210 215 220
Leu Leu Pro Asp Arg He Gly His Gly Thr Phe Leu Asn Ser Gly Glu 225 230 235 240
Gly Gly Ser Leu Asp Leu Val Asp Phe Val Arg Gin His Arg He Pro 245 250 255
Leu Glu Leu Cys Leu Thr Ser Asn Val Lys Ser Gin Thr Val Pro Ser 260 265 270
Tyr Asp Gin His His Phe Gly Phe Trp Tyr Ser He Ala His Pro Ser 275 280 285
Val He Cys Thr Asp Asp Lys Gly Val Phe Ala Thr His Leu Ser Gin
290 295 300
Glu Tyr Gin Leu Ala Ala Glu Thr Phe Asn Leu Thr Gin Ser Gin Val 305 310 315 320
Trp Asp Leu Ser Tyr Glu Ser He Asn Tyr He Phe Ala Ser Asp Ser 325 330 335
Thr Arg Ser Glu Leu Arg Lys Lys Trp Asn His Leu Lys Pro Arg Val 340 345 350
Leu His He 355
<210> 5
<211> 1068
<212> DNA
<213> Modified human deaminase sequence
<400> 5 atgcatgagg cagaagagca acagccttgc aagtcagact tctattctga attgccaaaa 60 gtggaacttc atgcccactt gaatggctcc attagttctc ataccatgaa gaaattaata 120 gtccagaagc cagatcttaa aatccacgat cagatgactg tgattgacaa gggaaagaaa 180 cgcactttgg aagaatgttt ccagatgttt caaactattc atcagcttac tagtagccct 240 gaagatattc taatggtcac aaaagatgtc ataaaagaat ttgcagatga cggcgtcaag 300 tacctggaac tacgtagcac accccgtcgc gaaaatgcta ccggaatgac taaaaagact 360 tatgtggaat ctatacttga aggtataaaa cagtccaaac aagaaaactt ggacattgat 420 gttcgctatt tgatagcagt tgaccgtcgt ggtggccctt tagtagccaa ggagactgta 480 aaacttgccg aggagttctt cctttctact gagggtacag ttcttggcct tgacctcagt 540 ggagacccta ctgtaggaca agcaaaagac ttcttggaac ctcttttaga agctaagaaa 600 gcaggtctga agttagcatt gcatctttca gagattccaa accaaaaaaa agaaacacaa 660 atactcctgg atctgcttcc tgaccgtatc gggcatggaa catttctcaa ctccggtgag 720 ggaggttccc tggatctggt ggactttgtg aggcaacatc ggataccact ggaactctgt 780 ttgacctcaa acgtcaaaag tcagacagtc ccatcttatg accagcacca tttcggattc 840
tggtacagca ttgcacatcc ttctgtgatc tgtactgatg ataagggtgt ttttgcaaca 900 cacctttctc aagagtacca gctggcagct gaaacattta atttgaccca gtctcaggtg 960 tgggatctgt cttatgaatc catcaactac atctttgctt ctgacagcac ccgctctgaa 1020 ctgcgcaaga aatggaatca cctgaagccc cgtgtgttac atatttaa 1068
<210> 6
<211> 55
<212> DNA
<213> nucleotide probe
<400> 6 gctgaattcg catatgcatg aggcagaaga gcaacagcct tgcaagtcag acttc 55
<210> 7
<211> 62
<212> DNA
<213> nucleotide probe
<400> 7 gcaagcttgg atcctattaa atatgtaaca ctctgggctt caggtgattc catttcttcc 60 tc 62