22
`
`Kisliuk
`
`Chapter 2 I Folate Bioch1
`
`factor may tether it to the enzyme and allow access of the site to nucleotide substrate and
`release of product. The bifunctional DHFR-TS from Leishmania major has an unusual
`charge distribution that could account for channelling of folate cofactors between active
`sites (55). Human TS has been cloned, sequenced, expressed (56,57), and crystallized
`( 58 ). The crystal structure suggests a mechanism for docking of substrates involving the
`pivoting of an active site loop.
`Purified cellular human and rat thymidylate synthases are acetylated at their N-termi(cid:173)
`nal amino acids and have a lower specific activity than the corresponding recombinant
`enzymes ( 57). In H35 rat hepatoma cells, TS has been localized to the nucleolar region
`but appears in the cytoplasm when overexpressed (59). TS was also present in the mito(cid:173)
`chondria of H35 cells and a small amount of phosphorylated TS was identified. TS binds
`to its own mRNA as a negative regulator (60). This binding requires that TS be blocked
`at its N-terminal position. Elements in the promoter region of the human TS gene have
`been identified and the nuclear factor Spl is a major contributor to promoter activity, but
`other positive and negative regulators have been identified (61). Further studies of these
`modifications and interactions should elucidate the relationship of thymidylate synthase
`to the cell division cycle.
`Accumulation of dUTP and its misincorporation into DNA is a major factor in the cy(cid:173)
`totoxicity resulting from the inhibition of TS. dUTPase catalyzes the conversion of
`dUTP to dUMP and therefore acts to counteract the toxic action of dUTP (62). Con(cid:173)
`versely, inhibitors of dUTPase should enhance the toxicity of TS inhibitors. X-ray crys(cid:173)
`tallographic studies show that the active site of human dUTPase, a trimeric enzyme,
`consists of residues from all three subunits (63). The human dUTPase gene codes for
`both nuclear and mitochondrial isoforms of the enzyme (62).
`
`3.4 Dihydrofolate Reductase
`In contrast with SHMT (a tetramer) and TS (a dimer), human DHFR is monomeric
`(22 kDa). It catalyzes the reduction of 7,8-H2PteGlun to 5,6,7,8-H4PteGlun (64). The hu(cid:173)
`man enzyme has been cloned, expressed, and crystallized (65) and the lH and 15N nu(cid:173)
`clear magnetic resonance assignments obtained (66). Again in contrast with SHMT and
`TS, the primary structures of eukaryotic DHFRs are not highly homologous, only 20%
`of the residues of human DHFR are identical to those found in eight other eukaryotic
`DHFRs (64).
`Site-directed mutagenesis studies have led to the production of variants of human
`DHFR resistant to methotrexate (67,68). Current studies in mice are testing the concept
`that cDNA coding for methotrexate-resistant DHFR transduced into bone marrow pro(cid:173)
`genitor cells will lead to improved curability of mice bearing a methotrexate-sensitive
`tumor (67,68).
`Folate polyglutamates and antifolate polyglutamates often have a modest two- to 10-
`fold enhanced affinity for human DHFR as compared with monoglutamate forms
`(64,69). An interesting exception is 2-desamino-2-methylaminopterin, which has an
`IC50 value greater than 50 µM but the addition of four -y-linked glutamyl residues low(cid:173)
`ers the IC50 >200-fold to 0.25 µM (70).
`The cellular synthesis of human DHFR is negatively regulated by the binding of the
`enzyme to its cognate mRNA (71,72). Methotrexate binding to DHFR prevents this in(cid:173)
`teraction and promotes DHFR production.
`
`C1THF
`synthase
`
`Fig. 6. Interconversion of
`
`The activity of the rr
`01" the cell cycle, media
`
`Cl-THF-synthase i:
`mammalian cells (74).
`pendent 5,10-CHrHJ
`CHO-H4PteGlun synth
`and S for dehydrogem:
`functional enzyme has
`34-kDa DC portion o1
`convenient for kinetic
`likely necessary to cat
`and methyl groups in
`cleotide synthesis and
`thesis (76-78). This :
`metabolic studies.
`The second form is
`34-kDa mitochondrial
`zymes have been clor
`gested to serve as the
`tRNA required for prol
`sistent with its resemb
`to the DC portion of ti
`quirement for Mg2 + a
`phosphate on the cider
`quence identity with ti
`
`'-'
`
`Sandoz Inc.
`Exhibit 1002-00541
`
`JOINT 1002-0541
`
`

`
`1·~D_f;p
`
`\
`j 1 l :!,,_L,Fd
`Ii
`j ·~·1/iL)?
`
`)
`
`'(
`CH'·H4Pt<0Glun
`
`l
`f l,\f:,ff! >I
`ii
`:}, .!U~:n,:::>t1:1y1~n9···.rltffi' !
`I /
`t t \
`
`S,10-1,te·thenyl-'l'HF
`cyclohydrolase
`-
`\ ·~)
`
`.
`10-CHO-H4PteGiu0
`
`~-CHO-H4PteGlun
`
`) 5,10-methenyl'lHF
`
`synthetase
`'-----"'
`;("' ATP
`(MTHFS)
`ADP
`
`J l
`
`(
`I
`
`C1THF
`3ynC.hase
`
`0/'
`
`~~i~l t1il!J,~1l :li
`
`n.3 t:t'l\"c··;':•~"'ll ;).i
`l, 3nd crystallized
`ates involving the
`
`d at their N-termi-
`1ding 1ecombinant
`_e nucleolar region
`;resent in the mito(cid:173)
`ientified. TS binds
`that TS be blocked
`man TS gene have
`)rooter activity, but
`her studies of these
`ymidylate synthase
`
`~or factor in the cy(cid:173)
`; the conversion of
`1f dUTP (62). Con-
`1ibitors. X-ray crys(cid:173)
`a trimeric enzyme,
`=>ase gene codes for
`
`>HFR is monomeric
`' 'teGlu0 ( 64). The hu(cid:173)
`the lH and 15N nu(cid:173)
`rast with SHMT and
`mologous, only 20%
`_ght other eukaryotic
`
`lf variants of human
`re testing the concept
`tto bone marrow pro-
`1ethotrexate-sensiti ve
`
`• a modest two- to 10-
`tonoglutamate forms
`1pterin, which has an
`lutamyl residues low-
`
`by the binding of the
`HFR prevents this in-
`
`ATP l ~ 10-formyl'rHF
`
`10-formyl-THF
`synthetase
`
`(S) ADP)
`
`ADP
`
`NADP dehydrogenase
`\,. NADPH
`( FDH)
`
`(
`
`ATP
`
`C02 + H4PteGiu0
`
`Fig. 6. Interconversion of methylene and formyl tetrahydrofolate derivatives.
`
`The activity of the murine DHFR gene promoter increases at the G 1-S-phase boundary
`of the cell cycle, mediated by a member of the E2F family of transcription factors (73 ).
`
`4. Cl-THF-SYNTHASE
`C 1-THF-synthase is a homodimeric enzyme complex that occurs in two forms in
`mammalian cells (74). One form is a trifunctional, cytoplasmic, 100 kDa, NADP+ -de(cid:173)
`pendent 5, 1 O-CHz-H4PteGlun dehydrogenase-5, 1 O-CH-H4PteGlun cyclohydrolase- lO(cid:173)
`CHO-H4PteGlun synthetase (Fig. 6). The three enzyme activities are abbreviated D, C,
`and S for dehydrogenase, cyclohydrolase, and synthetase, respectively. The human tri(cid:173)
`functional enzyme has been cloned, sequenced, and expressed (75). The amino-terminal
`34-kDa DC portion of the trifunctional enzyme has been expressed separately and is
`convenient for kinetic studies. The trifunctional cytoplasmic DCS complex is most
`likely necessary to catalyze the incorporation of formate, arising from serine, glycine,
`and methyl groups in mitochondria, into 10-CHO-H4PteGlun for use in purine nu(cid:173)
`cleotide synthesis and into 5,10-CHz-H4PteGlun for use in dTMP and methionine syn(cid:173)
`thesis (76-78). This role for the trifunctional enzyme is supported by kinetic and
`metabolic studies.
`The second form is bifunctional NAD+ -dependent DC, which is a nuclear-encoded
`34-kDa mitochondrial enzyme. The human and murine bifunctional mitochondrial en(cid:173)
`zymes have been cloned, sequenced, and expressed (79). This system has been sug(cid:173)
`gested to serve as the source of formyl groups for the synthesis of formylmethionyl
`tRNA required for protein synthesis in mitochondria. Its location in mitochondria is con(cid:173)
`sistent with its resemblence to DC enzyme complex found in bacteria (79 ). In contrast
`to the DC portion of the cytosolic trifunctional enzyme complex, it has an absolute re(cid:173)
`quirement for Mg2+ and Pi (80). It is proposed that Mg2 + and Pi substitute for the 2'
`phosphate on the adenosine portion of NADPH because it has a 44% amino acid se(cid:173)
`quence identity with the DC domain of yeast mitochondrial NADP-dependent trifunc-
`
`Sandoz Inc.
`Exhibit 1002-00542
`
`JOINT 1002-0542
`
`

`
`24
`
`Kisliuk
`
`-c -dependent turnover
`
`Lional enzyme, the human l'TAD-dependent ::nzyme has a low, ;;Ig2
`with NADP, and P 1 competes for NADP binding.
`Kinetic studies provide an explanation of the mechanism by which the mitochondrial
`NAD-clependent DC enzyme functions to convert 5,10-CH2-H4PteGlun to lO-CHO(cid:173)
`H4PteGlu110 whereas the cytosolic NADP-dependent enzyme functions in the reverse
`direction (78). In the cytosolic system, where the NADPH/NADP ratio is high, the rate(cid:173)
`limiting C-reaction is stimulated by NADPH analogs, and presumably by NADPH as
`well, but technical difficulties prevent a direct test. The 10-CHO-H+PteGlun is 100%
`channeled for reduction to 5,10-CHi-H4PteGlu. In mitochondria, the NADH/NAO ratio
`is low, favoring the oxidative reaction and the conversion of 5,10-CHrH4PteGlun to 10-
`CHO-H4PteGlu11 which is not stimulated by nucleotides. Both cytosolic and mitochon(cid:173)
`drial DC activities are carried out at a single active site.
`In contrast with cytosolic tdfunctional NAOP-dependent OCS, where high activity is
`found widely distributed among various tissues, the NAO-dependent system is usually
`not detectable in most tissues of adult animals, but its cognate mRNA is detectable (81 ).
`NAO-dependent DC activity is found in embryonic tissue and in most all transfonned
`cultured cells.
`The affinity of the DC complexes for folate substrates is not greatly enhanced by
`increasing the polyglutamate chain length. Monoglutamate fom1s function in substrate
`channeling as well as polyglutamates. This contrasts with the fonniminotetrahydrofolate
`transferase-formiminotetrahydrofolate cyclodearninase system involved in histidine
`catabolism, in which affinity and channeling are enhanced with polyglutamate substrates.
`The 1 O-CHO-H4PteGlun synthetase domain of cytoplasmic OCS catalyzes the forma ·
`tion of 10-CHO-H4PteGlun from formate and H4PteGlu11 accompanied by the hydroly(cid:173)
`sis of MgATP to MgAOP. In contrast with DC activities, S has a very high affinity for
`its polyglutamate substrate. The Km value for H4PteGlu5 for the rabbit liver enzyme is
`0.1 µMand the binding of H4PteGlu11 and MgA TP enhance the binding of formate. The
`activity of S from bacteria and mammals is stimulated by K+ or by NH4 j-, but Na+ and
`u+ have no effect (82). Spermine stimulates S from Lac1obocil111s orabinosus and L.
`casei by lowering the Km of H4PteGlu 1 ( 83 ). This observation is noteworthy because
`spennine reduces the amount of thyrnidine cequired to reverse lhe inhibidon of growth
`of L. arabinosus by aminopterin and other antifolates (84 ). Therefore, spermine might
`play a regulatory role at the formyl levels::; well ns at the methyl level mentioned abo"e.
`Both of i:hese stimulations could benefit growing cells by stimulating rhe formation 1)f
`single-carbon units for methionine, ihymidylate, and purine imcleotide synthesis.
`In vitro kinetic studies with rabbit liver DC3 coupled to SHl'v:IT s11ggesc ihac 1he two
`:}rotcins int.:ract lo facilltaie £he con'iersion
`fJCS plns SHl"l.T
`fcirn1:ite •o
`~ 1rovid,~ :111,~srini<:1led in 11ivo •:0nc,~ntr:>1.ion <Jf'/.5 !-•,M CoJ;if,~ :Jr:dve sii·':S. ··vhich inc!ic:11es
`rh0t JJt<JSf of U1~:: foJarc c~)enzy(l1·~S ju cc> Us 8re prctei11 boun(t.
`
`/ i '
`
`C-te1
`conn
`catal
`as th
`lular
`func
`dom
`alyz1
`com
`re inc
`drog
`C02
`of fc
`F
`H4P
`ties
`H4P
`H4P
`I1
`to a
`ti on
`68 i
`lrati
`est ii
`GN
`
`hi bi
`ing
`::-.ho
`inat
`acti
`in le
`tn g
`: lllil
`nm
`fr.-11
`If,)
`j,, ...
`'.
`'(l!
`
`Sandoz Inc.
`Exhibit 1002-00543
`
`JOINT 1002-0543
`
`

`
`:;;.
`
`1
`v • ;
`
`'
`
`li~j ,;\J;i [:
`
`' jlj
`
`r:-1
`
`( hond fr1 l
`10-CHO-
`1e reverse
`t, che rate-
`ADPH as
`11 is 100%
`'.'l'ADratio
`]lu11 to 10-
`mitochon-
`
`'
`
`1 activity is
`1 is usually
`;table ( 81 ).
`:ansformed
`
`'
`
`1hanced by
`in substrate
`hydrofolate
`ln histidine
`:! substrates.
`s the forma-
`he Jwdroly-
`ty for
`ta
`~r enzyme is
`ormate. The
`but Na+ and
`wsus and L.
`rthy because
`)fl of growth
`:rmine might
`ioned above.
`formation of
`1thesis.
`t that the two
`i plus SHMT
`iich indicates
`
`ed.
`
`.J'ASE
`cytosolic en-
`; been cloned,
`~-terminal do-
`·ltransferase, a
`
`' ) ' ' t'
`
`1
`
`i(U1_c;_ (',;,._ .._'~H1L,,u,
`
`e - _ _ _ ,_ - - - · - - - - - - - -----~--~-·-----•-·-------~---~----- --~ - - - - - - - - - - - - -
`
`•
`
`(~l~i1!_-, ,-1.-·1qs \ tbai lcsen1bles aklehyd,:;
`!·'-111,~ti\1 ~ :~1 ifl
`t~ t n!,,-1
`-ti1i
`l·1.Y1 ·-qfitno ::;i:1~Js_ lt Is Hk1:-l_·,1 ih~ll the •r~:-tjcif hl11i-=:tJ0i1 cf FL)l-i L~. ~-~J
`1-,=-rii~h: ~--i-
`,_, 1 ,,. ii'
`·iii·~ o\io.Lt1i(•li vf r;ic:-
`l 61UU(' i:·l !
`'t-10-HJ-'te(rlnr, 10 1~~(J2 nsing NADf'
`.• t,\I\
`"'''~~·:-11 '.<•:•_eptor This 'Nould provide egress of si11gle-carbon units from the eel~
`th·:
`Jubr \l!k'-c•rbon pool as well as providing NADPH for reductive reactions (Fig. 6). This
`; uncr10n n::q uires rhe nppropri,1te j uxt::i.position of the NH::i-terminal and COOH-tenninal
`d\.imains provided by the connecting peptide. In addition, the NHrterminal domain cat(cid:173)
`alyze<> the hydrolysis of lO-CHO-H4PteGlun to H4PteGlun and formate. This activity,
`1.:ombinecl with that of C 1 THF synthase, leads to a futile cycle for the production and
`reincorporation of formate. The isolated COOR-terminal domain has aldehyde dehy(cid:173)
`Jrugenase activity and serves this function in the conversion of lO-CHO-H4PteGlu11 to
`C0 2 and R+PteGlun by FDR, the 10-CHO group coITesponding to the aldehyde portion
`of formic acid.
`FDH has a very high affinity ( Kct = 20 nM) for both 10-CHO-H4PteGlu5 and
`H4PteGlu5, the latter being the product of both the dehydrogenase and hydrolase activi(cid:173)
`ties and a likely product inhibitor (88). Both SHMT and ClTHF synthase discharge
`H4PteGlu5 from FDH, thus FDH could serve as a distribution point for 10-CHO(cid:173)
`H4PteGlu5 and H4PteGlu5.
`fo rat and rabbit liver FDR accounts for 1.2% of the soluble protein. This is equivalent
`to a concentration of FDH subunits of 42 µMin vivo (88). Adding this to the concentra(cid:173)
`tion of folate-binding sites provided by SHMT plus C 1 THF synthase (26 µM) yields
`68 µM. This reinforces the suggestion that most all the folate coenzymes, whose concen(cid:173)
`tration in rabbit liver is estimated at 26 µM, are enzyme bound in vivo especially since the
`estimate of 68 µM for folate-binding sites does not include other folate enzymes or
`GNMT.
`Since the N-terminal portion of FDR shows homology with GARFT, the GARFT in(cid:173)
`hibitors DDATHF (5, 10-dideazatetrahydrofolate) and 5, DACTHF (a folate analog lack(cid:173)
`ing the tetrahydropyrazine ring) were tested as inhibitors of rat liver FDH ( 89 ). DDATHF
`showed an IC50 of 48 µM but 5-DACTHF showed no inhibition at 340 µM. Polygluta(cid:173)
`mate derivatives were not tested, but this work opens the possibility of influencing FDH
`activity, and thus one-carbon metabolism, with folate analogs. In this connection it is of
`interest to consider mice that are totally lacking FDH (90). Although these mice are able
`to grow and reproduce, their breeding time is greatly extended. The liver folates of these
`animals were compared with those of normal mice and 1 O-CHO-H4PteGlu went from 2.8
`nmol/g in normal mice to 7 .3 nmol/g in the FDR-deficient mice, whereas H4PteGlu went
`from 19.0 nmol/g in normals to 4.4 nmol/g in the deficient strain. Levels of 5-CHO(cid:173)
`RiPteGlu and 5-CHr~PteGlu were unchanged. These results are compatible with the
`loss of FDH in that the increase in 1 O-CHO-H4PteGlu could be because of diminished
`ability to metabolize the CHO group and the decrease in H4PteGlu could be caused by the
`loss of a major liver H4PteGlu-binding protein.
`
`6. 5,10-METHENYLTETRAHYDROFOLATE SYNTHETASE
`5.10-methenyltetrahydrofolate synthetase (MTHFS) (formerly 5-fom1yltetrahydrofo(cid:173)
`late cyclodehydrase) catalyzes the irreversible MgA TP-dependent conversion of 5-
`CHO-H4PteGlu11 to 5,10-CH-H4PteGlu11 (91). As discussed above, 5-CHO-H4PteGlun is
`formed from 5,10-CH-R1PteGlu0 in a reaction catalyzed by SHMT. The reactions cat(cid:173)
`alyzed by SHMT and MTHFS therefore constitute a futile cycle (Fig. 6) that is proposed
`
`Sandoz Inc.
`Exhibit 1002-00544
`
`JOINT 1002-0544
`
`

`
`an ;nt1il•it0c 0f ::.;I-Hvff (47) :.wcl
`-~- 1 'l-IU-t-L,Ptei
`tJ ,·.:gu1ate r:cil 1Jl<lr le'.tr:L
`d·:::J>.f.:.fT (-/.')). 11I'TI-IF3 is the oaly 1u10 1vn tf;:::ymatic 1··~11ction carable of returning S(cid:173)
`CHO-R+PleGlu11 to the rnajor pathways of one-carbon metabolism and therefofe is a key
`e11zyme in the clinical uses of 5-CHO-H.+PteGlu for prevention of methotrexate toxiciry
`and for enhancing the antitumor activity of fluorouracil.
`Human MTHFS has been cloned, sequenced, and expressed (91,92). It is a cytosolic
`23-kDa protein with little homology to other folate enzymes except for an SLLP se(cid:173)
`quence found in most enzymes having 10-CHO-H+PteGlun as a substrate. It is highly ho(cid:173)
`mologous to rabbit liver MTHFS which was chemically sequenced earlier (93). Some
`MTHFS has been found in human mitochondria (91 ), but none in rabbit liver mitochon(cid:173)
`dria (92), which is surprising because 5-CHO-H4PteGlun is probably formed in vivo by
`rabbit liver mSHMT and would require a mechanism to re-enter the pool of mitochon(cid:173)
`drial folate coenzymes. However, folate polyglutamates can leave mitochondria (4),
`which might replace the need for a mitochondrial MTHFS.
`Human cytosolic and mitochondrial MTHFS have similar molecular weights and sub(cid:173)
`strate affinities. Both forms show a much higher affinity for 5-CHO-H4PteGlu5 than for
`5-CHO-H4PteGlui, as does the rabbit liver cytosolic enzyme. A cDNA isoform for
`MTHFS encoding a mitochondrial signal sequence has not been reported.
`5-CHO-H4 homofolate (having an additional methylene group between the 9 and 10
`positions of H4PteGlu) is a competitive inhibitor of MTHFS. The Ki values are 0.1 µM
`for the rabbit enzyme (94) and 1.4 µ,M for human cytosolic enzyme (91 ). 5-CHO-H4 ho(cid:173)
`mofolate also behaves as a poor substrate for the reaction. The inhibition of MTHFS by
`5-CHO-H4 homofolate in MCF-7 cells provided important evidence that 5-CHO(cid:173)
`H4PteGlun inhibits AICARFT in vivo as well as in vitro (49).
`
`7. GLYCINAMIDE RIBONUCLEOTIDE
`FORMYLTRANSFERASE (GARFT)
`
`The de novo pathway for purine nucleotide biosynthesis consists of 10 enzyme-cat(cid:173)
`alyzed reactions starting from 5-phosphoribosyl-1-pyrophosphate, leading to inosinic
`acid, the precursor of AMP and GMP (95). Two reactions in this pathway, the third and
`the ninth, require 10-CHO-H4PteGlun as a formyl donor: glycinamide ribonucleotide
`formyltransferase and aminoimidazolecarboxamide ribonucleotide formyltransferase
`(AICARFT) (Fig. 7). The gene for mouse and human GARFT encodes a trifunctional
`protein of 110 kDa, the GARFT activity residing in the carboxy-terminal 29-kDa por(cid:173)
`tion (96). The other two activities on the trifunctional protein catalyze the second and
`fifth steps on the purine biosynthetic pathway, synthesis of glycinamide ribonucleotide
`and aminoimidazole ribonucleotide, respectively. The genes for both mouse and human
`trifunctional protein have been cloned and expressed and a fully functional 23-kDa hu(cid:173)
`man GARFT segment has been expressed as well (95-97). The mouse and human genes
`are very similar.
`10-formyl-5,8-dideazafolate and its polyglutamate derivatives are usually employed
`as substrates in enzymatic studies because they are more stable than the natural substrate,
`10-CHO-H4PteGlu11 • 10-formyl-5,8-dideazaPteGlu6 binds to mouse GARFT 10 times
`more tightly than the monoglutamate (98). These substrate analogs, their deformylated
`products, as well as the corresponding derivatives of the inhibitor, 5-10-dideazatetrahy(cid:173)
`clrofolale (DDA Tfff), all bind to the enzyme very tightly with dissociation constants in
`
`Sandoz Inc.
`Exhibit 1002-00545
`
`JOINT 1002-0545
`
`

`
`'•,liuk
`
`· 147) and
`:turning 5-
`ire is a key
`tte toxicity
`
`a cytosolic
`1 SLLP se(cid:173)
`. highly ho-
`193). Some
`·mitochon(cid:173)
`l in vivo by
`f mitochon-
`1ondria ( 4 ),
`
`h~andsub­
`;Ju~ than for
`i&oform for
`;:
`
`(hapter,;,, I folate Biochemisrry
`
`~~~--~~~~~-
`
`glycinamide
`ribonucleotide
`formyltransferase
`(GARFT)
`
`aminoimidazole(cid:173)
`carboxamide
`ribonucleotide
`formyltransferase
`(AICARFT)
`
`aminoimidazole(cid:173)
`carboxamide
`ribonucleotide
`
`inosinic acid
`
`l !
`
`GMP
`
`AMP
`
`Fig. 7. Folate enzymes involved in purine nucleotide synthesis.
`
`the nanomolar range. Therefore Ki values determined under the standard assay condi(cid:173)
`tions do not reflect true dissocation constants. These studies (98) also show that the or(cid:173)
`der of binding of the folate and GAR substrates is random sequential rather than ordered
`sequential, with the folate substrate binding first as was suggested by studies carried out
`under the standard conditions (97).
`Site-directed mutagenesis studies have identified putative residues for the binding of
`the polyglutamate chain (99 ). These residues are located on the opposite lobe of GARFT
`from that which binds the pteridine portion of the cofactor. The poly glutamate substrate
`therefore appears to span the active site cleft of the enzyme.
`
`8. AMINOIMIDAZOLECARBOXAMIDE RIBONUCLEOTIDE
`FORMYLTRANSFERASE (AICARFT)
`
`The ninth and tenth steps on the pathway of the conversion of 5-phospho-ribosyl-1-
`pyrophosphate to inosinic acid are catalyzed by a bifunctional protein having AICARFT
`and inosine monophosphate cyclohydrolase (IMPCH) activity, respectively (Fig. 7). The
`human 64-kDa AICARFT has been cloned, sequenced, and expressed and the two ac(cid:173)
`tivities have been expressed separately, a 39-kDa carboxy-terminal fragment containing
`AICARFT activity and a 25-kDa amino-terminal fragment containing IMPCH activity
`( 100). Although both AICARFT and GARFT utilize 10-CHO-H4PteGlun as the formyl
`donor, there is very little sequence homology between the two enzymes. However, there
`is a high degree of homology between AICARFT/IMPCH amino acid sequences from
`different sources.
`Polyglutamate forms of coenzymes and inhibitors are more effective with AICARFT
`than the monoglutamate forms. For example, methotrexate plus four glutamate residues
`is more than 2000-fold more inhibitory than methotrexate for AICARFT from MCF-7
`cells with 10-CHO-H4PteGlu 1 as substrate (101). With lO-CHO-H4PteGlu5 as substrate,
`however, the methotrexate polyglutamate was only sixfold more inhibitory than
`methotrexate. The true Kct values for folate and antifolate polyglutamates with AICARFT
`have not been detennined as they have for GARFT (98). 10-CH0-5,8,10-trideazapteroic
`acid ( 102) is reported to be an effective inhibitor of human AICARFT ( 103).
`
`:1
`
`'I
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`28
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`Kisliuk
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`9. METHIONYL tRNAret FORMYLTRANSFERASE
`In animal mitochondria and in prokaryotes, the initiation of protein synthesis utilizes
`formyl tRNAret ( 104). The tRNAret formyltransferase of animal mitochondria has not
`been studied extensively. 10-CHO-H4PteGlun is the formyl donor for the reaction for the
`E. coli enzyme which has a strong structural resemblence to E. coli GARFT (105). An
`alternative system to initiate protein synthesis in mammalian mitochondria must be
`available since cultured cells grow in folate-free RPMI 1640 medium supplemented with
`thymidine and inosine. The human dietary requirement for folate therefore results from
`in vivo metabolite deficiencies.
`
`10. FORMIMINOTRANSFERASE-CYCLODEAMINASE
`The two activities of this protein serve to catalyze the conversion of the formimino
`group, arising as formiminoglutamic acid in histidine catabolism, to formimino
`~PteGlun and then to 5,10-CH-~PteGlun· The porcine enzyme has been cloned, se(cid:173)
`quenced, and expressed (106). It is a 480-kDa tetramer of dimers that channels
`formiminoH4PteGlu 5 between the formiminotransferase and cyclodeaminase sites. Both
`activities require the formation of specific subunit interfaces ( 107).
`
`11. GLYCINE CLEAVAGE SYSTEM
`The glycine-cleavage system is a tetrafunctional enzyme complex found in mito(cid:173)
`chondria that converts glycine to C02, NH3, and 5,10-CHrH~teGlun (37,40). In the
`first step, P-protein, a pyridoxal phosphate enzyme, catalyzes the decarboxylation of
`glycine to C02 and an enzyme-bound methylamine group. In the second step, the en(cid:173)
`zyme-bound methylamine is transfered to lipoic acid (S-S) attached to H-protein. Dur(cid:173)
`ing this transfer, the lipoic acid is reduced to the SH level with the methylamine group
`still attached. In the third step, T-protein catalyzes the conversion of the attached methy(cid:173)
`lamine to NH3 and 5,10-CHz-H4PteGlun. The fourth step, the reoxidation of reduced
`lipoic acid by NAD is catalyzed by L-protein, which is dihydrolipoyl dehydrogenase, an
`enzyme shared among several mitochondrial a-keto acid dehydrogenases ( 108, 109 ).
`The four protein components, P,H, T, and L can be separated from one another by molec(cid:173)
`ular-sieve chromatography.
`The glycine cleavage system is the principle route for the catabolism of glycine in
`mammals and the system is stimulated by glucagon in rat hepatocytes ( 110 ). Metabolic
`leisons in the glycine cleavage system are associated with nonketotic hyperglycinemia,
`a condition causing severe neurological symptoms in neonates ( 11 l ). It is suggested that
`the glycine cleavage system plays a role in regulating glycine levels near N-methyl-o(cid:173)
`aspartate (NMDA) receptors in the central nervous system ( 112) that contain a glycine(cid:173)
`:;pecific site. Deficiency of the glycine cleavage system leads to increased levels of
`o-sro;rine in mammalian brain. o-serine occurs naturally in mammalian brain and binds to
`,:i.-" ~:belne cite of NMDA receptors ( 112) .
`. ~H•'odi11g 1-he P, r-t
`11;d L com1)Cn•-'>11;-.; ('" •h" h<J11F111 ·~iyr::ine de:wage
`(} 1 f) Cipnyhtr:d
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`,id1•~iH.,-'3 '
`,;_it»: ~t;<t. l!i ?. '_iii ( //)/'})
`i_:1-',:'t1
`
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`
`Chapter 2 I Folate Biochemistry
`
`29
`
`12. DIMETHYLGLYCINE DEHYDROGENASE
`AND SARCOSINE DEHYDROGENASE
`These two mitochondrial enzymes provide a pathway for the conversion of the
`methyl groups of choline, betaine, and methionine to 5,10-CHrH4PteGlun. Both rat
`liver enzymes contain covalently bound FAD, have kDa values near 100 and, as iso(cid:173)
`lated, contain H4PteGlu5 ( 113 ). Whereas sarcosine dehydrogenase is very specific for
`sarcosine, dimethylglycine dehydrogenase shows activity with many N-methyl com(cid:173)
`pounds including sarcosine ( 113 ). In the absence of H4PteGlun or if the folate site is
`blocked chemically, both enzymes continue to oxidize methyl groups unabated, yield(cid:173)
`ing free formaldehyde stoichiometrically ( 114, 115 ). Dimethylglycine dehydrogenase
`from rat liver and from rabbit liver bind both H4PteGlu1 and H4PteGiu5 very tightly
`with Kd values< 1 µM (44,115). Rat liver dimethylglycine dehydrogenase has been
`cloned ( 116). The enzyme is present in highest amounts in liver and kidney, but low
`levels are found in many tissues (117). FAD spontaneously binds covalently to rat
`dimethylglycine dehydrogenase and this binding aids in protein folding and mito(cid:173)
`chondrial import ( 118). Sarcosinemia is found in mice lacking sacrcosine dehydroge(cid:173)
`nase (119).
`
`13. FOLYLPOLY--y-GLUTAMATE SYNTHETASE
`Folylpoly-'Y-glutamate synthetase (FPGS) catalyzes the MgATP and K+-dependent
`attachment of glutamate residues to the 'Y-position of folates and folate analog (4). Cells
`lacking this enzyme cannot retain folates after their transport through the cell membrane
`and therefore cannot grow. FPGS activity in cells controls the level of folate polygluta(cid:173)
`mates in cells as well as the glutamate-chain length. Most folate enzymes have a higher
`affinity for polyglutamate forms of folate coenzymes and folate analogs. FPGS is found
`in the mitochondria and in the cytosol. Mitochondrial folate accumulation and cytosolic
`folate accumulation require the activity of mFPGS and cFPGS, respectively. However,
`pteroyltriglutamates synthesized in mitochondria can move to the cytoplasm and func(cid:173)
`tion there, whereas the reverse does not occur, indicating a unidirectional flow of mito(cid:173)
`chondrial folate triglutamates. Cells lacking mFPGS can synthesize thymidylate and
`purine nucleotides in the cytoplasm but require glycine for growth. Cells lacking cFPGS
`require thymine and purines for growth (methionine is routinely added to tissue-culture
`media) because the mitochondrial Glu chain lengths are longer than three and cannot
`pass into the cytosol. FPGS activity is increased in proliferating tissues and activity as
`well as mRNA levels increase after mitogen stimulation and decline during differentia(cid:173)
`tion.
`'n1e 60-kDa human FPGS has been cloned, sequenced, and expressed. A single gene
`with an alternative splice site codes for cytosolic and mitochondrial FPGS, the mito(cid:173)
`d1ondriaJ transc1ipt coding for a 42-residue amino-terminal leader sequence ( 120-123 ).
`H;PteGlun and I O-CHO-H4PteGlu 11 are much better substrates than the corresponding
`PteGJu. 5-CHO-H4PteGlu. and 5-CH3-H.1PteGlu derivatives ( 121 ). Thus, under concli(cid:173)
`tions in which methionine synthase activity is low, 5-CHrH4PteGlu 1 the major circu(cid:173)
`hting form nf folare produced in the liver is poorly polyglutarnybted and is not retained
`llh.~r eme-ring cells, leading to folace coenzyme deficiency.
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`Lowered expression of FPGS is associated with resistance to polyglutamylatable an(cid:173)
`tifolates (124).
`
`14. GLUTAMYL HYDROLASE
`)'-Glutamyl hydrolase (GH) catalyzes the hydrolytic cleavage of )'-linked polygluta(cid:173)
`mates ( 125). A role for GH in regulating the levels of ptereoylpolyglutamates in cells is
`indicated since cells expressing high levels of this enzyme show resistance to the poly(cid:173)
`glutamylatable antifolate DDATHF (126,127). The levels of methotrexate polygluta(cid:173)
`mates in human blast cells in vivo can be related to their sensitivity to treatment with
`methotrexate (128). The extent of accumulation of methotrexate polyglutamates has
`been attributed to the relative activities of FPGS and GH ( 129).
`The gene encoding human GH has been cloned, sequenced, and expressed ( 130). The
`35-kDa protein product has four potential asparagine-containing glycoyslation sites and
`is a glycoprotein when purified from tissues. Human GH shows 74% homology with rat
`GH. However the two enzymes show a different pattern of poly glutamate products with
`4-NHr 10-CH3PteGlu5 as a substrate. Human GH behaves like an exopeptidase, yield(cid:173)
`ing a series of products containing from one to four Glu residues, whereas the rat enzyme
`is an endopeptidase yielding 4-NHrlO-CH3PteGlu1 (methotrexate) as the product. GH
`is found in lysosomes that have a transport system for methotrexate polyglutamates
`( 131). GH is also excreted from tumor cells (132). Prostate-specific membrane antigen
`has GH activity ( 133 ).
`
`15. CONCLUSIONS
`Advances in studies of the genes encoding folate enzymes are empowering investi(cid:173)
`gators with knowledge of the expression of these genes in specific tissues, and tumors,
`during the cell cycle and during development. Further development of mathematical
`models of folate and antifolate transport and metabolism will aid in predicting the con(cid:173)
`sequences of inhibiting a given enzyme or combination of enzymes. The interaction of
`folate enzymes with messenger RNA, the phosphorylation of TS, the potential role of
`polyamines as regulators, mechanisms of antifolate-induced apoptosis, and levels of
`DNA methylation are examples of exciting phenomena that could aid the understanding
`of antifolate selectivity. We eventually should be able to address such problems as:
`
`I. Why do the target cells involved in methotrexate treatment of psoriasis or of rheumatoid
`arthritis not become resistant to methotrexate?
`2. What is the metabolic basis of methotrexate selectivity in the treatment of choriocarci-
`noma?
`3. What is the metabolic basis of the effect of diurnal rhythms on antifolate sensitivity?
`4. What is the basis of lipophilic, nonpolyglutamylatable antifolate antitumor selectivity?
`5. Why is methotrexate toxic to the liver where cells are not dividing?
`6. What folate system is pa1ticularly sensitive to folate deprivation in the genesis of neural
`tube defects?
`!. How can agents superior to methotrexate be designed bcised on knowledge of folate a11d
`:1111ifo!ate metabolism, .;;nzymes, and pharmacology?
`'-\f;::: 1hi~~ .. ~ 1~G 1T~bj11nlions \~{ fol0,i.17'S nnd 211:~