A major detoxification mechanism of the cell involves the glutathione transferase (GST)‐catalyzed formation of glutathione (GSH) conjugates with various xenobiotics Based on the same mechanism, GST overexpression may lead to multidrug resistant phenotypes Therefore, several compounds with inhibitory potency against GSTs have been developed as potential tools fortackling GST-­‐attributed MDR. Several individual compounds and prodrugs have been proposed as GST‐inhibiting substances. In addition, GSH analogues have been considered as specific GST inhibitors, with particular attention been directed towards the synthesis of GSH analogues stable against γ‐glutamyltranspeptidase (γGT) and peptidases, as GST inhibitors

Clonis, Yannis

Cordopatis, Paul

Georgakis, Nikolaos

Labrou, Nikolaos

Lamari, Fotini

Pairas, Giorgos

Pappa, Eleni V.

Tsoungas, Petros

Zompra, Aikaterini A.

Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

SYNTHESIS	  OF	  GLUTATHIONE	  ANALOGUES	  AND	  SCREENING	  AS	  SUBSTRATES	  &	  INHIBITORS	  FOR	  HUMAN	  GLUTATHIONE	  TRANSFERASE	  P1-­‐1	  Eleni	  V.	  Pappa1,	  Aikaterini	  A.	  Zompra1,	  Nikolaos	  Georgakis2,	  Giorgos	  Pairas1,	  Fo;ni	  Lamari1,	  	  Petros	  Tsoungas3,	  Nikolaos	  	  Labrou2,	  Yannis	  Clonis2	  and	  Paul	  Cordopa;s1	  	  	  	  The	  role	  of	  GSTs	  in	  medicine:	  A	  major	  detoxiﬁca;on	  mechanism	  of	  the	  cell	  involves	  the	  glutathione	  transferase	  (GST)-­‐catalyzed	  forma;on	  of	  glutathione	  (GSH)	  conjugates	  with	  various	  xenobio;cs	  [1].	  Based	  on	  the	  same	  mechanism,	  GST	  overexpression	  may	  lead	  to	  mul;drug	  resistant	  phenotypes	  [2].	  Therefore,	  several	  compounds	  with	  inhibitory	  potency	  against	  GSTs	  have	  been	  developed	  as	  poten;al	  tools	  fortackling	  GST-­‐aVributed	  MDR	  [3-­‐8].	  Several	  individual	  compounds	  and	  prodrugs	  have	  been	  proposed	  as	  GST-­‐inhibi;ng	  substances.	  In	  addi;on,	  GSH	  analogues	  have	  been	  considered	  as	  speciﬁc	  GST	  inhibitors,	  with	  par;cular	  aVen;on	  been	  directed	  towards	  the	  synthesis	  of	  GSH	  analogues	  stable	  against	  γ-­‐glutamyltranspep;dase	  (γGT)	  and	  pep;dases,	  as	  GST	  inhibitors	  [9-­‐11].	  Aim	  of	  the	  present	  work:	  we	  aimed	  to	  iden;fy	  GSH-­‐analogues	  that	  discriminate	  between	  the	  expression	  products	  of	  the	  polymorphic	  human	  GSTP1	  gene	  locus	  involved	  in	  MDR.	  	  These	  proteins,	   designated	   as	   hGSTP1*A	   (Ile104/Ala113),	   hGSTP1*B(Val104/Ala113)	   and	   hGSTP1*C	   (Val104/Val113),	   are	   expressed	   diﬀeren;ally	   in	   normal	   and	   cancer	   cells,	   also	   metabolizing	  diﬀeren;ally	  electrophilic	  substrates,	  including	  chemotherapeu;c	  agents	  [12].	   What	  we	  did:	  We	   synthesized	  25	  GSH	  analogues,	  using	   solid	  phase	   technology,	   and	   studied	   them	  with	   the	  human	  GSTP1	  gene	  products.	   In	  par;cular,	  we	   iden;ﬁed	  GSH	  analogues	  displaying	  selec;ve	  reac;vity	  with	  the	  hGSTP1	  allozymes.	  These	  analogues	  may	  prove	  useful	  tools	  for	  the	  selec;ve	  probing	  of	  the	  hGSTP1	  ac;vi;es	  as	  well	  as	  for	  the	  design	  of	  novel	  conjugates	  bearing	  a	  GSH-­‐mime;c	  moiety	  linked	  to	  an	  hGST-­‐inhibi;ng	  heterocyclic	  one. RESULTS 1Department	  of	  Pharmacy,	  University	  of	  Patras,	  Rion,	  Greece.	  	  2Laboratory	  of	  Enzyme	  Technology,	  Department	  of	  Biotechnology,	  Agricultural	  	  	  University	  of	  Athens,	  Athens,	  Greece.	  	  3Department	  of	  Biochemistry,	  Hellenic	  Pasteur	  InsGtute,	  Athens,	  Greece.	  INTRODUCTION MATERIALS AND METHODS CONCLUSIONS  SelecOon	  of	  GSH	  analogues	  with	  human	  GSTP1-­‐1	  acOviOes	  Table	  3	  summarizes	  GSH	  analogues	  able	  to	  discriminate	  between	  the	  three	  hGSTP1-­‐1	  allozymes,	  by	  ac;ng	  as	  selec;ve	  substrates	  (data	  taken	  from	  Table	  2).	  Analogues	  II,	  IV	  and	  XIII	  are	  substrates	  only	  with	  allozyme	  A.	  Analogues	  V	  and	  XVI	  are	  substrates	  only	  with	  allozyme	  C.	  Analogues	  III	  and	  XIV	  are	  weak	  substrates	  with	  both	  allozymes	  A	  and	  C.	  	  Therefore,	  it	  appears	  that	  certain	  GSH	  analogues,	  from	  groups	  A	  and	  C	  (Table	  1),	  act	  selec;vely	  as	  substrates	  for	  either	  A	  or	  C	  allozymes	  or	  both	  of	  them,	  however,	  no	  analogue	  showed	  selec;vity	  exclusively	  with	  allozyme	  B.	  This	  is	  the	  ﬁrst	  report	  on	  this	  mode	  of	  behavior	  for	  GSH	  analogues.	  The	  engagement	  of	  molecular	  modeling	  and	  dynamic	  approaches	  may	  ra;onalize	  these	  encouraging	  ﬁndings.	  Nevertheless,	  the	  iden;ﬁed	  GSH	  analogues	  of	  Table	  3	  may	  prove	  useful	  for	  the	  selec;ve	  probing	  of	  the	  hGSTP1	  allozyme	  ac;vi;es	  and	  the	  design	  of	  novel	  conjugate	  inhibitors	  against	  hGSTP1s.	  	  Pep$de	  Synthesis	  	  25	  new	  pep;des	  analogues	  of	  GSH	  (Table	  1)	  were	  synthesized	  manually	   following	   the	   Fmoc/tBu	   strategy	   [13].	   H-­‐Gly-­‐2-­‐2-­‐chlorotrityl	   resin	   (CTC)	   (0.5	   mmol/g	   capacity)	   or	   H-­‐Ala-­‐2CTC	  resin	  (0.5	  mmol/g	  capacity)	  was	  used	  as	  a	  solid	  support	  for	  the	  analogues	  possessing	  Gly	  or	  Ala	  at	  posi;on	  3	  while,	  CTC	  resin	  (1.2	   mmol/g	   capacity)	   was	   used	   for	   the	   synthesis	   of	   all	   the	  other	  pep;des.	  	  Puriﬁca;on	  of	  crude	  pep;des	  was	  achieved	  by	  semiprepara;ve	  HPLC	   	   on	   Supelcosil	   C18	   Analy;cal	   HPLC	   equipped	   with	   a	  Waters	   C18	   column	   at	   a	   1	   mL/min	   ﬂow	   rate	   with	   the	   same	  solvent	   system	   as	   in	   the	   prepara;ve	   HPLC	   produced	   single	  peaks	  with	  at	  least	  98%	  of	  the	  total	  peak	  integrals	  (integrated	  with	   Empower	   sohware).	   Electron	   Spray	   Ioniza;on-­‐Mass	  Spectrometry	  was	   in	  agreement	  with	   the	  expected	  mass.	  The	  structures	   and	   some	   physicochemical	   proper;es	   of	   the	   GSH	  analogues	  are	  shown	  in	  Table	  1.	  	  Screening	  the	  GSH	  analogues	  as	  substrates	  for	  hGSTP1-­‐1.	  The	  enzyme	  assay	  was	  performed	  by	  adding	  the	  ingredients	  in	  the	  following	   order	   (1	   mL	   assay	   volume):	   potassium	   phosphate	  buﬀer	   (100	   mM,	   pH	   6.5),	   an	   appropriate	   quan;ty	   of	   GSH	  analogue	  (in	  water	  or	  DMSO),	  equal	  to	  the	  Km(GSH)	  value	  for	  the	  par;cular	  allozyme	   (see	  Table	  2)	  and	  puriﬁed	  enzyme	   (≈	  0.06	  ΔΑ340/min).	  The	  mixture	  was	  incubated	  at	  25	  0C	  for	  1	  min,	  prior	  to	  adding	  1	  μmol	  of	  CDNB	  (in	  ethanol).	  The	  observed	  rate	  was	  used	   to	   calculate	   the	   frac;onal	   ac;vity	   (%),	   taking	   as	   100%	  ini;al	  ac;vity	  value	  the	  rate	  observed	  aher	  replacing	  the	  GSH	  analogue	  by	  an	  equal	  concentra;on	  of	  GSH.	  	  Screening	  the	  GSH	  analogues	  as	   inhibitors	  for	  hGSTP1-­‐1.	  The	  enzyme	   assay	  was	   implemented	   by	   adding	   the	   ingredients	   in	  the	  following	  order	  (1	  mL	  assay	  volume):	  potassium	  phosphate	  buﬀer	  (100	  mM,	  pH	  6.5),	  1	  μmol	  of	  CDNB	  (in	  ethanol)	  and	  25	  nmol	  of	  GSH	  analogue	  and	  puriﬁed	  enzyme	  (≈	  0.06	  ΔΑ340/min).	  The	  mixture	  was	  incubated	  at	  25	  0C	  for	  1	  min,	  prior	  to	  adding	  an	  appropriate	  quan;ty	  of	  GSH	  (in	  water),	  equal	  to	  the	  Km(GSH)	  value	   for	   the	   par;cular	   allozyme	   (see	  Table	   2).	   The	   observed	  rate	  was	  used	  to	  calculate	  the	  remaining	  ac;vity	  (%),	  taking	  as	  100%	  ini;al	  ac;vity	  value	  the	  rate	  observed	  aher	  replacing	  the	  GSH	  analogue	  by	  an	  equal	  volume	  of	  buﬀer	  or	  DMSO.	  	  REFERENCES	  1.	  Oakley,	  A.	  	  Drug	  Metab.	  Rev.	  2011,	  43,	  138.	  	  2.	  Morrow,	  C.S.,	  Smitherman	  P.K.,	  Townsend	  A.J.	  Biochem.	  Pharmacol.,	  1998,	  56,	  1013.	  3.	  Sau,	  A.;	  Trengo,	  F.	  P.;	  Valen;no,	  F.;	  Federici,	  G.;	  Caccuri,	  A.	  M.	  Arch.	  Biochem.	  Biophys.	  2010,	  500,	  116.	  4.	  	  Mahajan,	  S.;	  Atkins,	  W.	  M.	  Cell.	  Mol.	  Life	  Sci.	  2005,	  62,	  1221.	  5.	  Adang,	  A.	  E.;	  Brussee,	  J.;	  van	  der	  Gen,	  A.;	  Mulder,	  G.	  J.	  J.	  Biol.	  Chem.	  1991,	  266,	  830.	  6.	  Koutsoumpli,	  G.	  E.,	  Dimaki,	  V.	  D.,	  Thireou,	  T.	  N.,	  Eliopoulos,	  E.	  E.,	  Labrou,	  N.	  E.,	  Varvounis,	  G.	  I.,	  Clonis,	  Y.	  D.	  	  J.	  Med.	  Chem.	  2012,	  55,	  6802.	  7.	  Zoi,	  O.G.;	  Thireou,	  T.N.;	  Rinotas,	  V.E.	  ;	  Tsoungas,	  P.G.;	  Eliopoulos,	  E.E.;	  Douni,	  E.K.;	  Labrou,	  N.E.	  and	  Clonis,	  Y.D.	  J.	  Biomol.	  Screen.,	  2013,	  18,	  1092.	  	  8.	  Perperopoulou	  F.D.,	  Tsoungas	  P.G.,	  Thireou	  T.N.,	  Rinotas	  V.E.,	  Douni	  E.K.,	  Eliopoulos	  E.E.,	  Labrou	  N.E.	  and	  Clonis	  Y.D.	  Bioorg.	  Med.	  Chem.,	  2014,	  in	  press;	  DOI:	  10.1016/j.bmc.2014.06.007.	  9.	  Cacciatore,	  I.;	  Caccuri,	  A.	  M.;	  Cocco,	  A.;	  De	  Maria,	  F.;	  Di	  Stefano,	  A.;	  Luisi,	  G.;	  Pinnen,	  F.;	  Ricci,	  G.;	  Sozio,	  P.;	  Turella,	  P.	  Amino	  Acids	  2005,	  29,	  255.	  10.	  Burg,	  D.;	  Riepsaame,	  J.;	  Pont,	  C.;	  Mulder,	  G.;	  van	  der	  Water,	  B.	  Biochem.	  Pharmacol.	  2006,	  71,	  268.	  11.	  Razza,	  A.;	  Galili,	  N.;	  Smith,	  S.;	  Godwin,	  J.;	  Lancet,	  J.;	  Melchert,	  M.;	  Jones,	  M.;	  Keck,	  J.	  G.;	  Meng,	  L.;	  Brown,	  G.	  L.;	  List,	  A.	  Blood,	  2009,	  113,	  6533.	  12.	  Ali-­‐Osman	  F.,	  Akande	  O.,	  Antoun	  G.,	  Mao	  J.X.,	  Buolamwini	  J.	  J.	  Mol.	  Chem.,	  1997,	  272,	  10004.	  13.	  P.	  Lloyd-­‐Williams,	  F.	  Albericio,	  E.	  Giralt,	  Chemical	  Approaches	  to	  the	  Synthesis	  of	  Pep;des	  and	  Proteins,	  CRC,	  Boca	  Raton,	  FL	  1997.	  PepOde	  Code	  Sequence	  	  RetenOon	  Ome;	  tR	  (min)*	  Yield	  (%)	  GSH	  	   γ-­‐Glu-­‐Cys-­‐Gly	   2.36	   -­‐	  Group	  A	   I	  	   pGlu-­‐Cys-­‐Gly	  	   2.86	   84	  II	  	   Tic-­‐Cys-­‐Gly	  	   4.72	   72	  III	  	   Sar-­‐Cys-­‐Gly	  	  	   1.48	   85	  IV	  	   Hyp-­‐Cys-­‐Gly	  	   1.52	   82	  V	  	   Ac-­‐γ-­‐Glu-­‐Cys-­‐Gly	   3.11	   92	  VI	  	  	   NMeGlu-­‐Cys-­‐Gly	  	   1.55	   81	  VII	   Pro-­‐Cys-­‐Gly	  	   1.45	   93	  Group	  B	   VIII	  	   γ-­‐Glu-­‐Met-­‐Gly	   3.55	   89	  IX	   γ-­‐Glu-­‐Thi-­‐Gly	   4.91	   87	  X	   γ-­‐Glu-­‐Cys(tButhio)-­‐Gly	   7.35	   93	  XI	   γ-­‐Glu-­‐Cys(Acm)-­‐Gly	   1.52	   86	  XII	   γ-­‐Glu-­‐NMeCys-­‐Gly	  	   1.47	   89	  Group	  C	   XIII	   γ-­‐Glu-­‐Cys-­‐Nva	   4.75	   91	  XIV	   γ-­‐Glu-­‐Cys-­‐Nle	   6.07	   88	  XV	   γ-­‐Glu-­‐Cys-­‐Sar	  	   3.03	   91	  XVI	   γ-­‐Glu-­‐Cys-­‐Aib	   3.80	   82	  XVII	   γ-­‐Glu-­‐Cys-­‐a-­‐t-­‐butyl-­‐Gly	   5.27	   91	  Group	  D	   XVIII	   pGlu-­‐Cys(Trt)-­‐Gly	   9.75	   68	  XIX	   Sar-­‐Cys(Trt)-­‐Gly	   7.94	   71	  XX	   Tic-­‐Cys-­‐Ala	  	   5.20	   88	  XXI	   Hyp-­‐Cys-­‐Ala	  	   2.11	   87	  XXII	   Gly-­‐Cys-­‐Hyp	   2.12	   79	  XXIII	   Tic-­‐Cys-­‐Aib	   5.77	   86	  XXIV	   Tic-­‐Cys(Acm)-­‐Gly	   4.51	   92	  XXV	   Tic-­‐Cys-­‐Sar	   4.94	   93	  pepOde	  code	  	  hGSTP1-­‐A	   hGSTP1-­‐B	   hGSTP1-­‐C	  KmGSH	  =	  0.3	  ±	  0.02	  mM	   KmGSH	  =	  0.074	  ±	  0.008	  mM	   KmGSH	  =	  0.15	  ±	  0.01	  mM	  Inhibitor;	  RA	  (%)a	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  =	  0.3	  mM,	  [analogue]	  =	  25	  μΜ)	  Substrate;	  	  FA	  (%)b	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  or	  [analogue]=	  0.3	  mM)	  Inhibitor;	  RA	  (%)	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  =	  0.074	  mM,	  [analogue]	  =	  25	  μΜ)	  Substrate;	  	  FA	  (%)	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  or	  [analogue]=	  0.074	  mM)	  Inhibitor;	  RA	  (%)	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  =	  0.15	  mM,	  [analogue]	  =	  25	  μΜ)	  Substrate;	  	  FA	  (%)	  (Assays:	  [CDNB]	  =	  1	  mM,	  [GSH]	  or	  [analogue]=	  0.15	  mM)	  Group	  A	  I	   NAc	   11.5±	  10.4	   79.2	  ±	  4.8	   0	   80.0	  ±	  3.6	   0	  II	   NA	   41.3±	  2.9	  	   83.1	  ±	  1.5	   0	   78.0	  ±	  1.9	   0	  III	   NA	   15.8	  ±	  3.8	  	   83.5	  ±	  1.6	   0	   NA	   7.0	  ±	  14.0	  	  IV	   NA	   39.5	  ±	  3.8	   89.9	  ±	  3.2	   0	   88.0	  ±	  1.5	   0	  V	   102.3	  ±	  2.8	   0	   85.3	  ±	  1.2	   0	   NA	   10.1	  ±	  0.7	  VI	   NA	   3.9±	  4.7	   81.0	  ±	  1.4	   0	   72.3	  ±	  3.5	   0	  VII	   NA	   21.6±	  4.0	   82.6	  ±	  3.0	   0	   NA	   10.4	  ±	  5.0	  Group	  B	  VIII	   79.4±	  1.1	   NA	   70.1	  ±	  0.8	   NA	   70.7	  ±	  2.6	   NA	  IX	   67.4±	  4.2	  	   NA	   92.5	  ±	  3.9	   NA	   67.4	  ±	  3.6	  	   NA	  X	   85.6	  ±	  0.4	   NA	   79.5	  ±	  3.7	   NA	   81.0	  ±	  1.2	   NA	  XI	   85.2±	  1.7	   NA	   64.0	  ±	  2.9	   NA	   59.4	  ±	  2.7	   NA	  XII	   100.0	   0	   100.0	   0	   100.0	   0	  Group	  C	  XIII	   NA	   16.0±	  2.7	   80.1	  ±	  4.8	   0	   85.2	  ±	  0.1	   0	  XIV	   NA	   26.4±	  2.7	   80.9	  ±	  4.1	   0	   NA	   11.6	  ±	  5.4	  XV	   NA	   17.0	  ±	  4.3	   NA	   60.8	  ±	  3.3	   NA	   67.7	  ±	  1.2	  XVI	   100.0	   0	   82.7	  ±	  1.9	   0	   NA	   15.8	  ±	  3.5	  XVII	   100.0	   0	   80.4	  ±	  5.0	   0	   79.1	  ±	  3.7	   0	  Group	  D	  XVIII	   82.3±	  0.2	  	   NA	   95.8	  ±	  2.1	   NA	   99.1	  ±	  2.3	   NA	  XIX	   100.0	   NA	   102.3	  ±	  5.0	   NA	   92.2	  ±	  4.4	   NA	  XX	   100.0	   0	   100.0	  ±	  1.1	   0	   98.1	  ±	  2.6	  	   0	  XXI	   98.7±	  2.0	  	   0	   81.3	  ±	  1.0	   0	   76.6	  ±	  3.5	  	   0	  XXII	   87.4±	  0.2	   0	   81.6	  ±	  5.0	   0	   65.9	  ±	  3.1	   0	  XXIII	   80.1±	  4.8	   0	   75.2	  ±	  5.0	   0	   84.2	  ±	  2.1	   0	  XXIV	   68.5±	  4.5	   0	   84.2	  ±	  5.0	   0	   68.6	  ±	  2.0	  	   0	  XXV	   53.3±	  	  0.0	   0	   72.0	  ±	  2.2	   0	   74.6	  ±	  3.6	   0	  GSH	  analogue	   hGSTP1-­‐1	  allozyme	  A	   B	   C	  II	  	  	  	  	  	  	  Tic-­‐Cys-­‐Gly	   +	  (41	  %)	   -­‐	   -­‐	  III	  	  	  	  	  Sar-­‐Cys-­‐Gly	   +	  (16	  %)	   -­‐	   +	  (7	  %)	  IV	  	  	  	  	  Hyp-­‐Cys-­‐Gly	   +	  (40	  %)	   -­‐	   -­‐	  V	  	  	  	  	  	  Ac-­‐γ-­‐Glu-­‐Cys-­‐Gly	   -­‐	   -­‐	   +	  (10	  %)	  XIII	  	  γ-­‐Glu-­‐Cys-­‐Nva	   +	  (16	  %)	   -­‐	   -­‐	  XIV	  	  	  γ-­‐Glu-­‐Cys-­‐Nle	   +	  (26	  %)	   -­‐	   +	  (12	  %)	  XVI	  	  	  γ-­‐Glu-­‐Cys-­‐Aib	   -­‐	   -­‐	   +	  (16	  %)	  Table	  3.	  SelecOvity	  of	  GSH	  analogues	  (as	  substrates)	  against	  the	  	  hGSTP1-­‐1	  allozymes	  (%	  values	  are	  relaOve	  to	  GSH	  as	  substrate) Table	  1.	  Yield	  of	  synthesis	  and	  physicochemical	  	  properOes	  of	  GSH	  analogues	  Table	  2.	  Screening	  of	  GSH-­‐analogues	  as	  substrates	  and	  inhibitors	  for	  hGSTP1-­‐1	  allozymes.	  	  

Synthesis of glutathione analogues and screening as substrates & inhibitors for human glutathione transferase p1‐1

http://repository.edulll.gr/edulll/bitstream/10795/2622/2/2622_Poster%2010.pdf

Synthesis of glutathione analogues and screening as substrates & inhibitors for human glutathione transferase p1‐1

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Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)

Synthesis of glutathione analogues and screening as substrates & inhibitors for human glutathione transferase p1‐1

Abstract

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Full text

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Digital Repository of Hellenic Managing Authority of the Operational Programme &quot;Education and Lifelong Learning&quot; (EDULLL)

Digital Repository of Hellenic Managing Authority of the Operational Programme "Education and Lifelong Learning" (EDULLL)