Structure-based Inhibitor Design for an Enzyme That Binds Different Steroids

Human type 5 17β-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30Aå. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9 ± 1.4 nm, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy. Human type 5 17β-hydroxysteroid dehydrogenase plays a crucial role in local androgen formation in prostate tissue. Several chemicals were synthesized and tested for their ability to inhibit this enzyme, and a series of estradiol derivatives bearing a lactone on the D-ring were found to inhibit its activity efficiently. The crystal structure of the type 5 enzyme in complex with NADP and such a novel inhibitor, EM1404, was determined to a resolution of 1.30Aå. Significantly more hydrogen bonding and hydrophobic interactions were defined between EM1404 and the enzyme than in the substrate ternary complex. The lactone ring of EM1404 accounts for important interactions with the enzyme, whereas the amide group at the opposite end of the inhibitor contributes to the stability of three protein loops involved in the construction of the substrate binding site. EM1404 has a strong competitive inhibition, with a Ki of 6.9 ± 1.4 nm, demonstrating 40 times higher affinity than that of the best inhibitor previously reported. This is observed despite the fact that the inhibitor occupies only part of the binding cavity. Attempts to soak the inhibitor into crystals of the binary complex with NADP were unsuccessful, yielding a structure with a polyethylene glycol fragment occupying the substrate binding site. The relative crystal packing is discussed. Combined studies of small molecule inhibitor synthesis, x-ray crystallography, enzyme inhibition, and molecular modeling make it possible to analyze the plasticity of the substrate binding site of the enzyme, which is essential for developing more potent and specific inhibitors for hormone-dependent cancer therapy. According to the American Cancer Society, prostate cancer is the most common malignant tumor, excluding skin cancers, in American men. It is estimated that ∼232,090 new cases of prostate cancer will be diagnosed in the US during 2005 with 30,350 deaths (www.cancer.org). The growth and function of the prostate is dependent on androgens, which play important roles in the pathogenesis of prostate cancer (1Lopez-Otin C. Diamandis E.P. Endocr. Rev. 1998; 19: 365-396Crossref PubMed Scopus (182) Google Scholar). Androgen withdrawal triggers the programmed cell apoptosis in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen withdrawal; however, they do retain the cellular machinery necessary to activate the apoptotic cascade when sufficiently damaged by exogenous agents (2Denmeade S.R. Lin X.S. Isaacs J.T. Prostate. 1996; 28: 251-265Crossref PubMed Scopus (389) Google Scholar). In prostate cancer, the balance between cell proliferation and programmed cell death is lost, thus more cell proliferation results in net cell growth. An important finding in prostate cancer research is that ∼50% of dihydrotestosterone, the most potent natural androgen, remains in the prostatic tissue of patients who have undergone surgical or chemical castration (3Labrie F. Dupont A. Belanger A. Giguere M. Lacoursiere Y. Emond J. Monfette G. Bergeron V. J. Steroid Biochem. 1985; 23: 833-841Crossref PubMed Scopus (140) Google Scholar). In fact, prostatic tissue is able to efficiently transform the inactive adrenal steroid precursor dehydroepiandrosterone, into the active androgen dihydrotestosterone, in a new androgen biosynthetic pathway in which human type 5 17β-hydroxysteroid dehydrogenase (17β-HSD5) 4The abbreviations used are: 17β-HSD5, 17β-hydroxysteroid dehydrogenase type 5; AKR, aldoketo reductase; PEG, polyethylene glycol; EM1404, 3-carboxamido-1,3,5-(10)-estratrien-17(R)-spiro-2′-(5′,5′-dimethyl-6′-oxo)tetrahydropyran, C25H30O3N; E2, estradiol. 4The abbreviations used are: 17β-HSD5, 17β-hydroxysteroid dehydrogenase type 5; AKR, aldoketo reductase; PEG, polyethylene glycol; EM1404, 3-carboxamido-1,3,5-(10)-estratrien-17(R)-spiro-2′-(5′,5′-dimethyl-6′-oxo)tetrahydropyran, C25H30O3N; E2, estradiol. is involved (4Pelletier G. Luu-The V. Tetu B. Labrie F. J. Histochem. Cytochem. 1999; 47: 731-738Crossref PubMed Scopus (92) Google Scholar, 5Pelletier G. Li S. Luu-The V. Tremblay Y. Belanger A. Labrie F. J. Endocrinol. 2001; 171: 373-383Crossref PubMed Scopus (81) Google Scholar, 6El-Alfy M. Luu-The V. Huang X.F. Berger L. Labrie F. Pelletier G. Endocrinology. 1999; 140: 1481-1491Crossref PubMed Google Scholar, 7Labrie F. Cusan L. Gomez J.L. Candas B. Belanger A. Luu-The V. Labrie C. Simard J. Med. Sci. (Paris). 2003; 19: 910-919Crossref PubMed Scopus (4) Google Scholar). In practice, hormonal therapy with combined androgen blockade could lead to even longer term control of localized prostate cancer (8Labrie F. Dupont A. Belanger A. Cusan L. Lacourciere Y. Monfette G. Laberge J.G. Emond J.P. Fazekas A.T. Raynaud J.P. Husson J.M. Clin. Invest. Med. 1982; 5: 267-275PubMed Google Scholar, 9Labrie F. Candas B. Gomez J.L. Cusan L. Urology. 2002; 60: 115-119Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). 17β-HSD5 has also been known as type 2 3α-HSD (10Lin H.K. Jez J.M. Schlegel B.P. Peehl D.M. Pachter J.A. Penning T.M. Mol. Endocrinol. 1997; 11: 1971-1984Crossref PubMed Google Scholar), prostaglandin 11-ketoreductase (prostaglandin F synthetase) (11Matsuura K. Shiraishi A. K. Y. M. S. J. Biochem. 1998; PubMed Scopus Google Scholar), and dehydrogenase Penning T.M. 1998; PubMed Scopus Google Scholar). It is a of the aldoketo with a of J.M. Schlegel B.P. M. Penning T.M. Biochem. J. 1997; PubMed Scopus Google Scholar, J.M. Penning T.M. Biochem. 1997; PubMed Scopus Google and was human prostate and (10Lin H.K. Jez J.M. Schlegel B.P. Peehl D.M. Pachter J.A. Penning T.M. Mol. Endocrinol. 1997; 11: 1971-1984Crossref PubMed Google Scholar, Huang X.F. Luu-The V. Endocrinology. 1999; 140: PubMed Scopus Google Scholar). It has been that this enzyme has a activity that a to potent This enzyme has been localized in human prostate and it has been to to local androgen formation in prostate (4Pelletier G. Luu-The V. Tetu B. Labrie F. J. Histochem. Cytochem. 1999; 47: 731-738Crossref PubMed Scopus (92) Google Scholar, 5Pelletier G. Li S. Luu-The V. Tremblay Y. Belanger A. Labrie F. J. Endocrinol. 2001; 171: 373-383Crossref PubMed Scopus (81) Google Scholar, 6El-Alfy M. Luu-The V. Huang X.F. Berger L. Labrie F. Pelletier G. Endocrinology. 1999; 140: 1481-1491Crossref PubMed Google Scholar, H.K. S. Penning T.M. PubMed Scopus Google Scholar, Y. M. M. K. Y. A. S. S. Y. Endocr. PubMed Scopus (81) Google Scholar). The enzyme during to was by the crystal of 17β-HSD5 in complex with or on Human and 5 2002; Google Scholar, M. Labrie F. Lin Mol. Endocrinol. PubMed Scopus Google Scholar). The 11-ketoreductase activity of this enzyme has also been in with prostaglandin and inhibitors J. K. F. PubMed Scopus Google Scholar, J.P. Cancer PubMed Scopus Google Scholar). of this enzyme could lead to the of a which cell and results in apoptosis in cell and M. G. C. Mol. 2002; PubMed Scopus (140) Google Scholar, M. J.P. Cancer 2003; Google Scholar). The that 17β-HSD5 was to human and Luu-The V. 1999; PubMed Google Scholar). 17β-HSD5 was also to be in prostate cancer J. M. Prostate. 2002; PubMed Scopus Google Scholar). In a 17β-HSD5 was in of cells prostate cancer and was with the of the Y. M. M. K. Y. A. S. S. Y. Endocr. PubMed Scopus (81) Google Scholar). This that 17β-HSD5 be involved in the of the local of and in prostate tissue J. J. Biochem. 28: PubMed Scopus Google Scholar, C. M. C. J. Luu-The V. Simard J. Labrie F. J. Steroid Biochem. Mol. PubMed Scopus Google Scholar), by of the the prostate cancer cell as a cell to the of prostate cancer, it has been that in androgen and in the cells during this in the 17β-hydroxysteroid dehydrogenase activity was whereas the activity 17β-HSD5 and to M. S. L. Li Y. A. A. V. J. Steroid Biochem. Mol. PubMed Scopus Google Scholar). It has also been that 17β-HSD5 was higher in than in normal tissue of by tissue and normal Li Y. A. Y. Cancer PubMed Scopus Google Scholar). This that the ability of 17β-HSD5 to play important role in cancer It is also to that 17β-HSD5 plays important role in androgen in tissue in with M. B. J. Endocrinol. PubMed Scopus Google Scholar). to the of this enzyme, not only in prostate cancer research also in the of inhibitor this enzyme has been A. G. J. Med. 2002; PubMed Scopus Google Scholar, T.M. Jez J.M. Lin H.K. M. K. Mol. Endocrinol. 2001; 171: PubMed Scopus Google Scholar, Y. S. Penning T.M. Mol. PubMed Scopus Google Scholar, F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; Scholar, A. G. J. Mol. Endocrinol. 2001; 171: PubMed Scopus Google Scholar). The crystal structure of 17β-HSD5 is a necessary for The enzyme was in M. A. Lin 2002; PubMed Scopus Google Scholar), and in complex with or were determined on Human and 5 2002; Google Scholar, M. Labrie F. Lin Mol. Endocrinol. PubMed Scopus Google Scholar). crystal of in have been on Human and 5 2002; Google Scholar, J. K. F. PubMed Scopus Google Scholar, J.P. Cancer PubMed Scopus Google Scholar). The plasticity of its substrate binding site in and its in to the of this The plasticity of the as as and has been by Penning and previously T.M. Jez J.M. Lin H.K. M. K. Biochem. J. PubMed Scopus Google Scholar). that at the active site could be important for inhibitor have the active site of the enzyme in by the of inhibitors and in the In this the crystal structure of 17β-HSD5 in complex with a potent inhibitor EM1404 and structure with a of polyethylene glycol glycol in the active site. by EM1404 was also and the with inhibitors has been discussed. to the interactions between this potent inhibitor and its enzyme, 17β-HSD5, and to for the of inhibitor on the crystal and was in and by affinity and a as previously M. A. Lin 2002; PubMed Scopus Google Scholar). the protein was in of in the of The was by at with EM1404 was with 17β-HSD5 and to the of EM1404, it was in to a of and in to a of as a It was to complex of this into 2 of to a of The was times to by the EM1404 was which could be The was to the enzyme substrate binding site by the The protein was to The of the EM1404 ternary complex has been the and M. A. Lin 2002; PubMed Scopus Google Scholar). The and Human crystals were at the by of protein and a of enzyme in the binary complex crystals were with of EM1404 inhibitor and crystal were and of crystal of and cell of the resolution in of the resolution in and the of and the for the with and the and and excluding The of the of in EM1404, and PEG, and of the resolution in and the of and the for the with and the and and excluding The of the of in a new and of and were at the in were the on and 1997; PubMed Scopus Google Scholar). The in and of was a molecular the 1999; PubMed Scopus Google Scholar). to resolution was used in molecular The of the protein structure the complex in the group were used as the of and of were The of were a the molecular the 2003; PubMed Scopus Google and the PubMed Scopus Google were used to a was with to was on a to to were the and was by the K. 60: PubMed Scopus Google on The were with to The inhibitor was in the substrate binding site the An molecule was also the substrate and binding and was during the The was also used to into the In the were with hydrogen bonding have been in the the for structure is that for is was used for the enzyme in the of EM1404 at for its Ki 17β-HSD5, at in small has of The were in the of of NADP at and ± The for in the of and in the of and were were by the of enzyme to a of in the of the were and the was at by the with of The were by in and for a and by were with substrate The were with of to were used for substrate for 2 for for and for were in the inhibitor to of the enzyme activity the in F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; The were is the in the of substrate is the and and the substrate and inhibitor J. PubMed Scopus Google Scholar). The in the of inhibitor were as by the of the of which J. PubMed Scopus Google Scholar), on the of 2 were on the crystal structure for the complex EM1404 and the structure for the complex with the inhibitor were with the in the The were for the inhibitor EM1404 H.K. Jez J.M. Schlegel B.P. Peehl D.M. Pachter J.A. Penning T.M. Mol. Endocrinol. 1997; 11: 1971-1984Crossref PubMed Google was a chemical F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; it was to have in of ± nm, the best the inhibitors for The strong affinity of EM1404 is in the The ternary complex structure of was determined by molecular a the complex on Human and 5 2002; Google Scholar). structure is to the crystal M. Labrie F. Lin Mol. Endocrinol. PubMed Scopus Google Scholar, J. K. F. PubMed Scopus Google Scholar, J.P. Cancer PubMed Scopus Google Scholar), with a structure of the aldoketo Luu-The V. 1999; PubMed Google Scholar, J. M. Prostate. 2002; PubMed Scopus Google Scholar, J. J. Biochem. 28: PubMed Scopus Google Scholar, C. M. C. J. Luu-The V. Simard J. Labrie F. J. Steroid Biochem. Mol. PubMed Scopus Google Scholar, M. S. L. Li Y. A. A. V. J. Steroid Biochem. Mol. PubMed Scopus Google Scholar, Li Y. A. Y. Cancer PubMed Scopus Google Scholar, M. B. J. Endocrinol. PubMed Scopus Google Scholar, A. G. J. Med. 2002; PubMed Scopus Google Scholar, T.M. Jez J.M. Lin H.K. M. K. Mol. Endocrinol. 2001; 171: PubMed Scopus Google Scholar, Y. S. Penning T.M. Mol. PubMed Scopus Google Scholar), and to the and binding and The and for this ternary complex were and with of the in the most of the to The with the of most the NADP the EM1404 inhibitor, and it be that have and thus to be the the for the inhibitor EM1404 at the substrate binding site. The of EM1404 was with of whereas the for the protein is The is higher in the as with the The is observed in most for by ring accounts for the strong between the EM1404 and the in the lactone ring a strong hydrogen with The of has with the lactone The at the in the lactone ring also strong interactions with enzyme and the of the the of the amide group of EM1404 a hydrogen to the which to the of the amide group hydrogen with It is of to that the of has in it with in it a hydrogen with and with amide group of have been in interactions in and hydrogen bonding in of the in the inhibitor EM1404 of a steroid a lactone and amide In the substrate binding the lactone ring of EM1404 is at the of the substrate binding whereas the amide group is the of the The EM1404 binding is and has with a of The of the binding is and the of EM1404 is in this complex. the fact that the inhibitor occupies only part of the binding the inhibitor affinity at by which is by the hydrogen bonding and hydrophobic interactions The the of the binding is with hydrophobic which a to the of In the steroid of EM1404 is to the of with of and the three that the of It be that and hydrogen bonding the enzyme at the or at the of the binding and and of hydrogen and hydrophobic interactions were between the inhibitor EM1404 and 17β-HSD5 than found in the and The in the lactone ring a strong hydrogen with and is with group The in the lactone ring also a with group The at the in the lactone ring also strong interactions with enzyme and the of the The lactone ring of EM1404 a strong of hydrophobic interactions with the of and as as and a binding at the of the substrate binding site that to the lactone ring of The hydrogen the inhibitor the of the active site. the amide group of the end of EM1404, three more hydrogen were the is between the of the amide group of EM1404 to the which to the is between the of the amide group to a molecule which also a hydrogen to the thus the inhibitor and the the is between the of the amide group and It is to that has at the end of its as in have on the In the a of hydrogen with and the EM1404 amide group In the a of hydrogen with the group of the in with were on the in the which the most the the crystal for that the despite the fact that they to of as small as to as as The in the loops that the substrate binding site and to the three and the of and on by on is by and on by In the of and the is local at the active site were as for the the is The is also a role in the of the substrate binding In steroid androgens, and their with the ring as the their in on their function S. J. Endocrinol. PubMed Scopus Google Scholar, A. Huang Lin J. Full Text Full Text PDF PubMed Scopus Google Scholar). The in the binding site of 17β-HSD5 is when with the binding site of In the of the it is a hydrophobic and its structure in the complex with estradiol is the as the enzyme structure of for A. Labrie F. Lin 1996; PubMed Scopus Google Scholar). The type 5 enzyme the type enzyme by a and steroid binding site. This is estimated to be or in 17β-HSD5 ternary complex with or whereas only in complex A. M. Mol. Endocrinol. Scopus Google Scholar). This in complex is that in complex. and This of the 17β-HSD5 binding site the of in which in a of and to prostaglandin In the ternary complex with the steroid is as with the complex with This contributes to steroid in to the of 17β-HSD5 in both and prostate the structure as a complex were on the the of loops to binding in the substrate binding site bonding is to be the that the of and in the complex the of a hydrogen with that to the substrate binding site. In this the of is to more for the of and to the at the of the a the substrate binding more on Human and 5 2002; Google Scholar). In the of the complex hydrogen at the of the steroid at the binding site on Human and 5 2002; Google Scholar). is observed in the the of prostaglandin in hydrogen bonding with and J. K. F. PubMed Scopus Google Scholar). The is involved in a hydrogen with a molecule in a whereas the in a hydrogen bonding It has also been that prostaglandin and involved in a hydrogen bonding with even they have chemical J. K. F. PubMed Scopus Google Scholar). The of a in the the a complex of in the substrate binding site in of crystal This in to soak the inhibitor into the binary complex crystals with of EM1404 and and and the glycol be with at the of the substrate binding whereas the glycol the to the substrate binding site has possible be found with the the the substrate binding site have more to their the of the The interactions between the enzyme and hydrogen bonding interactions be In the observed only the fragment in the substrate binding site. the A. Lin J. 1996; Scopus Google Scholar), the ternary complex of and the results have been of crystals have the group and cells. into the crystal packing the substrate and found that the packing of in both crystal The for is in both could be the substrate molecule is and the of at the of the substrate binding site in both ternary The of is in more 5 by in the for to in the and the of EM1404 at the on the the to inhibitor the in the of inhibitor in a higher the on the control This a and competitive inhibition, in with The results also with the inhibitor binding in the substrate site by the Ki of 6.9 ± 1.4 was the by the of the on the of the which was thus determined to be more than 40 times than inhibitor in the demonstrating a strong enzyme by The of 17β-HSD5 for was determined as ± EM1404 affinity is higher than with the of EM1404 with the 17β-HSD5 binding of enzyme and molecular with the fact that the lactone ring of EM1404 accounts for the strong between the inhibitor and the inhibitors with a lactone ring EM1404, and have a higher affinity than F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; Scholar). is in with the of EM1404 in the chemical F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; Scholar), with the the inhibitors for A. G. J. Med. 2002; PubMed Scopus Google Scholar, T.M. Jez J.M. Lin H.K. M. K. Mol. Endocrinol. 2001; 171: PubMed Scopus Google Scholar, Y. S. Penning T.M. Mol. PubMed Scopus Google Scholar, F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; Scholar, A. G. J. Mol. Endocrinol. 2001; 171: PubMed Scopus Google Scholar). inhibitors into the crystal structure of complex the J. 1998; 19: Scopus Google Scholar). It is to that inhibitors a with the lactone ring at the of the binding as observed for the crystal structure not the for EM1404, and it that amide group at the contributes to the higher affinity of EM1404 to 17β-HSD5 than the of 5 to of that only human 17β-HSD5 has a at whereas in this is by a In the is in a strong hydrogen with the in the lactone ring of EM1404 and of human with human 17β-HSD5 in the inhibitor binding site that the of in will the of the lactone ring of EM1404 to the of the substrate binding that this will 17β-HSD5 inhibitor with a lactone the lactone ring will of inhibitors to the 17β-HSD5 and results lead to do inhibitor studies molecular modeling and as of the more the of of the 17β-HSD5 binding in the binding a the binding of the lactone and the binding of the as the only the structure occupies both The structure of the complex was with the to the between the of the found that the group of occupies a to the in the complex as in whereas the ring is the of the EM1404 lactone ring as in on this a new inhibitor is by the ring by a lactone The were into the active site the The is to a interactions with a The of the as observed by the molecular studies and for more is higher for the EM1404 complex than the new inhibitor whereas the protein the This that the inhibitor has a higher affinity with the EM1404, a potent inhibitor for 17β-HSD5 was a chemical F. Belanger A. S. Luu-The V. Y. L. of 5 and and for 1999; Scholar). the enzyme has been by with 17β-HSD5 as by the crystal The new has in of the inhibitor molecular the the of a combined of small molecule and protein both in the of structure and the of new The for the affinity of EM1404 the of a lactone ring at the D-ring to the binding the amide group at the end of EM1404 with and the of and strong hydrophobic interactions between the enzyme and inhibitor, which and the of the binding the of EM1404 EM1404 and the molecule a of the binding in this complex. for the binding of EM1404 is in the of the the of EM1404 is to that of its In in the the of the is higher than the of the that the binding of not or have the and and and were the for steroid with hydrophobic or were in the of PEG, the binding of the into the hydrophobic thus the by the of steroid into the in In fact, complex at or was was a to the of estradiol in the binary complex A. Lin J. 1996; Scopus Google Scholar). to steroid is the of the of or their in a of with or was able to be and used for complex to A. Lin J. Steroid Biochem. Mol. 1999; PubMed Scopus Google Scholar, A. V. Lin J. 2003; PubMed Scopus Google Scholar, A. Tremblay Lin J. 2002; PubMed Google Scholar, and C. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In the crystals by be the the binary complex to soak the inhibitor EM1404 into this binary complex to a ternary complex not the this the be by EM1404, which is the enzyme The of EM1404 in the was whereas the enzyme is in the for as and In the crystal a molecule is and thus this to to the of the substrate binding site in the ternary complex of in It is that this the of for EM1404 In the complex crystal most in be by its for in and they substrate in the the of the steroid has not been by as was in the of A. Lin J. 1996; Scopus Google Scholar), the of a for EM1404 be for the formation of ternary complex the binary complex molecule is defined at the site the ring of This molecule is to be the the of the molecule at the substrate binding site not with EM1404 binding on the binding of be used to a new inhibitor that could inhibit the enzyme more The group of the is the of inhibitor that is more than EM1404, a of group and EM1404 could its and affinity with the enzyme, to the of the inhibitor, for A. Tremblay Lin J. 2002; PubMed Google Scholar). The substrate binding is of that the of this could be to the of In of complex the to the of a such as In it is than the thus to on Human and 5 2002; Google Scholar). In the binding even a such as J. K. F. PubMed Scopus Google Scholar). The stability of the substrate binding is dependent on the of the of also on the end group by the amide of EM1404 with a group that could make more hydrogen bonding interactions with the protein to Y. for the of the of for on the of new inhibitor and M. and for of the with

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