Rediscovery of PF-3845 as a new chemical scaffold inhibiting phenylalanyl-tRNA synthetase in Mycobacterium tuberculosis

Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 μM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl–thiazolylurea–sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl–pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine–piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 μM, and the potency of PF-3845 increased against an engineered strain Mtb pheS–FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine–piperazine urea moiety may result in the identification of a selective antibacterial lead compound. Mycobacterium tuberculosis (Mtb) remains the deadliest pathogenic bacteria worldwide. The search for new antibiotics to treat drug-sensitive as well as drug-resistant tuberculosis has become a priority. The essential enzyme phenylalanyl-tRNA synthetase (PheRS) is an antibacterial drug target because of the large differences between bacterial and human PheRS counterparts. In a high-throughput screening of 2148 bioactive compounds, PF-3845, which is a known inhibitor of human fatty acid amide hydrolase, was identified inhibiting Mtb PheRS at Ki ∼ 0.73 ± 0.06 μM. The inhibition mechanism was studied with enzyme kinetics, protein structural modeling, and crystallography, in comparison to a PheRS inhibitor of the noted phenyl–thiazolylurea–sulfonamide class. The 2.3-Å crystal structure of Mtb PheRS in complex with PF-3845 revealed its novel binding mode, in which a trifluoromethyl–pyridinylphenyl group occupies the phenylalanine pocket, whereas a piperidine–piperazine urea group binds into the ATP pocket through an interaction network enforced by a sulfate ion. It represents the first non-nucleoside bisubstrate competitive inhibitor of bacterial PheRS. PF-3845 inhibits the in vitro growth of Mtb H37Rv at ∼24 μM, and the potency of PF-3845 increased against an engineered strain Mtb pheS–FDAS, suggesting on target activity in mycobacterial whole cells. PF-3845 does not inhibit human cytoplasmic or mitochondrial PheRS in biochemical assay, which can be explained from the crystal structures. Further medicinal chemistry efforts focused on the piperidine–piperazine urea moiety may result in the identification of a selective antibacterial lead compound. Protein synthesis is the cellular process targeted by many commercial antibiotics. It has always been a focal point of modern antibacterial drug discovery (1Fischbach M.A. Walsh C.T. Antibiotics for emerging pathogens.Science. 2009; 325: 1089-1093Crossref PubMed Scopus (1199) Google Scholar). Aminoacyl-tRNA synthetases (aaRSs), a family of ∼20 essential enzymes, ligate amino acids to the corresponding tRNAs that decode messenger RNA to produce protein at the translating macromolecular ribosome (2Schimmel P.R. Söll D. Aminoacyl-tRNA synthetases: general features and recognition of transfer RNAs.Annu. Rev. Biochem. 1979; 48: 601-648Crossref PubMed Scopus (455) Google Scholar). Inhibition of bacterial aaRS blocks the translation and ultimately shuts down protein synthesis, which is crucial for pathogens to survive in host or inside host cells (3Francklyn C.S. Mullen P. Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.J. Biol. Chem. 2019; 294: 5365-5385Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 4Kwon N.H. Fox P.L. Kim S. Aminoacyl-tRNA synthetases as therapeutic targets.Nat. Rev. Drug Discov. 2019; 18: 629-650Crossref PubMed Scopus (55) Google Scholar). Between a bacterial aaRS protein and its human counterpart, either the large sequence difference or small variation of key residues in the catalytic core explains the high selectivity of successful and promising aaRS inhibitor drugs, as exemplified by the mupirocin used for the treatment of staphylococcal infection (4Kwon N.H. Fox P.L. Kim S. Aminoacyl-tRNA synthetases as therapeutic targets.Nat. Rev. Drug Discov. 2019; 18: 629-650Crossref PubMed Scopus (55) Google Scholar). Tuberculosis (TB) caused by the single agent Mycobacterium tuberculosis (Mtb) has surpassed AIDS/HIV, becoming a leading infectious disease worldwide (https://www.who.int/news-room/fact-sheets/detail/tuberculosis). Most TB drugs were discovered in the past century and are losing efficacy because of the resistance inevitably arisen in bacteria. New chemical scaffold with novel inhibition mechanism against Mtb is being actively sought. An oxaborole compound GSK3036656 that inhibits Mtb leucyl-tRNA synthetase is currently undergoing clinical trial (5Tenero D. Derimanov G. Carlton A. Tonkyn J. Davies M. Cozens S. Gresham S. Gaudion A. Adeep P. Muliaditan M. Rullas-Trincado J. Mendoza-Losana Alfonso Skingsley A. Barros-Aguirre D. First-time-in-human study and prediction of early bactericidal activity for GSK3036656 , a potent leucyl-tRNA synthetase inhibitor for tuberculosis treatment.Antimicrob. Agents Chemother. 2019; 63: 1-15Crossref Scopus (21) Google Scholar). Bacterial phenylalanyl-tRNA synthetase (PheRS) is a member of class II aaRS based on the active site topology (6Safro M. Moor N. Lavrik O. Phenylalanyl-tRNA synthetases.in: Ibba M. Francklyn C. Cusack S. The Aminoacyl-tRNA Synthetases. Landes Bioscience/Eurekah.com, Georgetown, TX2005: 265-280Google Scholar). A functional PheRS is typically made of two heterodimers (αβ)2, with a whole molecular weight around 250 kD. In Mtb H37Rv genome, two consecutive essential genes, Rv1649 and Rv1650 (pheST), encode the protein subunits (7Dejesus M.A. Gerrick E.R. Xu W. Park S.W. Long J.E. Boutte C.C. Rubin E.J. Schnappinger D. Ehrt S. Fortune S.M. Sassetti C.M. Ioerger T.R. Comprehensive essentiality analysis of the Mycobacterium tuberculosis genome via saturating transposon mutagenesis.MBio. 2017; 8: 1-17Crossref Scopus (203) Google Scholar). Like other aaRSs, PheRS catalyzes the formation of phenylalanyl-tRNAPhe in two steps: (1) activation of phenylalanine (Phe) by hydrolyzing ATP to form Phe-AMP and pyrophosphate (PPi) and (2) subsequent transfer of Phe to the 2′-OH group of adenosine ribose at the 3′-terminal of tRNAPhe and simultaneous release of AMP. In addition to the synthetic site on the α subunit, PheRS has a dedicated domain on the β subunit that can hydrolyze improperly charged tRNA, for example, tyrosinyl-tRNAPhe, to maintain the fidelity of aminoacylation and translation (8Roy H. Ling J. Irnov M. Ibba M. Post-transfer editing in vitro and in vivo by the β subunit of phenylalanyl-tRNA synthetase.EMBO J. 2004; 23: 4639-4648Crossref PubMed Scopus (116) Google Scholar). From a drug discovery standing point, PheRS synthetic activity that involves binding of the three substrates and the editing activity be PheRS binding of that the because of its molecular and other A. The phenylalanyl-tRNA synthetase binds Biol. PubMed Scopus Google Scholar). PheRS was a antibacterial target in a of screening P. J. D. P. The identification of antibacterial drug discovery a study with aminoacyl-tRNA Scholar, D. G. S. M. J. M. D. C. H. New class of bacterial phenylalanyl-tRNA synthetase with high potency and Agents Chemother. 2004; 48: PubMed Scopus Google Scholar, for the challenges of antibacterial Rev. Drug Discov. PubMed Scopus Google Scholar, P.L. J. C. and of as of bacterial phenylalanyl-tRNA Chem. 2009; PubMed Scopus Google Scholar, J. The and of a high screening for of Mycobacterium tuberculosis phenylalanyl-tRNA Scholar, A. P.R. N. J. S.M. The of a novel pocket in bacterial phenylalanyl-tRNA synthetase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, the of antibacterial Rev. Drug Discov. PubMed Scopus Google Scholar, M. and analysis of inhibiting protein synthesis in Agents Chemother. PubMed Scopus Google Scholar). Bacterial as from synthetic compound the of of were not for TB drug that identified a to inhibit the growth of Mtb strain H37Rv of the tuberculosis phenylalanyl-tRNA synthetase from Chem. Google Scholar). PheRS from which is of has become and target for in to novel for and other 2019; 1-17Crossref PubMed Scopus Google Scholar). PheRS with novel at the molecular and cellular were discovered N. A. M. M. J. D. E.R. S. S. synthesis novel PubMed Scopus Google to a discovery Mtb PheRS with new and compound as of small J. N.H. S. as a drug and its to the treatment of S. A. PubMed Scopus Google Scholar). In Mtb PheRS enzyme was and with a compound screening was to a against the PheRS. screening PF-3845, was to inhibit Mtb PheRS with Ki ∼ 0.73 μM. It is a inhibitor of human fatty acid amide to inhibit growth of H37Rv is ∼24 μM, and the activity increased against an engineered strain Mtb pheS–FDAS, which a The mechanism of inhibition was studied with enzyme and crystal structures. PF-3845 was to be a non-nucleoside bisubstrate competitive inhibitor of the PheRS. The medicinal chemistry to the the and with TB In Mtb H37Rv genome, encode the α and β subunits of The amino acid sequence of is to to human cytoplasmic or mitochondrial The mitochondrial PheRS does not a β The of is of class II aaRS are identified in Mtb sequence The was based on the in the was for in were made for of the The a of to with the and an Mtb from It was that of the and be the G. S. D. G. J. and of and of the in Biol. PubMed Scopus Google Scholar, of by the of synthetase in PubMed Scopus Google Scholar). used aminoacylation that the of Phe tRNAPhe to the protein and were functional of Mtb PheRS were with the aminoacylation and in The and with on and other bacterial are example, and the with to Phe at ± μM, is the to of other bacterial J. The and of a high screening for of Mycobacterium tuberculosis phenylalanyl-tRNA Scholar, A. P.R. N. J. S.M. The of a novel pocket in bacterial phenylalanyl-tRNA synthetase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. P. H. is by amino acid binding pocket in phenylalanyl-tRNA PubMed Scopus Google Scholar, N. Bacterial and phenylalanyl-tRNA synthetases of Phe with Biol. 18: Full Text Full Text PDF PubMed Scopus Google Scholar). The are to in of PheRS and amino acid biochemical were for screening for PheRS inhibitor and the subsequent analysis of of In a Ibba M. Söll D. A for aminoacyl-tRNA synthetase 23: PubMed Scopus Google the is to of by the as a in a to compound into and that has at the assay, that the is as was in the of PheRS at a in the first of the the of the ATP with to ATP was to be ± μM, Phe ± μM, and ± were used as in the screening and compound used a commercial which the of ATP in a PheRS is by of in the of the The is with the of PheRS ATP at to the PheRS is its the is C. C. S. M. synthetase as a drug target in and S. A. 2019; PubMed Scopus Google Scholar). the in and the of PheRS was to be at the first A known bacterial PheRS inhibitor D. G. S. M. J. M. D. C. H. New class of bacterial phenylalanyl-tRNA synthetase with high potency and Agents Chemother. 2004; 48: PubMed Scopus Google was as and in against Mtb PheRS was to be ± μM. A 2148 small with and The was in which at to the was to inhibition with were identified a A PF-3845 is a potent inhibitor of human enzyme with Ki to be ± M. D. Long N. M. S. C. S. and of a selective inhibitor that Biol. 2009; Full Text Full Text PDF PubMed Scopus Google and many are in PF-3845 and an were The of the two were with the two and other A for of aminoacyl-tRNA synthetase 18: PubMed Scopus Google the assay, the of PF-3845 was ± ± and the of was ± 0.73 ± PF-3845, at to μM, not inhibit the of and that were used in the The inhibition was Mtb PheRS was in the identified drug PF-3845 as a new PheRS PF-3845 has a scaffold from known PheRS The of inhibition of PF-3845 was with enzyme in comparison with was used to the of PheRS which of two substrates of a at inhibitor was into the of a of with to the was for a of or inhibition and PF-3845 are Phe competitive of PheRS as the of inhibitor not In the mode, Ki of is ± and Ki of PF-3845 is ± μM, with the It was that was ATP D. G. S. M. J. M. D. C. H. New class of bacterial phenylalanyl-tRNA synthetase with high potency and Agents Chemother. 2004; 48: PubMed Scopus Google and In PF-3845 is in a inhibition with to ATP a of PF-3845 PF-3845 is an inhibitor with to tRNAPhe was made to first the of Mtb PheRS protein and its complex with at and Like other bacterial PheRS Mtb PheRS is an The α subunit catalytic and β subunit editing site for the residues of α subunit, other residues were The of PheRS was to form a a in interaction with N. O. D. M. M. The crystal structure of the complex of phenylalanyl-tRNA synthetase with tRNAPhe and a a of the PubMed Scopus Google Scholar). It was and in the Mtb PheRS was The binding of was by into the catalytic site of the α subunit, that the amino acid (Phe) pocket and ATP pocket, is an pocket between the two The key residues the pocket are and occupies the Phe pocket and the pocket, whereas the ATP pocket is and The group of into the amino acid pocket a Phe In pocket, can be between the of and are between the urea group of and the of and In the with the of and the of and The moiety of is to the and into the pocket, which has interaction with the with the two differences were in the form the of has two with that occupies the amino acid pocket, and the of the in the which occupies of the with the of a amino acid pocket of the complex the form the to a binding and has the is binds the other analysis a that PF-3845 two binding the In PF-3845 is to with the pocket Phe pocket by is that PF-3845 into the ATP Further is to which binding is the binding of PF-3845, the structure of Mtb PheRS in complex with PF-3845 at a of of PF-3845 with is in PF-3845 binds in the amino acid pocket as well as the ATP pocket, whereas the pocket is An and interaction network in two can be between PF-3845 and Mtb PheRS. 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In the the with corresponding to in Mtb PheRS. the and in the ATP pocket and to PF-3845 binding into the ATP pocket of Mtb PheRS. has been at the active site of other aaRS of to the binding of a as in the of PF-3845 S. C. M. S. S. and of synthetase from the Biol. PubMed Scopus Google Scholar, M. N. C. J. of new into the mechanism of activation and for drug PubMed Scopus Google Scholar). the of the and the PF-3845 inhibition with in the PheRS enzyme PF-3845 as an ATP competitive inhibitor with the Ki to be 0.73 ± μM, which is Ki ± with to ATP was The that sulfate increased the binding of PF-3845, a bisubstrate competitive inhibitor of Mtb PheRS. The of PF-3845 against Mtb H37Rv by a for high-throughput screening of against Mycobacterium tuberculosis and Mycobacterium Agents Chemother. PubMed Google was with the of at μM. It that PF-3845 has into Mtb cells its Ki to PheRS target is The of PF-3845 or as inhibition of the of from to which is not for a drug discovered as a human protein to PF-3845 can PheRS target in Mtb used Mtb in which was to encode a The of PF-3845, by an at down from in H37Rv to in the suggesting that PF-3845 can inhibit PheRS in Mtb to the phenyl–thiazolylurea–sulfonamide class of PheRS discovered in by at D. G. S. M. J. M. D. C. H. New class of bacterial phenylalanyl-tRNA synthetase with high potency and Agents Chemother. 2004; 48: PubMed Scopus Google Scholar). an group the of PheRS in complex with and identified an pocket that is an of the pocket A. P.R. N. J. S.M. The of a novel pocket in bacterial phenylalanyl-tRNA synthetase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It was that the pocket is a for drug discovery because compound binding in the Phe pocket of bacterial PheRS to high screening resistance and protein Further to the of by the pocket in In by a screening with a commercial of bioactive compounds, identified a chemical PF-3845, as a inhibitor of Mtb PheRS. PF-3845 binds to Mtb PheRS in a from Most PF-3845 into the ATP pocket of PheRS with the of an sulfate ion. It represents the first non-nucleoside bisubstrate inhibitor of PheRS. on the 2.3-Å the group on the of PF-3845 be to its binding to the pocket, whereas the on the is of the of the chemical and for of TB drug as well as new the has a in and as well as in vitro with human D. N. P. S. C. and of a potent and selective fatty acid amide inhibitor that and PubMed Scopus Google other from PF-3845 be promising clinical for TB the piperidine–piperazine urea group of PF-3845 occupies the ATP pocket, does not as as or Phe-AMP Moor N. Lavrik O. of Phenylalanyl-tRNA synthetase with phenylalanine and a Biol. PubMed Scopus Google Scholar, N. H. and in the crystal structure of phenylalanyl-tRNA synthetase with phenylalanine and PubMed Scopus Google Scholar). The with a between PheRS and PF-3845 are through or sulfate in The sulfate an in an interaction and that the inside the ATP of the complex can be example, to the binding the compound is to the the sulfate is to a from PF-3845 to its for binding losing cellular to the sulfate be made in because because of a of sulfate and in The of in Scopus Google Scholar). PF-3845 does not inhibit either human cytoplasmic or mitochondrial PheRS in biochemical at to and the are by protein structural the catalytic site of Mtb PheRS with Moor N. N. of human phenylalanyl-tRNA for of the active and binding 18: Full Text Full Text PDF PubMed Scopus Google and by structural and sequence a difference is in the amino acid pocket in and in which form the pocket at the are the corresponding in Mtb in the amino acid pocket of of the that with the of can be from binding to human analysis with D. G. S. M. J. M. D. C. H. New class of bacterial phenylalanyl-tRNA synthetase with high potency and Agents Chemother. 2004; 48: PubMed Scopus Google Scholar, A. P.R. N. J. S.M. The of a novel pocket in bacterial phenylalanyl-tRNA synthetase Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and explains the selectivity and antibacterial potency of is not in to PF-3845 its two the as the of the two are not by The of PF-3845 against human has been A. J. acid amide inhibitor PF-3845 and of human an in vitro 2017; PubMed Scopus Google Scholar). In a medicinal chemistry at PF-3845 for the treatment of and other the for inhibition of human increased from to C. M. D. N. of a and selective urea Chem. PubMed Scopus Google Scholar). result that with at to against two human is PF-3845 with at to It that cellular other the are by PF-3845 The be for the of the human cells. to be into the PF-3845 scaffold is a TB drug The enzyme in the PF-3845 has its molecular weight of and of are at the of of that the drug of a compound based on active drugs for A is that the of PF-3845 to cells is its against Mtb at a to become a TB drug as of synthetic of PF-3845 been in the M. D. Long N. M. S. C. S. and of a selective inhibitor that Biol. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar, W. M. J. M. S. S. C.S. J. M. as of fatty acid amide Chem. 18: PubMed Scopus Google Scholar, D. A. N. S. S. S. S. D. A. and of novel in Chem. 2019; PubMed Scopus Google the PF-3845 scaffold can be used as an chemical to study the and of bacterial and human The of Mtb for and for were by and into the and of Between the two a site was to the of the of was to be to a for of the two subunits The was to and were by was into or and the was for at A of was used to for and at the at to The was down to and was to the of of at cells were by and with and inhibitor cells were into two and to through by was through by against and the was two in The was with and were with and on with and with the were and to with two in with and were with and based on analysis were and to and to for The were and and to of PheRS was on of PheRS was by into on PheRS used in was with of of and the protein was by It was into and at for biochemical and of with at the and at the 3′-terminal was An site was of and a site was of the The was into the and of The was to cells. on single was to and for at A of was used to for and at the at to was to of at cells were by and in of was and at for the was to be with of The was and the was for a transfer the into new of were and acid was at for was A was in and at for acid was as and a was two with by in and in The acid was with to a of was with The acid was the and with by was with by at the that The be by urea and were by of and of for at The was with by and in to of It was to and at The was based on of aaRS (3Francklyn C.S. Mullen P. Progress and challenges in aminoacyl-tRNA synthetase-based therapeutics.J. Biol. Chem. 2019; 294: 5365-5385Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, Ibba M. Söll D. A for aminoacyl-tRNA synthetase 23: PubMed Scopus Google Scholar, C. C. S. M. synthetase as a drug target in and S. A. 2019; PubMed Scopus Google Scholar, H. A for the synthetic and of aminoacyl-tRNA synthetases for high-throughput drug Biol. PubMed Scopus Google Scholar). was in and the were on The of in the is In the was with in a well Mtb and at was for was made with and to and the were for at was to the was used for used for was based on The with aaRS was C. C. S. M. synthetase as a drug target in and S. A. 2019; PubMed Scopus Google Scholar, C. M.A. M. and structural of selective of the drug 2017; PubMed Scopus Google Scholar). was in The in the are and from bioactive were in to of μM. The compound is in was into and compound was in Mtb PheRS in was to and the was for and was to the at and for down the with was and for was was in in a of were of and enzyme were of with Mtb the was used as and were with compound at for the as that was used at were for as In analysis with the the enzyme activity was was PheRS in was the from and was from inhibitor The activity was against inhibitor the was to was the the assay, of Mtb PheRS with two saturating substrates and were used in the of enzyme at a of of PF-3845 were for of inhibition or the Ki for competitive the with against were the Ki for the were Mtb PheRS was with or at a of for of the form and two were by at The were by with well The and were in at and were to the group and PheRS The form structure was by molecular with the in PheRS as a search The sequence between the α subunits of Mtb and PheRS is with the of whereas that between the β subunits is with the of The and were and of the two were the of the form The are in were The and structure been in Protein with and and PheRS in are for the in are for the in a new and cells were in and at with of were with well and for compound in of was and the were at for in the was with The were with The Mtb were in with and and for in a at and growth was The were with and to an at of The were to and at drug in in for a of were with in of three and at and in a The at of the was were to and were by the to a in study Protein and the been in Protein with and The that of with the of The the of at for and with the and at Tuberculosis and for with in the S. C. and H. W. the by in and in of of and H. M. C. A. M. and to H. M. C. A. D. and S. C. to H. M. C. A. and S. C. to S. D. and S. C. to H. M. C. A. M. and S. C. to and H. M. C. A. and S. C. to is by and Drug in