title: Phthalein derivatives as a new tool for selectivity in thymidylate synthase inhibition.
abstract: A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were designed to specifically complement structural features of a bacterial form of thymidylate synthase (Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds were screened for their activity and their specificity against TS enzymes from different species, namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and human thymidylate synthase (hTS). Apparent inhibition constants (Ki) for all the compounds against LcTS were determined, and inhibition factors (IF, ratio between the initial rates of the enzymatic reaction in the presence and absence of each inhibitor) against each of the four TS species were measured. A strong correlation was found between the two activity parameters, IF and Ki, and therefore the simpler IF was used as a screening factor in order to accelerate biological evaluation. Compounds 5b, 5c, 5ba, and 6bc showed substantial inhibition of LcTS while remaining largely inactive against hTS, illustrating for the first time remarkable species specificity among TSs. Due to sequence homology between the enzymes, several compounds also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H, 3H-naphtho[1,8-c,d]pyran-1-one (6bc) showed an IF < 0.04 for CnTS (Ki = 0.45 microM) while remaining inactive in the hTS assay at the maximum solubility concentration of the compound (200 microM). In cell culture assays most of the compounds were found to be noncytotoxic to human cell lines but were cytotoxic against several species of Gram-positive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several compounds also had selective activity against Cr. neoformans in cell culture assay. In general, the most active and selective compounds against the Gram-positive bacteria were those designed and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound was least selective against most of the cell lines tested. To our knowledge these compounds are the first TS inhibitors selective for bacterial TS with respect to hTS.
authors.affiliation: Dipartimento Scienze Farmaceutiche and Dipartimento Scienze Chimiche, Università di Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy.
authors.forename: P M
authors.initials: PM
authors.lastname: Costi
authors.forename: M
authors.initials: M
authors.lastname: Rinaldi
authors.forename: D
authors.initials: D
authors.lastname: Tondi
authors.forename: P
authors.initials: P
authors.lastname: Pecorari
authors.forename: D
authors.initials: D
authors.lastname: Barlocco
authors.forename: S
authors.initials: S
authors.lastname: Ghelli
authors.forename: R M
authors.initials: RM
authors.lastname: Stroud
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
authors.forename: T J
authors.initials: TJ
authors.lastname: Stout
authors.forename: C
authors.initials: C
authors.lastname: Musiu
authors.forename: E M
authors.initials: EM
authors.lastname: Marangiu
authors.forename: A
authors.initials: A
authors.lastname: Pani
authors.forename: D
authors.initials: D
authors.lastname: Congiu
authors.forename: G A
authors.initials: GA
authors.lastname: Loi
authors.forename: P
authors.initials: P
authors.lastname: La Colla
mesh_terms: D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002460:Cell Line; D002733:Chlorophenols / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D002867:Chromones / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D003455:Cryptococcus neoformans / Q000201:enzymology; D018360:Crystallography, X-Ray; D015195:Drug Design; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006801:Humans; D007780:Lacticaseibacillus casei / Q000201:enzymology; D008958:Models, Molecular; D020113:Phenolphthalein / Q000737:chemistry; D011010:Pneumocystis / Q000201:enzymology; D013045:Species Specificity; D013329:Structure-Activity Relationship; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors*
chemical_list: C119794:3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H,3H-naphtho(1,8-c,d)pyran-1-one; D000900:Anti-Bacterial Agents; D000890:Anti-Infective Agents; D002733:Chlorophenols; D002867:Chromones; D004791:Enzyme Inhibitors; D020113:Phenolphthalein; D013940:Thymidylate Synthase