Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT): Cocrystal Structures of Pyrazolopyrans with Potent Blood- and Liver-Stage Activities

Abstract

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Conformational Aspects in the Design of Inhibitors for Serine Hydroxymethyltransferase (SHMT): Biphenyl, Aryl Sulfonamide, and Aryl Sulfone Motifs

“…The pyrazolopyran core and its alkyl substituents (methyl and isopropyl) were kept intact. [25] Intermolecularh ydrogen bonding is indicated by red dashed lines. a) The one-carbon-unit transfer reaction catalyzed by SHMT.Dihydrofolate reductase (DHFR), SHMT,and thymidylate synthase(TS) are the three enzymes involved in the folate cycle.…”

“…The same synthetic pathway [25,26] was followed to preparea ll pyrazolopyran-based ligands ((AE)-2-22)r eported in this manuscript (Scheme 1; for the full structures of all ligands, see Ta bles 1a nd 2b elow). Brominated 23 was either converted into the pinacol boronate ester 24 prior to aS uzuki crosscoupling [43] (for (AE)-2-20)o rd irectly subjected to aB uchwald-Hartwig cross-coupling [44] (for (AE)-21 and (AE)-22)l eading to intermediates 25 a-u.As ubsequent Knoevenagel condensation [45] gave access to the dinitrile precursors 26 a-u needed for the one-pot synthesis of the pyrazolopyranc ore involving a Michael addition of 3-methyl-1H-pyrazol-5(4H)-one, followed by an intramolecular cyclization to provide (AE)-2-22 (for details, see the Supporting Information, Section S1).…”

“…The two enantiomers of the benzyl sulfonamide( AE)-17 were separated by chiral-phase HPLC to afford pure (+ +)-17 and (À)-17.A sa lready observedw ith other pyrazolopyran-based ligands, [25,26] there is ah ighl evel of chiral recognition at the active site of PfSHMT and large discrepancies in biologicala ctivity were measured for (+ +)-17 and (À)-17.L igand (+ +)-17 inhibited PfSHMT (IC 50 = 150 nm)m uch more efficientlyt han (À)-17 (only 38 %i nhibition at 250 mm)( Supporting Information, SectionS2, Ta ble S1). The preference for the (+ +)-enantiomer is not surprising when considering the binding mode of pyrazolopyran-based ligands.…”

“…Recently Cottrell et al expanded this CSD search to various ring fragments. [25] Lately,w es howedt hat optimized inhibitors targeting SHMT induce as ignificant reduction of parasitemia in vivo in a mouse model. SHMT is ap yridoxal 5'phosphate (PLP)-dependent enzyme catalyzing the onecarbon-unit transfer from serine to tetrahydrofolate (H 4 F) ( Figure 1).…”

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold

“…Amino acid analogues, such as d ‐serine, d ‐threonine, and thiosemicarbazide, were also investigated, but no significant inhibition was observed either . We recently demonstrated that pyrazolopyran‐based inhibitors, such as carboxylate (±)‐ 18 (Figure ), could inhibit Pf SHMT in the lower nanomolar IC 50 range and were highly effective (half‐maximal effective concentration (EC 50 )) in cell‐based assays against the Pf NF54 strain . Several co‐crystal structures with Pv SHMT showed that these highly preorganized ligands nicely occupy the H 4 F binding pocket of the enzyme.…”