Identification of Resistance Determinants for a Promising Antileishmanial Oxaborole Series

Abstract

Current treatment options for visceral leishmaniasis have several drawbacks, and clinicians are confronted with an increasing number of treatment failures. To overcome this, the Drugs for Neglected Diseases initiative (DNDi) has invested in the development of novel antileishmanial leads, including a very promising class of oxaboroles. The mode of action/resistance of this series to Leishmania is still unknown and may be important for its further development and implementation. Repeated in vivo drug exposure and an in vitro selection procedure on both extracellular promastigote and intracellular amastigote stages were both unable to select for resistance. The use of specific inhibitors for ABC-transporters could not demonstrate the putative involvement of efflux pumps. Selection experiments and inhibitor studies, therefore, suggest that resistance to oxaboroles may not emerge readily in the field. The selection of a genome-wide cosmid library coupled to next-generation sequencing (Cos-seq) was used to identify resistance determinants and putative targets. This resulted in the identification of a highly enriched cosmid, harboring genes of chromosome 2 that confer a subtly increased resistance to the oxaboroles tested. Moderately enriched cosmids encompassing a region of chromosome 34 contained the cleavage and polyadenylation specificity factor (cpsf) gene, encoding the molecular target of several related benzoxaboroles in other organisms.

DNDI-6148: A Novel Benzoxaborole Preclinical Candidate for the Treatment of Visceral Leishmaniasis

“…These data are also consistent with a recently published study reporting CPSF3 as amongst the top "hits" following the selection of the Cos-Seq genome-wide overexpression library with DNDI-6148. 23 Molecular Modeling. A homology model of the L. donovani CPSF3 was generated using the crystallographic structure of the Thermus thermophilus TTHA0252 homologue as a template (PDB code 3IEMFigure 3).…”

“…Overexpression of CPSF3 WT in L. donovani promastigotes did not substantively affect susceptibility to either acoziborole (1) (Figure 2C) or DNDI-6148 (23) (Figure 2D). However, promastigotes overexpressing the mutated version of this enzyme were significantly less sensitive to both compounds, demonstrating a 5-fold and 3.6-fold reduction in susceptibility to acoziborole (1) and DNDI-6148 (23), respectively (Figure 2C,D). The shift in potency observed with parasites overexpressing mutated CPSF3 was found to be specific for benzoxaboroles.…”

“…Compound 3 elicited a similar shift in potency, while the established N-myristoyltransferase inhibitor DDD100097 22 did not (Figure S3). The fact that this single Asn 219 His mutation in the active site of CPSF3 has a marked effect on the potencies of both acoziborole (1) and DNDI-6148 (23) provides strong evidence that this endonuclease is the molecular target of this preclinical candidate.…”

“…The Leishmania enzyme shares 30% sequence identity with this bacterial homologue. The proposed binding mode for DNDI-6148 (23) in the model is consistent with the binding of the benzoxaborole acoziborole to the catalytic site of CPSF3 located at the interface of the metallo-β-lactamase and β-CASP domains in T. brucei CPSF3. 19 The site comprises two zinc atoms coordinated by a network of conserved histidine and aspartic acid residues.…”

“…Interaction with a zinc-activated water molecule leads to the formation of a negatively charged tetrahedral boronate species that coordinates the zinc atoms (Figure S4), mimicking the transition state of the phosphate of the RNA substrate. The amide in position 6 of the benzoxaborole moiety directs the pyridyl-triazole moiety of DNDI-6148 (23) toward the area occupied by the terminal uracil base of the RNA substrate and establishes a π-stacking interaction with Tyr 370 (Figure 3A). Additionally, the amide NH forms a hydrogen bond with the hydroxyl group in the Thr 218 side-chain.…”