5’‐Phosphorylation Increases the Efficacy of Nucleoside Inhibitors of the DNA Repair Enzyme SNM1A

Abstract

Certain cancers exhibit upregulation of DNA interstrand crosslink repair pathways, which contributes to resistance to crosslinking chemotherapy drugs and poor prognoses. Inhibition of enzymes implicated in interstrand crosslink repair is therefore a promising strategy for improving the efficacy of cancer treatment. One such target enzyme is SNM1A, a zinc co-ordinating 5'-3' exonuclease. Previous studies have demonstrated the feasibility of inhibiting SNM1A using modified nucleosides appended with zinc-binding groups. In this work, we sought to develop more effective SNM1A inhibitors by exploiting interactions with the phosphate-binding pocket adjacent to the enzyme's active site, in addition to the catalytic zinc ions. A series of nucleoside derivatives bearing phosphate moieties at the 5'-position, as well as zinc-binding groups at the 3'-position, were prepared and tested in gel-electrophoresis and real-time fluorescence assays. As well as investigating novel zinc-binding groups, we found that incorporation of a 5'-phosphate dramatically increased the potency of the inhibitors.

The 5′-phosphate enhances the DNA-binding and exonuclease activities of human mitochondrial genome maintenance exonuclease 1 (MGME1)

“…We demonstrate unambiguously that MGME1 prefers to interact with the 5′-end of an ssDNA substrate and that a 5′-phosphate contributes to the optimal DNA binding and catalysis of MGME1. A close examination of the X-ray crystal structures of MGME1 did not show any apparent phosphate-binding pocket, unlike other 5′-exonucleases, such as human SNM1B ( 29 ). Rather, we found that several motifs could potentially mediate the interactions with phosphates on the basis of the electrostatic potential analysis ( Fig.…”

Two‐Step Validation Approach for Tools To Study the DNA Repair Enzyme SNM1A

“…[11c] Fragments identified during this proof-of-concept assay correspond to metal-binding groups that have previously been recognised by SNM1A. [7][8]11] This demonstrates that fragment-based screening is a suitable approach for the identification of metal-binding groups suitable for targeting this enzyme.…”

“…Although screens of bioactive molecules have previously succeeded in identifying several inhibitors of SNM1A, [6,10] there is currently no screening method focused solely on the identification of metalbinding groups, which have been demonstrated as being key for targeting SNM1A. [7][8]11] Fragment-based screening approaches evaluate the ability of low-molecular-weight molecules to exert a desired biological action. [12] Although the fragments themselves may only have low binding affinity for the desired target, they can be combined with other scaffolds or functional groups to create a final drug molecule or tool compound with high binding affinity.…”

“…The β‐lactam antibiotics cephalosporins, prototypical MBL inhibitors, show strong SNM1A inhibition [6] . Incorporation of metal‐binding groups into phosphorylated nucleoside derivatives is an effective method of generating inhibitors of SNM1A [7] . The incorporation of a nucleoside/oligonucleotide scaffold has proven to enhance substrate recognition and increase binding affinity [8] .…”

“…New, facile synthetic and screening methodologies are therefore required to rapidly evaluate the efficiency of substrate binding and to access non‐native SNM1A substrates. Although screens of bioactive molecules have previously succeeded in identifying several inhibitors of SNM1A, [6,10] there is currently no screening method focused solely on the identification of metal‐binding groups, which have been demonstrated as being key for targeting SNM1A [7–8,11] …”