New Insights Into DNA Helicases as Druggable Targets for Cancer Therapy

Abstract

Small molecules that deter the functions of DNA damage response machinery are postulated to be useful for enhancing the DNA damaging effects of chemotherapy or ionizing radiation treatments to combat cancer by impairing the proliferative capacity of rapidly dividing cells that accumulate replicative lesions. Chemically induced or genetic synthetic lethality is a promising area in personalized medicine, but it remains to be optimized. A new target in cancer therapy is DNA unwinding enzymes known as helicases. Helicases play critical roles in all aspects of nucleic acid metabolism. We and others have investigated small molecule targeted inhibition of helicase function by compound screens using biochemical and cell-based approaches. Small molecule-induced trapping of DNA helicases may represent a generalized mechanism exemplified by certain topoisomerase and PARP inhibitors that exert poisonous consequences, especially in rapidly dividing cancer cells. Taking the lead from the broader field of DNA repair inhibitors and new information gleaned from structural and biochemical studies of DNA helicases, we predict that an emerging strategy to identify useful helicase-interacting compounds will be structure-based molecular docking interfaced with a computational approach. Potency, specificity, drug resistance, and bioavailability of helicase inhibitor drugs and targeting such compounds to subcellular compartments where the respective helicases operate must be addressed. Beyond cancer therapy, continued and new developments in this area may lead to the discovery of helicase-interacting compounds that chemically rescue clinically relevant helicase missense mutant proteins or activate the catalytic function of wild-type DNA helicases, which may have novel therapeutic application.

Keywords: DNA repair; cancer; genomic instability; helicase; replication; small molecule; therapy.

Figure 1

Mechanisms of DNA helicase inhibitors and therapeutic strategies. (A) Small molecule helicase inhibitors may interfere with the catalytic activities of DNA helicase proteins and their molecular and cellular functions by a variety of mechanisms. A helicase-interacting compound may disrupt protein oligomerization, binding to DNA substrate, or compete with ATP binding. Small molecules may alter helicase interactions with other proteins (e.g., DNA repair/replication factors) by orthosteric or allosteric mechanisms. Helicase-interacting compounds may also cause the protein to become trapped on DNA, resulting in a toxic complex or lead to the hijacking of other proteins. (B) Two potential strategies for helicase inhibitors (that are not mutually exclusive) are (i) Chemical-based synthetic lethality whereby pharmacological helicase inhibition compromises the cancer cell to chemotherapy DNA damaging drugs or radiation; (ii) Genetic-based synthetic lethality whereby the defined genetic mutant background of the cancer cell is hypersensitive to pharmacological helicase inhibition. See text for details.

Figure 2

Flow diagram for discovery, optimization, and validation of DNA helicase inhibitors. See text for details.

Figure 3

A dimer of RECQL1 molecules, bound to two forked DNA molecules. The first RECQ1 monomer is marked with standard lettering, the second monomer is with primes. The two RecA binding domains (D1, D2) and Zn²⁺ binding domain (Zn) are indicated. The white asterisk denotes the strand-separating beta hairpin. 3' end of DNA strand is indicated. The locations of the conserved aromatic-rich loops (ARL) implicated in the coupling of single-stranded DNA binding to ATP hydrolysis are indicated by the gray shadow. The region comprising the dimer interface between RECQL1 monomers is indicated by gray shadow. Docking small molecules at the ARL or dimer interface may provide a strategy to modulate RECQL1's catalytic function. Image was modified from one kindly provided by Dr. Opher Gileadi, University of Oxford.

Figure 4

Conformations of the BLM helicase core and winged helix domains. (A) A co-crystal structure with a nanobody (orange), which shifts the WH domain out of its helicase-active position (PDB:4CDG). (B) A superposition of the nanobody-bound conformation (WH domain in blue, nanobody omitted), with a DNA-bound conformation (WH domain in cyan, DNA omitted; PDB:4CGZ). The RecA domains (D1, D2) and the conserved HRDC are indicated with a region of the HRDC residing close to the ATP binding pocket shown by gray shadow. Small molecules which bind to the HRDC may modulate ATP binding and/or hydrolysis by BLM. Image was modified from one kindly provided by Dr. Opher Gileadi, University of Oxford.

Figure 5

Structure of human RECQL4 (residues 449-1111). The ATPase domain, comprising HD1 and HD2, are shown in dark blue and light blue, respectively. RECQL4 features a structurally unique domain, termed RECQL4-Zn²⁺-binding domain (R4ZBD), shown in olive. The R4ZBD coordinates a Zn²⁺-ion (cyan sphere). The gray shadow regions represent the upper and lower halves of the R4ZBD that may be suitable for molecular docking of small molecules to the unique Zn²⁺ binding domain of RECQL4. RECQL4 harbors the Sld2-homology domain at its N-terminus (not shown). Image was modified from one kindly provided by Drs. Sebastian Kaiser and Caroline Kisker, University of Wuerzburg.

Figure 6

Closed and open conformations of RECQL5 helicase domain. (A) Closed state, no nucleotide (PDB ID: 5LB8); (B) Open state, with Mg²⁺-ADP (PDB: 5LB3). Note the absence of the WH and HRDC domains in the C-terminal region of the protein which can be found in WRN, BLM, and RECQL1. The unique “wedge” α-helix of RECQL5 critical for helicase activity is indicated and may be targeted by small molecules to modulate ATP-dependent strand separation. Also indicated by gray zone is the inter-domain cleft which widens significantly between the open and closed conformations. The inter-domain cleft may be a useful site for molecular docking of small molecule interfacial compounds that perturb open-closed conformational switches of RECQL5. Image was modified from one kindly provided by Dr. Opher Gileadi, University of Oxford.

Figure 7

Overall structure of the taXPD-DNA complex. Shown are the two RecA-like domains in yellow orange and ruby, the FeS cluster domain in deep teal, and the Arch domain in forest green. The DNA is shown in orange/yellow/blue. The Arch and Fe-S domains, both implicated in strand separation and only found in Fe-S helicases, are proposed sites for docking small molecules to modulate helicase function. Image was modified from one kindly provided by Drs. Jochen Kuper and Caroline Kisker, University of Wuerzburg.

Figure 8

Therapeutic avenues for helicase-interacting small molecules. Compounds that bind DNA helicase proteins may be useful for (i) targeted helicase inhibition to deter DNA damage response pathways to enhance chemotherapy drug or radiation strategies (ii) chemical rescue of catalytically defective and/or misfolded helicase proteins due to missense mutations linked to hereditary chromosomal instability disorders, or (iii) functional activation of a wild-type helicase that has an overlapping role with a DNA repair pathway that is defective due to a genetic inactivating mutation linked to a DNA repair disorder.