Despite many attempts to find specific inhibitors of HR, no direct inhibitors of proteins catalyzing the HR reactions are available to date. Nevertheless, many classes of novel anticancer drugs that are currently in clinical trials have recently been shown to target HR, and although they were not developed as inhibitors of HR their activity may in fact be related to their ability to inhibit HR.
The many potential ways that HR may be inhibited can be appreciated by considering the large and growing number of proteins known to be involved in the various aspects of HR. Compounds known to modulate HR activity interfere with the expression, nuclear localization, recruitment, and/or activation of HR. For example, inhibitors of the tyrosine kinases c-Abl (Imatinib or Gleevec) and EGFR (Erlotinib) affect nuclear localization of Rad51 and BRCA1, while inhibitors of histone deacetylation (HDAC) (e.g., valproic acid, PCI-24781), and of HSP90 protein (e.g., 17-AAG) decrease the levels of active HR components, affecting either expression or maturation of key HR gene products,40,42
like Rad51 and BRCA1. On the other hand, proteasome inhibitors (β-lactacystin, MG132, bortezomib, nelfinavir) do not interfere with expression of Rad51, but impair HR through inhibition of proteasome-dependent degradation of mediator proteins, such as MDC1,43
acting downstream of the DSB sensors ATM and MRN, but before the formation of a ssDNA/RPA recombinogenic intermediate.44
Inhibition of the more upstream targets has become possible with the identification of mirin, a compound affecting the Mre11-associated exonuclease activity.45
In light of the recent finding that Rad51 is phosphorylated in a Mec (an ATR homolog)-dependent manner in response to DNA damage in yeast,46
it seems attractive to give closer consideration to compounds that inhibit kinases that directly regulate the HR process. NU6027, originally developed as a CDK2 inhibitor, has recently been shown to inhibit ATR.47
Consistent with a key function of ATR resulting in resolution of stalled replication forks through signaling to the HR pathway, NU6027 has been shown to inhibit Rad51 foci formation. The downstream target of ATR, the CHK1 protein, has also been identified as a regulator of HR,14
and the inhibitor of CHK1, AZD7762, has been shown to hamper assembly of Rad51 foci, as well as to inhibit gene conversion.48
BRCA1 has been recently identified as a phosphorylation target of the CDK1 kinase,49
and this phosphorylation has been shown to be essential for the formation of BRCA1 and Rad51 foci, and for gene conversion.49,50
Consistent with impaired HR, CDK1 inhibition (by RO-3306 and AG024322) decreased formation of Rad51 and BRCA1 foci and gene conversion.50
As expected of cells with defects in HR, all groups of the aforementioned inhibitors were found to sensitize cells to radiation, chemotherapy drugs, and PARP inhibitors.47,48,50
In principle, screens aimed at identifying HR inhibitors can either be target-based, i.e., they search for inhibitors of a specific enzyme known to be involved in HR, or they can be phenotypic, i.e., based on observing inhibition of a cellular phenotype that is dependent on HR. Because of the great complexity of HR, most high-throughput chemical screens are justifiably based on the phenotypic approach. This enables them to detect compounds that may have unknown or multiple enzymatic targets, rather than requiring the screen to identify only inhibitors of one or more chosen targets. Other reasons contributing to the success of the phenotypic approach are in the utilization of cell-based assays relevant to the human disease. Examples of successful phenotypic screens that resulted in identification of small molecule inhibitors of HR include a screen based on phosphorylation of a peptide derived from histone H2AX in cell-free extracts derived from Xenopus laevis
Compounds from this screen, which potentially target any step leading to H2AX phosphorylation, have been narrowed down to the compound mirin, which has been shown to interfere with the nuclease activity of Mre11 and subsequently with the MRN-dependent activation of ATM, impairing HR repair.45
Another fruitful approach was based on inhibition of the assembly of FANCD2 foci in response to cross-linking drugs and ionizing radiation.51
The FANCD2 foci formation happens after FANCD2 is monoubiquitinated by a complex of Fanconi Anemia proteins, and the foci are believed to be the sites of HR repair. Among new compounds inhibiting formation of FANCD2 foci were protein kinase inhibitors (wortmannin, H-9 and alsterpaulone) and a natural compound, curcumin.51
Further expansion of the screening library identified more compounds, which included proteasome inhibitors, CDK inhibitors, and an HSP90 inhibitor, well representing the main categories of compounds known to inhibit HR (see above). Significant overlap in terms of proteins involved must exist among the different subpathways of HR, as compounds known to sensitize cells to interstrand crosslinking (ICL) agents often impair Rad51 foci formation and/or gene conversion, and vice versa (refs. 41, 47, 51
and our observations). Our own strategy to screen for inhibitors of HR was based on increased sensitivity to the ICL-inducing agent chlorambucil (Chernikova and Brown, unpublished observations). To screen against compounds that would cause sensitivity to ICL by inhibiting other pathways involved in repair of ICL, i.e., nucleotide-excision repair (NER) and/or translesion synthesis, we re-screened compounds testing positive for sensitization using a gene conversion assay and a Rad51 foci formation assay. Both the gene conversion assay and Rad51 foci formation assay are amenable to the high-throughput format, therefore other approaches may include screening compound libraries using these assays initially. As more HR proteins are characterized and more compounds identified that impair each subpathway of HR, we will have a better overview of the complex HR system.
Although, as mentioned, no inhibitors of proteins directly catalyzing the HR reactions are yet available, the situation might change soon as several potential inhibitors of the Rad51 recombinase activity have recently been identified in an in vitro target-based screen.52
Still at the initial stages of characterization, these compounds have been shown to inhibit IR-induced formation of Rad51 foci and gene conversion (A. V. Mazin, personal communication).