Potential Strategies to Target ProteinâProtein Interactions in the

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Potential Strategies to Target Protein−Protein Interactions in the DNA Damage Response and Repair Pathways Naoaki Fujii* Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS1000, Memphis, Tennessee 38105, United States ABSTRACT: This review article discusses some insights about generating novel mechanistic inhibitors of the DNA damage response and repair (DDR) pathways by focusing on protein−protein interactions (PPIs) of the key DDR components. General requirements for PPI strategies, such as selecting the target PPI site on the basis of its functionality, are discussed first. Next, on the basis of functional rationale and biochemical feasibility to identify a PPI inhibitor, 26 PPIs in DDR pathways (BER, MMR, NER, NHEJ, HR, TLS, and ICL repair) are specifically discussed for inhibitor discovery to benefit cancer therapies using a DNA-damaging agent.

1. INTRODUCTION: WHY PPI TO TARGET DDR? Many studies have shown the potential for developing new anticancer drugs by focusing on DNA damage response and repair (DDR) pathways. Regardless, still relatively few molecules in DDR pathways have been targeted for therapeutic purpose. Most strategies for targeting DDR pathways remain rather indirect approaches (e.g., targeting ATM or ATR kinases for DDR-mediated cell cycle). So many papers conventionally address that “protein A is a great drug target for disease B”, but few of them propose how to actually target the protein, especially for DDR. One reason for this is that many DDR proteins are unconventional drug targets, therefore, targeting them is not easy. Drugs acting on traditional targets such as cell surface receptors (e.g., G-protein coupled receptor, ion channel) and conventional enzymes (e.g., proteases, kinases) could have been identified by an established screening protocol, in which many assay kits are commercially available. In contrast, the DDR enzymes are often functionally unconventional (e.g., DNA helicase, polymerase, nuclease, ligase, lyase, etc.), and their enzymatic assays are often difficult for inhibitor screening. In addition, several important DDR molecules are very large (e.g., ATM, 351 kDa; BRCA1, 214 kDa) and difficult to purify for large-scale screening. Furthermore, the instability of DDR enzymes often makes the assay intolerable to the DMSO vehicle required for screening of chemical compounds. This article proposes to targeting DDR by developing chemical inhibitors of protein−protein interaction (PPI). PPI is important for regulating DDR because DDR components, including enzymes and scaffolding proteins, must be organized at the damaged DNA on chromatin. Disruption of PPI in the multiprotein complex for DDR can make this process fail, leading to DDR inhibition. Possible advantages of the PPI inhibition strategy are as follows. First, PPI assays can be easier and more robust than enzymatic assays in vitro. Second, in principal, a PPI inhibitor can be used to target anything, including nonenzymatic proteins, which opens the door to © 2017 American Chemical Society

entirely novel therapeutics (“drugging the undruggable”). Third, specific PPI of a single protein can be targeted to modulate only a specific function that the PPI coordinates. This is important for considering functional outcome because many proteins are multifunctional, coordinating multiple PPIs to define downstream pathways. Furthermore, protein posttranslational modifications (PTMs) add another layer of PPI regulation that offers further options to specifically inhibit only a protein function activated by the PTM. This article focuses on specific PPIs of functionally important and potentially targetable molecules of each DDR pathway for therapeutic purposes. This article does not extensively overview DDR biology, which numerous excellent review articles have done. Instead, this article discusses specific PPIs as potential targets by indicating the feasibility of targeting them and the therapeutic rationale and justification. To avoid confusion, PPIs are indicated with a hyphen (e.g., A-B). The names of each DDR molecules are those of the molecule in mammalian cells, and the alternative name commonly used is indicated by parentheses (e.g., A(B)). When the name of an enzyme in this review could refer to either DNA or RNA, it is used to refer to the DNA enzyme unless specifically noted (e.g., polymerase means DNA polymerase). The word “molecule” is used for a protein component of a signaling pathway, “small molecule” for a chemical compound and drug, and DNA-damaging “agent” for anything that damages DNA (e.g., UV, ionic radiation (IR)).

2. THERAPEUTIC RATIONALE FOR TARGETING DDR PATHWAYS Numerous excellent articles have reviewed DDR pathways comprehensively from therapeutic perspective (for instance, ref Received: March 6, 2017 Published: June 27, 2017 9932

DOI: 10.1021/acs.jmedchem.7b00358 J. Med. Chem. 2017, 60, 9932−9959

Journal of Medicinal Chemistry

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Table 1. Drugs, Types of DNA Damage, and DDRa drug

a

DNA damaging mechanism

types of DNA damage

main DDR

temozolomide decarbazine procarbazine

monofunctional

monoalkyl

BER, MMR, TLS

cyclophosphamide ifosfamide busulfan carmustine melphalan

bifunctional

monoalkyl intrastrand-X ICL

BER, TLS, MMR? NER, TLS ICL repair

cisplatin carboplatin oxaliplatin

bifunctional


BER, MMR, TLS NER, TLS ICL repair

mitomycin C psoralens+UVA

bifunctional


BER,TLS, MMR? NER, TLS ICL repair

doxorubicin daunorubicin

intercalation

DSB (partly)

NHEJ, HR, others?

topotecan irinotecan

radiomimetic

DSB DPC?

NHEJ, HR DPC repair?

etoposide mitoxantrone

radiomimetic

DSB

NHEJ, HR

fluorouracil

incorporation

monoalkyl

BER, MMR?

gemcitabine cytarabine

incorporation

others

HR?

olaparib veliparib

indirect

SSB DPC

BER? DPC repair?

Psoralen+UVA is not a cancer drug but commonly used for studying ICL repair. Intrastrand-X: intrastrand crosslinking.

1), in which they are generally categorized as BER, MMR, NER, NHEJ, HR, and TLS. TLS is not a mechanism of DNA repair but DNA damage tolerance (DDT) that some literature uses this terminology. This article uses DDR to refer to TLS. ICL repair is a mechanistic combination of DDR pathways but is often studied as a single subject by focusing on the FA pathway. Some proteins are shared in multiple DDR pathways (e.g., PCNA in BER (long-patch), MMR, NER, and TLS). The DDR pathways can be functionally redundant and act as backup mechanisms to each other (e.g., NER and TLS for intrastrand DNA cross-link, NHEJ and HR for DSB). Functional redundancy is therapeutically very important because it can eliminate the therapeutic effect of an inhibitor of a molecule by another molecule as a backup mechanism (i.e., not “addictive” to the target molecule = resistance). Some considerations for targeting DDR are discussed below. Environmental DDR: An Unwanted Target. Several types of DNA damage are naturally generated in daily life. Such DNA damage includes base deamination, nucleoside hydrolysis, oxidative damage, and sunlight-induced damage. Base deamination results in sequence mismatching by base conversion, which is a substrate for MMR. The apurinic/apyrimidinic/ abasic (AP) site generated by hydrolysis of nucleoside glycosidic bond and 8-oxoguanine (oxoG) generated by

guanine oxidation are a substrate of BER. The cyclopyrimidine dimer (CPD) and (6−4) photoadduct of thymidine are generated by intrastrand guanine dimerization by sunlight UV and are substrates of NER and TLS. Ideally, DDR pathways responsible for such environmental DNA damage should remain intact. However, this could be difficult to achieve for therapy because the DDR mechanisms for environmental DNA damage and that for therapeutic DNA damage are often common. The presence of some common man-made chemical carcinogens (e.g., tobacco smoke) further complicates this dilemma because DNA damage by such carcinogens may also be repaired by a DDR for therapeutic DNA damage. It could be an open discussion whether disrupting such a DDR should be absolutely avoided or allowed under a need of urgent treatment as long as it is therapeutically efficacious and reasonably nontoxic. Cancer Therapy-Resistance DDR. Diverse structures of DNA damage by chemotherapeutic drugs that alkylate DNA bases are often classified into three types: monoadduct, intrastrand cross-link, and interstrand cross-link (ICL). This system makes it easy to determine which DDR mechanism repairs which type of damage (Table 1) and, thus, which DDR can be targeted to inhibit the repair. Although relatively few studies address the relationship between the structure of DNA 9933



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3. HOW TO JUSTIFY AND PROCEED WITH A PPI INHIBITOR PROJECT The PPI strategy requires some precautions that are obvious but often missed in many studies. Some key questions are asked below. Is the PPI Druggable for Cellular Functions? The PPI must be biochemically and f unctionally druggable. Here, “biochemically druggable” means that the PPI can be effectively disrupted by a small-molecule compound, and “functionally druggable” means that the PPI disruption disables the f unction of the target in the cells. Both are indispensable for the success of the PPI project and are very important to consider specifically. Genetic elimination of the target (e.g., siRNA) is not perfect evidence to support functional druggability of a PPI because PPI inhibition does not eliminate the target.4 Even if genetic elimination of a DDR molecule influences the DDR pathway activity, it is not sufficient as a PoC because the specific PPI of the protein to be targeted can be dispensable for the function. Functional disability of the PPI-deficient mutant of the target (possible by gene editing) is considerably the best PoC evidence. The second-best PoC would be observation of a similar severity of functional defect in cells in which either of two proteins of the PPI is genetically eliminated. Such information in the literature could be supportive, but the functional effect is often different among cells and experimental conditions, so that should be revalidated specifically in cells and/or models of interest. Is the Assay Right for PPI Inhibition? One must assay the compounds for inhibition of target PPI. This sounds a matter-of-course but is often missed. Direct binding (e.g., NMR, SPR, ITC, thermal stabilization) does not mean PPI inhibition. A compound could bind to another site of the target protein that does not contribute the PPI (e.g., nonspecific binding). Therefore, discovery of a PPI inhibitor is possible only when an interaction partner of the target protein is known and a reliable PPI assay is available. The initial assay for the PPI inhibition can be for acceptable surrogates for the full-length protein interaction, such as interaction of the truncated target protein domain with a short sequence peptide of the interaction partner. However, the affinity of such surrogates can be weaker than that of the full-length protein interaction because of extra interaction outside the surrogate sequence, conformational facilitation of the PPI by additional proteins existing in cells, and so on. Therefore, it is important to confirm the interaction inhibition of the full-length PPI in cells if possible (e.g., co-IP, FRET, PLA). Is a Structure-Based Strategy Reliable? Rational design of a PPI inhibitor from the known structure of a target protein is a common practice but is not always successful because a target structure can be different from the known structure when the target binds to a compound. Often, so-called “induced fit” by a compound is observed, in which some amino acid residues and polypeptide backbones are moved to uniquely host the compound by a new structure. Is a Hit Compound Able to Be Optimized? Affinity of some PPIs is intrinsically modest. Generating potent inhibitors of a modest PPI sounds easy but may not be because such a PPI could be inhibited by divergent compounds with modest affinity. This means that the structure−activity relationship (SAR) for inhibiting such a PPI is unclear, and thus potent inhibitors are not easily identified even if lots of modest inhibitors are soon identified. A common strategy for

damage and the DDR pathway, the size of the damage and difficulty of the repair seem to be important factors for selection of the DDR pathway. For instance, cisplatin generates all three types of DNA lesions, and BER, MMR, NER, and TLS coordinate as DDRs for cisplatin-induced damage. Some radiomimetic drugs (e.g., topotecan, etoposide) induce a double-strand break (DSB) rather than alkylate DNA. Accordingly, NHEJ and/or HR are responsible for repair after radiomimetic drugs. Ionic radiation also induces other DNA damage types (e.g., single-stranded break (SSB), oxidative damage), but they are much less lethal than DSB and, thus, receive less attention from a therapeutic perspective. Cell-Cycle-, Replication-, and Cell Proliferation-Dependent DDR. Because aggressive proliferation is a feature of cancer cells, cell-cycle inhibition has been a classical strategy for molecular cancer therapeutics. Similarly, there are possible strategies with which to target DDR unique in proliferating cells to selectively target cancers. The main driver of cell proliferation is DNA replication in S-phase; therefore, only DDRs in S-phase or later2,3 could be potential targets for selective elimination of cancer cells. TLS and HR can be such DDRs: TLS is a process of DNA replication over damaged template strand, and HR uses a replicated DNA strand as the template for homology search. Another possible strategy for targeting DDR selectively in cancer cells is to focus on transcription. Although transcription is not a cancer-specific activity, demanding transcription for aggressive cancer cell growth could justify this rationale. For instance, actinomycin D, a transcription inhibitor, has been used as an anticancer drug. The transcription-coupled NER (TC-NER) is known to be mechanistically linked to the transcription. However, DDRs that occur only in S-phase or coupled to transcription may not be efficacious because cancer cells could use other DDRs. Mutagenic DDR. An important aspect of DDRs is mechanistic mutagenicity. A new mutation generated from DNA damage by chemotherapy is a potential cause of neoplasm leading to secondary cancers. Therefore, a mutagenic DDR is a target for preventing this. The mutagenicity is due to loss of the intact template strand from which to recover the original sequence. TLS and NHEJ are possible without the intact template strand. Many TLS are thus mutagenic because they insert incorrect nucleotides counter to the damage. NHEJ can ligate two DSB ends irrespective of the sequence after trimming some nucleotides to enable the ligation and thus can generate the sequence deletion and even could translocate the chromosome. Polypharmacology Approach for DDR Targeting. An emerging concept for improving therapeutic efficiency is to concurrently target multiple signaling pathways (polypharmacology) that are not epistatic but functionally complement each other. Inhibition of two such pathways can be synergistic and especially useful for DDR-targeting therapeutics because many DDR molecules are functionally redundant, i.e., targeting a single DDR molecule could be inefficacious because another DDR molecule can serve as a backup, leading to therapeutic resistance. A possible strategy is to inhibit a molecule that acts in multiple DDR pathways, although this could cause some toxicity if such a DDR molecule is also functional in a nonDDR pathway. Because there are still few strategies to rationally create a “one stone, two birds” compound, the realistic polypharmacology approach would be to carry out two screenings for each DDR molecule to identify compounds active for both. 9934



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short-patch BER for selectively targeting cancer cells. However, some chemotherapy drugs induce a modified base that could be removed by short-patch BER. For instance, 5-FU is metabolically incorporated into the DNA as an artificial base, which is removed by a uracil glycosylase.9,10 Ideally, inhibitors of each short- or long-patch BER could be chosen for a specific drug to maximally sensitize cancer cells. At least 11 glycosylases (e.g., Ogg1) carry out the base removal in mammalian cells. These glycosylases seem functionally redundant: mice in which a single glycosylase is genetically eliminated are normal except that they accumulate 8-oxoG.11 Therefore, the glycosylases are less likely to be functionally druggable (unless a small molecule can inhibit multiple glycosylases that are functionally redundant). XRCC1 is the central scaffold containing domains for many PPI with several BER enzymes.12 Genetic elimination of XRCC1 decreases SSB repair activity and makes the cells ∼2-fold sensitive to MMS.5,13 XRCC1 also has the capability to interact with molecules for nonclassical NHEJ.14 Key XRCC1 PPIs for BER include those with APE1, Polβ, and LigIII. However, LigIII may not be functionally druggable because genetic elimination of LigIII did not alter comet-tailing of the cells treated with hydrogen peroxide or IR.15 XRCC1-APE1. A study revealed that the XRCC1-APE1 PPI and endonuclease activity of APE1 is enhanced by XRCC1 in vitro,16 suggesting a possible strategy to inhibit APE1 function by targeting this PPI. APE1 lacking the first 35 AA completely abolished the PPI,16 suggesting that the interaction site of APE1 for XRCC1 is in these 35 AA, which could be used as the screening probe to identify an inhibitor of the PPI. A GST pulldown assay revealed that XRCC1(141−572) is sufficient for interaction with APE1 and, therefore, could be used as the protein material for the PPI assay. Unfortunately, no structural information or detailed analysis of sequences required for the PPI is yet available, therefore, nonrational screening strategies will be required to identify a PPI inhibitor. A functional assay reasonable for this PPI would be accumulation of AP species in the cells, measurable by a standard protocol using a tagged hydroxylamine reagent, because inhibition of APE1 recruitment to XRCC1 should prevent the excision of the AP site. XRCC1-Polβ. Genetic elimination of Polβ sensitizes cells to oxaliplatin17 and temozolomide,18 which is supportive evidence to justify targeting Polβ for therapy.19 Given that few existing Polβ inhibitors are satisfactory in terms of potency and druglike property,20 PPI inhibition can be an alternative strategy for inhibiting the Polβ functions in BER. Biochemical and structural characterizations indicate that the N-terminal domain of XRCC1 binds to Polβ. Mutational analysis has identified several residues (F67, E69, Y136) of XRCC1 that are essential for the Polβ PPI21 and would be targetable for PPI inhibition. The nucleoside-binding loop of Polβ, approximately 25 AA containing V303 as a key interaction residue, binds to these XRCC1 residues,22,23 which could be exploited to validate the binding affinity and utilized as a Polβ peptide probe for screening by a biochemical binding inhibition assay (e.g., FP, FRET) to find inhibitors. A structure of this PPI22 reveals that a cavity of XRCC1 adopts the Polβ nucleoside-binding loop23 (Figure 2), which could be targeted by a compound. Functional verification of the hit compounds should include both XRCC1 and Polβ. Assays using Polβ only in vitro (e.g., enzymatic Polβ activity) are invalid for this PPI inhibition. The inhibition of this PPI theoretically accumulates SSB after excision of AP site, therefore, a reasonable functional assay for inhibiting this PPI

improving potency of a modest inhibitor is the addition of extra side chains that would induce additional interactions to the target site, but it increases the molecular size and may decrease ADMET property of the compound. Another strategy is to identify allosteric inhibitors, but it is often harder than finding on-site inhibitors, and importantly, it is uncertain whether an allosteric inhibitor can be efficacious as an on-site inhibitor in cells.

4. TARGETING PPI TO INHIBIT BER BER repairs DNA base damage that is small and does not distort the strand helix, such as damaged bases generated endogenously (e.g., 8-oxoG, deaminated A= hypoxanthine) and monoalkylated bases. Once such a damaged base is found, a glycosylase removes it and generates an AP site. APE1, an endonuclease specialized for AP sites, cuts the DNA backbone near the site to remove a few nucleotides and generate a singlestranded patch that is structurally a single-stranded break (SSB). The patch is next filled by a Polβ using the intact strand as the template, and the nick left in the backbone is sealed by a ligase. Polβ is a polymerase for BER and does not contribute to regular DNA replication. XRCC1 is the central scaffold protein that coordinates these BER components. (Figure 1) An

Figure 1. Simplified BER (short-patch) pathway and molecules discussed for PPI inhibition. Diamond (magenta) represents a damaged base. Molecules that are not discussed in this chapter are not shown.

important aspect of BER is that BER is also a mechanism for SSB repair because SSB is an intermediate species of BER. Indeed, XRCC1 is required for SSB repair.5 Because SSBs are commonly generated by DNA-damaging therapies (alkylating drugs and IR), BER is a potential mechanism for resistance of cancers to the therapies. There are two mechanisms at the later stage of BER: shortpatch BER and long-patch BER. For short-patch BER, the phosphoribose remnant from the AP site is removed by the 5′dRP lyase activity of Polβ to enable the ligation of the patchfilled strand. For long-patch BER, the cut strand is deannealed (not excised) by up to ∼10 nucleotides by the primer extension by Polβ,6 followed by trimming the short flap structure by FEN1 to enable the final ligation.7 The process in long-patch BER is mechanistically shared with the regular DNA replication and therefore could be favored at S-phase.8 A comprehensive analysis also supports this because long-patch BER is upregulated in G1/S-phase, whereas short-patch BER is cellcycle-independent. 3 Because active DNA replication is indispensable for cell proliferation, long-patch BER can be more active in growing cancer cells than in dormant normal cells and thus could be a better potential DDR target than 9935



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substrate.34 Therefore, inhibiting MMR induces mutagenesis and can be potentially harmful for immunologic response. MMR is required for the toxicity of some anticancer DNA damaging drugs that generate 6-methylguanine adducts,35 and MMR inactivation promotes resistance against such drugs.36 Therefore, MMR could be a less plausible therapeutic target. Another notable MMR function is HR control. A mechanism of this has been validated in yeast as an interaction of Mut with the intermediate of HR containing mismatches during the strand exchange.37 Functional defects in MMR can induce resistance of cancer cells against chemotherapeutic drugs, as seen in colon cancer cell lines.38 At a glance, the situation appears to be the opposite: DDR def iciency allows cells to survive with DNA damage. A model proposed to understand this contradiction is the function of an MMR protein as an apoptosis mediator.39−43 MMR-deficient colon cancers acquire resistance to surprisingly divergent chemotherapy drugs.42,44 Therefore, at a glance, MMR seems not to be warranted as a chemotherapeutic target, but this may not be true because the majority of DNA damage by those divergent drugs is not an MMR substrate; some of the drugs do not even induce DNA damage (e.g., antimetabolites). Therefore, the resistance against those drugs in colon cancers is largely irrelevant to MMR and likely due to a defect of the apoptosis function of an MMR component. Selective inhibitors of the PPI of a Mut heterodimer could be inhibitory for MMR but not the apoptosis (i.e., decouple the MMR and apoptosis functions). A functional assay for inhibiting PPI of the MMR components could be an MMR assay using a DNA substrate45 or a reporter plasmid46 containing a mismatch. MSH2-MSH6 (MutSα). MSH2 binds to MMR components (MSH3, MSH6) and DDR-mediating kinases (ATR, CHK1, CHK247−49) that may be upstream regulators of DDRmediated apoptosis (Figure 3). Targeting functions of MSH2

Figure 2. PPI structure (3lqc.pdb) of XRCC1 (gray, surfaced)-Polβ (light cyan, cartoon). The nucleoside-binding loop of Polβ containing P300, V303, and V306 (magenta sticks) interacts with a cavity of XRCC1 in which F67, E69, and Y136 (that are essential for the Polβ PPI, yellow) locate.

would be an alkaline comet assay controlled by a neutral comet assay that measures DSB only. XRCC1-REV1. XRCC1 interacts with REV1,24 which is a TLS component (see the TLS chapter) and also a backup BER component.25 This PPI has been both structurally and biochemically validated.24 XRCC1 has a short sequence named REV1-interacting region (RIR) motif that is found commonly in polymerases for TLS (see section for REV1-RIR in the TLS chapter and Figure 15). XRCC1 may compete with those TLS polymerases on REV1 to prevent REV1-mediated TLS activation. A short (∼20 AA) peptide containing XRCC1 RIR binds to REV1 C-terminus domain in Kd = 4.3 μM, which can be used as a probe for inhibitor screening (e.g., FP, FRET). Upon the REV1 binding, the RIR peptide forms a small helix that is structurally similar to the VP16-like transcription activation domain (TAD) found in many transcription factors. Numerous studies have generated small-molecule mimetics of TAD (e.g., a mimic of p53 TAD26); some of these compounds could be inhibitory to the XRCC1 REV1 PPI. A functional assay for XRCC1-REV1 PPI inhibitors could be a reporter reactivation assay using a plasmid that has been challenged by MMS that generates a DNA damage substrate for BER but not TLS.

5. TARGETING PPI TO INHIBIT MMR MMR corrects mismatched base pairs generated by either incorporation of a wrong nucleotide during DNA replication or extension/deletion of the sequence forming an unannealed loop. In mammalian cells, the DNA structure of the mismatched portion is first recognized by a Mut heterodimeric protein complex followed by cleavage of adjacent of the mismatched portion to form a single-stranded patch, which is then filled by the PCNA machinery shared with regular DNA replication. Three Mut heterodimers have been characterized well: MutSα (MSH2-MSH6), MutSβ (MSH2-MSH3), and MutLα (MLH1-PMS2); all are ATPase. MMR is a background DDR for oxidative DNA damage (e.g., 8-oxoG) and a process for chromatin quality control, together, they remove DNA replication stress and prevent mutagenesis by the mismatch. Indeed, deficiency of any proteins composing the Mut complex generate microsatellite instability in cells and a tumorigenic phenotype in animals and humans.27−33 In addition, GU mismatch being generated by activation-induced cytidine deaminase (AID) in Ig class switching is also a MMR

Figure 3. Two Mut complexes discussed for PPI inhibition and the downstream pathways for apoptosis induction.

only for the MMR pathway activation but not for the DDR kinase regulation could be therapeutically valuable. This strategy could be possible by inhibiting an MSH2 PPI specific to MSH6, leaving MSH2-mediated apoptosis intact. MSH2(G674A) and MSH6(T1219D) mutants are defective in MMR.45,50 By characterizing MSH2(G674A) for tumorigenesis and apoptosis, it was shown that MSH2’s MMR and apoptosis induction functions can be decoupled.51 These MSH2 mutants are a good model for the PoC validation. If cells expressing MSH2(G674A) are not resistant against chemotherapies, MSH2 PPI defected by G674A mutation can be functionally druggable to inhibit chemoresistance. A study has extensively 9936



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mapped domain structures of the MSH2-MSH6 complex,52 in which MSH2 G674 and MSH6 T1219 are facing each other (∼13 Å) at the MSH2-MSH6 PPI interface (Figure 4). This

were found in Lynch syndrome and truncation constructs of MLH1 and PMS2.59−61 These studies revealed the importance of the C-terminus region of both MLH1 and PMS2 for the PPI and MMR activity. MLH1(506−756) strongly binds to PMS2 but MLH1(531−756) does not, indicating the importance of MLH1(506−531) for the PPI.59 However, in another study, a bioinformatics approach suggested a hypothetical model in which MLH1(531−549) and (740−756) interact with PMS2.60 The truncation proteins for the C-terminus regions of MLH1 and PMS2 could be validated to establish the PPI assay by using a strategy similar to that described for MSH2-MSH6 in the previous section.

6. TARGETING PPI TO INHIBIT NER NER repairs large and helix-distorting damage. NER-defective cell lines [e.g., xeroderma pigmentosum (XP) cells] are hypersensitive to DNA damage. NER first excises the damaged portion to generate a 25−30-nucleotide single-stranded patch to resolve the distortion that demands endonucleases. The excision is performed by coordination of at least two key endonucleases: XPF cuts the 5′-side and XPG cuts the 3′-side of the damage (Figure 5). The single-stranded patch generated

Figure 4. PPI structure (2O8C.pdb) of MSH2 (gray, surfaced)-MSH6 (light cyan, cartoon). Regions that are not on the PPI interface are omitted for clarification. An ADP (yellow stick) and a magnesium ion (green) are on a cavity on MSH2 G674 (magenta). MSH6 T1219 (magenta stick) is close to the cavity.

conformation suggests that MSH2-MSH6 PPI could be disrupted by targeting MSH2 or MSH6 at this site. The ADP cavity in MSH2 is directly on G674. ATPγS significantly protects the yeast msh2-msh6 complex from trypsin digestion.53 These observations may suggest that the ADP induces conformational changes of each MSH2 and MSH6 to facilitate the PPI, which can be a clue to how to rationally target the PPI. For instance, large ADP analogous compounds that bind to these cavities but are bumped out from the cavity could efficiently block the PPI. The PPI interface is composed of partial structures of multiple regions in each MSH2 and MSH6. Thus, screening for the PPI inhibitor requires a protein probe for each of them. The probes could be chimeras of PPI regions of MSH2 and MSH6 as long as the interaction is verified (e.g., via AlphaScreen, HTRF, IP, SPR, or ITC). Ideally, the assay would be better so that it could be performed by using fulllength MSH2 and MSH6 and adding a mismatch-stranded oligonucleotide duplex. This method would allow one to find a compound that can inhibit the PPI on a mismatched DNA and, therefore, could be a bona fide inhibitor of MMR, although preparation of full-length MSH2 and MSH6 would be challenging. MLH1-PMS2 (MutLα). A strategy analogous to that for MSH2-MSH6 could be applied to another MMR complex organized by MLH1 PPI. Activations of ATR, ATM, and caspase-3/7 are significantly lower in MLH1-deficient cells than in MLH1-proficient isogenic cells after UVA irradiation,54 indicative of a role of MLH1 in DDR-mediated apoptosis (Figure 3). PMS2 forms the MutLα complex with MLH1, but other MMR components (e.g., PMS1, MLH3) can be substituted for PMS2 to form other complexes,55 which could be functional for MMR.56,57 Therefore, for therapeutic purposes, it is desirable to block all MLH1 PPIs that form MMR complexes. Genetic elimination of PMS2 also sensitizes cells not only to DNA-damaging chemotherapies (e.g., cisplatin, etoposide) but also to nonDNA-damaging anticancer drugs (e.g., taxanes).58 This indicates that the cell death promoted by suppression of PMS2 is likely to be independent of MMR and therefore could be irrelevant to MLH1, although it could be another therapeutic opportunity. The MLH1-PMS2 PPI has been characterized by analyzing point mutants of MLH1 that

Figure 5. Simplified NER machinery for excision of a DNA lesion (magenta star) and molecules discussed for PPI inhibition. Molecules that are not discussed in this chapter are not shown.

is then filled by the machinery shared with regular DNA replication (i.e., PCNA-guided patch filling62). In a time-course experiment, XPG was recruited to the damaged DNA template before the factors for patch-filling were recruited.63 XPG is a large multifunctional protein that also functions in HR.64 XPF forms a complex with ERCC1, and this complex’s function is indispensable for ICL repair (see the ICL repair chapter). Because NER is indispensable for removal of a DNA intrastrand cross-link and for unhooking of an ICL, targeting NER has received much attention for selectively sensitizing cancers to drugs that cross-link DNA (e.g., cisplatin, cyclophosphamide). In theory, NER is a process independent of the cell cycle, but later steps of NER are mechanistically shared with regular DNA replication as described above. Therefore, an NER inhibitor mechanistically could show some selectivity for sensitizing proliferative cancer cells rather than dormant noncancerous cells. Two mechanisms for NER initiation are known: globalgenome NER (GG-NER) and transcription-coupled NER (TCNER). GG-NER senses a helix distortion irrespective of transcriptional activity on the DNA lesion. In TC-NER, RNA PolIII detects the damage during transcription of the damaged DNA portion. Studies indicated that activity of GG-NER is reduced in differentiated cells.65,66 If differentiated cells demand GG-NER less than undifferentiated cells do, GG-NER could be a better tool than TC-NER to selectively eliminate dedifferentiated cancer cells. However, TC-NER can serve as a backup of GG-NER; therefore, targeting only GG-NER may not be therapeutically efficacious. Patients with xeroderma pigmento9937



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sum (XP) group C mutations (XPC mutant: disabling GGNER67) have a high incidence of skin cancer, suggesting a possible risk in selectively targeting GG-NER for tumorigenesis. Further, XPC facilitates apoptosis induced by DNA-damaging drugs,68 indicating that it is not plausibly druggable for chemotherapeutic purposes. Because of these functional outcomes and because there are only a handful of DDR molecules known to act selectively in GG-NER, few studies have intentionally targeted GG-NER for therapeutic development. Functional assays for NER inhibition have been developed ex vivo by using a DNA duplex containing NER substrate lesions (e.g., CPDs) to analyze the excision by gel electrophoresis69 or Comet tailing.70 An imaging-based assay using an engineered plasmid containing an NER substrate can quantify the NER activity by fluorescence emission.71 These assays are steady but low-throughput and unsuitable for inhibitor screening. Hostcell reactivation assays using a UV-damaged reporter plasmid72 are scalable and practical for screening purposes and can even be multiplexed to assay other DDR activities concurrently,73 but this assay generates signals either by NER and TLS. Therefore, it needs to be controlled for by either assaying in NER-deficient cells [e.g., XPA−/− cells] or by performing another assay using a hairpin DNA duplex containing a lesion for NER in the cells, with the recovered DNA analyzed by qPCR,74 which distinguishes between the NER and TLS because TLS leaves the lesion that prohibits the PCR. XPA-RPA. XPA interacts with RPA70 and RPA34, and a coIP study has shown that RPA70 binds more predominantly than RPA34 does.75 DNA- and XPA-binding regions of RPA70 have been structurally characterized.76 Chemical inhibitors of DNA-binding of RPA70 have been validated for chemotherapeutic sensitization.77−80 The XPA-RPA PPI has been shown to be essential for efficient NER81,82 and, therefore, may be another druggable target. This hypothesis has been preliminarily validated by using anti-XPA antibody83 and point mutants of XPA:84 both are inhibitory for the PPI and for NER. The PPI-defective XPA mutations indicate a possible PPI interface that could be targeted. XPA(4−29) and XPA(98− 187) are required to interact with RPA34 and RPA70, respectively.75,81 In an HSQC NMR titration experiment of the C-terminus region of XPA with RPA70ΔC327 [RPA70(1− 326)], an RPA truncate that was previously characterized as weakly binding to XPA,85 a zinc-binding core of XPA76 was shifted uniquely.86 Although no structures of the XPA-RPA complex are known to date, these NMR mapping studies show that some XPA residues are shifted by the RPA PPI. This information could be utilized to develop assays for inhibitor screening of this PPI by using constructs encoding each RPA70ΔC327 and the XPA C-terminus region containing the zinc-binding core. XPA-ERCC1. ERCC1 and XPA each interact through their N-terminus regions.87 DNA interaction of XPA is facilitated by the ERCC1 PPI.88 Biochemical characterization has identified XPA mutants that are defective in this PPI and in DNA repair synthesis ex vivo and sensitize cells to UV,89,90 serving as justification for targeting the PPI. Some compounds identified in silico were characterized for direct binding to the truncated ERCC1 protein and for sensitizing cancer cells to UV.91 Studies characterized the structural outcome of this PPI:92−94 regions of ERCC1 for XPA(59−99) PPI were concentrated in the central region containing Q107, L139-N147, and R156 (Figure 6), which are distinct from the residues for DNA binding and,

Figure 6. Structure (2jnw.pdb) of ERCC1 (gray, surfaced)-XPA(67− 77) peptide (light cyan). The XPA peptide forms a sharp turn structure containing F75, I76, and L77 (sticks) and interacts with a cavity of ERCC1 containing F140−L148 (yellow).

therefore, ERCC1 is capable of binding both XPA and DNA concurrently. The apparent Kd of the PPI is 1 μM.92 Another study solved the structure of the complex ERCC1(96−241), in which a short XPA(67−80) peptide binds to a V-shaped groove containing the ERCC1 amino acid residues described above.93 These structural and biochemical outcomes are desirable properties for PPI inhibitor discovery, in which both the ERCC1 structure-based virtual screening91 and conventional biochemical screening using the XPA peptide probe (e.g., FRET, FP) could be applied. XPF(ERCC4)-ERCC1. This PPI has received much attention as a target because many studies have shown that this complex is essential for the first incision step of NER and that genetic elimination of either XPF or ERCC1 dramatically sensitizes cancer cells to DNA-damaging drugs, especially those that generate ICL (e.g., cisplatin).95−97 Excellent review articles have extensively discussed strategies to target this PPI,98−100 and many studies have already reported chemical inhibitors of this complex including ones inhibiting the PPI (Figure 7).101−105 In the studies, chemical library screenings were performed based on PPI assays using appropriate truncates of each XPF and ERCC1, and in silico strategies based on the structures this PPI106 were also performed.

Figure 7. A docking pose of a XPF-ERCC1 PPI inhibitor102 (orange stick and lower right) on a structure (2a1j.pdb) of XPF(837−898) (gray, surfaced) complex with ERCC1(218−296) (cartoons). Regions that are not on the PPI interface are omitted for clarification. Each dimethoxyphenyl and acetoxyphenyl moiety mimics ERCC1 F293 and I264 (sticks) which exist on different α-helices (light blue and green, respectively) and interact to different cavities of XPF. This model demonstrates a potential mechanism for inhibiting a multiregion PPI by a single small molecule. 9938



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XPG(ERCC5)-TFIIH(p62). The responsible subunit of TFIIH for XPG PPI is p62. Functional implication for this PPI was originally suggested by the existence of Cockayne syndrome (XP-G/CS), in which XPG is mutated and defective in TC-NER. A series of papers reporting this study were unfortunately retracted, but this PPI was later characterized; the role of this complex in NER was demonstrated initially in yeast107 and later in mammals.108 The PPI complex also regulates transcriptional events. XP-G/CS shows defects in transactivation of nuclear receptors109 and expression of EGFresponsive genes.110 A region required for the PPI is XPG(225−231), as discovered by analyzing a unique CS genotype in which this region is missing. Cells expressing XPG truncated in this region are ∼10-fold sensitive to UV than are those expressing wild-type XPG, regardless of the fact that the 3′-incision activity and recruitment to the chromatin CPD foci of the mutant and wild-type protein are the same.111 TFIIH p62(1−108) contains a PH domain and responsible for XPG interaction, and deletion of this region impairs NER.112 These observations indicate that this PPI is necessary for NER and, therefore, can be a potentially druggable target. The p62 PH domain structure has been characterized, but no structural characterization of XPG in mammals has been made, although Rad2(642−690) (yeast homologue of mammalian XPG) was found to interact with p62 PH and the structure of this PPI is characterized.113 Several regions of XPG interact with p62 and are necessary for NER, but it is unknown which region is the most druggable (i.e., which is the most efficient target for inhibiting PPI of each full-length protein). Therefore, it may be better to start the approach by using each engineered XPG and TFIIH p62 protein probe that covers these regions in one construct to perform screenings by methods that are scalable and capable for protein probe (e.g., AlphaScreen, HTRF) followed by validation (e.g., co-IP inhibition, chromatin colocalization).

Figure 8. Simplified NHEJ pathways and molecules discussed for PPI inhibition. MRN complex and other molecules that are not discussed in this chapter are omitted for clarity.

encoding no genes or functional sequences. Also, cNHEJ can incorporate nascent RNA as a template for recovering the deleted sequence to carry out error-free repair.115 Alternative NHEJ (abbreviated as aNHEJ hereafter) is mechanistically less characterized than cNHEJ is. The MRN complex is required for both cNHEJ and aNHEJ, but how the cNHEJ vs aNHEJ decision is made on the MRN is not understood well. For initiating aNHEJ, CtIP endonuclease is recruited to excessively resect the DSB ends for microhomology search. Many of the end-resection processes and endonucleases used for aNHEJ are common to those used for HR.116 53BP1 promotes aNHEJ in the cells at G1-phase, and 53BP1 elimination suppresses aNHEJ.117 The excessive removal from the original sequence increases the odds for genetic alteration in aNHEJ other those in cNHEJ. Furthermore, aNHEJ can ligate DSB ends from different regions of a chromosome or even from different chromosomes, generating a chromosomal translocation that is highly mutagenic.118 The aNHEJ pathway could be a better therapeutic target than the cNHEJ pathway because of the mutagenicity of aNHEJ. Upregulation of some aNHEJ components was found in a cancer.119 However, both NHEJ can be independently and concurrently active in cells.120 It is possible that selective inhibition of aNHEJ equilibrates DSB repair to cNHEJ. Conversely, inhibiting cNHEJ can promote aNHEJ121 that could be even worse. Therefore, from a therapeutic perspective, targeting both NHEJ concurrently would be required. Unfortunately, these two pathways are mechanistically independent and share only a few DDR molecules (e.g., MRN) known to date; therefore, current options to target both of them by a single agent are limited. Classical assays developed for functional NHEJ inhibition ex vivo have been previously summarized in an excellent article.122 The assays analyze the status of a plasmid DNA substrate

7. TARGETING PPI TO INHIBIT NHEJ NHEJ repairs DSB and occurs irrespective of cell cycle. From a functional perspective, there are programed NHEJ for V(D)J recombination and class-switch recombination (not a DDR discussed in this article) and nonprogrammed NHEJ for DDR. These two are mechanistically almost indistinguishable and thus are not easy to discriminately target to selectively inhibit DSB repair in cancer. From a mechanistic perspective, there are classical (canonical) and alternative microhomology-mediated nonclassical (atypical) NHEJ, which are very different in both mechanism and function, namely, mutagenicity (Figure 8). Classical NHEJ (abbreviated as cNHEJ hereafter) is initiated from recognition of the DSB ends by the Ku70−Ku80 heterodimer (referred to simply as Ku hereafter) followed by subsequent recruitment of other components, including DNAPK and MRN complex. Eliminating enzymatic activity of DNAPK sensitizes cells to DSB-generating agents; therefore, DNAPK has received much attention for identifying many inhibitors.114 Ku binds to almost any DSB end and prevents DSB end resection on which HR depends, thereby promoting NHEJ and preventing HR. The cNHEJ ligates DSB ends that are in close proximity. A few nucleotides from the DSB ends are trimmed to enable the ligation, which generates a small deletion of the original sequence. Therefore, cNHEJ is mechanistically mutagenic on the DSB ends, but the deletion will be genetically inert and clones containing the deletion are still functionally normal as long as the nucleotide trimming was in a region 9939



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inhibitors of these PPIs should be characterized in each respective pathway. Ku70(XRCC6)-Ku80(XRCC5). Ku is the essential initiator of cNHEJ. Ku binds to a DSB end in very high affinity (Kd ∼ 2 nM150) and triggers recruitment of DNA-PK. A modeling analysis151 revealed that PPI in this heterodimeric complex contains substructures composed of multiple portions from both Ku70 and Ku80 (i.e., PPI via multiple regions). Among them, Ku80(449−477) is the minimal sequence for interacting with Ku70.152 This short peptide could be used as a probe for screening Ku PPI inhibitors. Ku80(A453H V454H) double mutants are deficient for Ku70 interaction,152 suggesting a clue for rational design of an inhibitor based on the structure of Ala−Val dipeptide. However, CtIP-mediated aNHEJ is active in cells in which Ku70 has been genetically eliminated,153,154 indicating that Ku inhibition could activate aNHEJ. XRCC4-LigIV. This complex has been characterized as a key platform for the final ligation step of cNHEJ. The PPI is required for V(D)J recombination and DSB repair and involves two BRCT domains of LigIV.155 Cells expressing an XRCC4 truncate that is deficient for LigIV PPI are ∼10-fold more sensitive to radiation than are those expressing a full-length XRCC4.156 Expression of a LigIV fragment containing the region for XRCC4 interaction impairs NHEJ activity and increases cells’ sensitivity to radiation,157 indicating a rationale for inhibiting the PPI. An inhibitor of DNA binding of LigIV also inhibits NHEJ and sensitizes cancer cells to radiation.158 The PPIs is in an unusual mode: LigIV(755−782) is located between two BRCT domains and interacts with two long helixes from each one XRCC4 (i.e., 1:2 stoichiometry, Figure 9157,159). The XRCC4 helix dimer seems stable, and only a

containing a DSB after being incubated with cell extract. As long as the sequence around the DSB is unique in the plasmid (i.e., no homologous sequence), there is no possibility for HR, so the DSB repair occurs exclusively by NHEJ. The protocol is flexible to enable many variations (e.g., DSB structure, origin of the cell extract, time-course, dose−response for inhibitor) but uses electrophoresis or qPCR that is not scalable enough for screening purposes. Variants of this assay use cells expressing an engineered GFP that generates the GFP reporter signal only after in situ generation of DSB (by exogenous I-SceI endonuclease) followed by HR (described in the chapter for HR) or NHEJ.123 It has been used in many studies121,124,125 and is flexible to be adapted for even different mechanisms among aNHEJ subpathways.126 The cellular assay protocol is scalable and should be a great choice for functional screening for NHEJ PPI inhibitors. A sophisticated DSB plasmid repair assay that is low-throughput but capable of measuring activity of cNHEJ and aNHEJ concurrently in cells or ex vivo was also developed,127 which could be used to verify the selectivity of an inhibitor even in the same cells. MRN. MRN is the central component of both cNHEJ and aNHEJ and, therefore, a potential target for inhibiting both. As abbreviated, MRN is a dimer of the heterotrimeric complex of MRE11, RAD50, and NBS1 and serves as a sensor of DSB.128 MRE11 is a nuclease, essential for preventing genomic instability by also being involved in HR.129,130 Interestingly, genetic elimination of MRE11 by RNAi also concurrently diminishes expressions of RAD50 and NBS1,131,132 hampering validation of therapeutic qualification specific to MRE11. MRE11 has received much attention for developing the inhibitors that have been extensively characterized.130,133,134 RAD50 is capable of binding to DNA at an internal sequence rather than at DSB ends. RAD50-DNA binding was found to be as important for both telomere maintenance and the DSB repair.135 NBS1(Nibrin) was identified as a protein responsible for Nijmegen breakage syndrome, a genetic disease causing chromosome instability. It is noteworthy that NBS1 is overexpressed in many cancers,136−139 indicating that either under- or overexpression of NBS1 could lead to genomic instability, probably due to loss of balance between the benefits and risks of NHEJ. Indeed, when NBS1 expression was eliminated in cancer cells by using siRNA, mutagenesis by radiation was increased and apoptosis was decreased,140,141 suggesting that NBS1 itself should not be targeted. However, NBS1 contributes DDR signaling via a variety of PPIs142 that could be potential targets, some of which were characterized to determine their interacting sites and structures. A PPI structure of yeast Mre11-Nbs1 fragments identified two short motifs in Nbs1(477−527), and mutations at this Mre11-Nbs1 PPI site in each proteins’ human equivalent are associated with AT-like and NBS-like diseases143,144 that include a cancer-prone phenotype with hypersensitivity to radiation and chromosome instability. This observation may justify the functional rationale to target this PPI for radiation sensitization. The NBS1 PPI with phosphor-MDC1145,146 is required to maintain MRN at the site of DNA damage. The NBS1 PPI with ATM147 and FANCD2148 regulates the radiation-induced S-phase checkpoint and stabilizes FANCD2 itself.149 Because NBS1 PPI inhibition is unlikely to change the NBS1 expression level, beneficial activity of NBS1 for genomic stability may be intact. However, these approaches perturb events outside the NHEJ pathways (e.g., ATM-mediated checkpoint), therefore, the

Figure 9. Structure (1ik9.pdb) of XRCC4 homodimer (light cyan and green, surfaced)-LigIV(755−782) (gray, cartoon). An α-helix of LigIV(755−782) containing L774 and F778 (magenta stick) interacts with both XRCC4 molecules. LigIV D763 (red stick) locates on K188 (blue) of one of the XRCC4 molecules.

short region of LigIV contributes to this PPI, which is a feature that makes it feasible to develop a PPI inhibition assay using a LigIV fragment. A study identified the minimal LigIV region capable of effective inhibition of the PPI,160 which can be used as a peptide probe. A virtual screening for the LigIV region (clamp domain) has been performed to identify a few compounds that inhibit the PPI in milimolar range of concentration.161 XRCC4-XLF. XLF(XRCC4-like factor) was identified as a molecule that directly interacts with the XRCC4-LigIV complex.162 The XRCC4-XLF complex forms multimeric high-molecular weight filament163,164 that binds to the DSB 9940



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8. TARGETING PPI TO INHIBIT HR HR repairs DSBs by utilizing a sister chromatid as a template that is available only after S-phase. Therefore, HR is error-free but possible only in cells replicating DNA. HR has been focused to selectively sensitize proliferating cancer cells for therapeutic DSBs, such as those generated by IR. BRCA1 is a component for HR. PARP inhibitors prevent repair of DNA single-stranded breaks (SSBs) that are ultimately converted to DSBs and, therefore, particularly sensitize breast cancer cells in which BRCA1 is mutated to the therapy.179 BRCA2 is another HR component and coordinates RAD51 recombinase enzyme (discussed later) (Figure 11). When BRCA1 or BRCA2 are

ends. By characterizing XLF mutants deficient in XRCC4 PPI, it was shown that this PPI is required for bridging the DSB ends.165,166 XRCC4 mutants deficient in XLF PPI are also characterized as sensitizing cells to radiation and being inhibitory for coding joint in V(D)J recombination.167 The PPIs in the XRCC4-XLF filament structure also have unusual stoichiometry: two XLFs from each different XLF dimer coordinate asymmetrically together to hold one XRCC4 of a XRCC4 dimer.168 It could be difficult for a single agent to target two distant interfaces of one PPI, but the mutational studies provide a clue for a hot spot of the interaction that could be focused upon. Specifically, XRCC4 E55, D58, M61, and F106 are essential for XLF PPI.164 These residues form a cleft in which two turn structures of XLF(61−67) and XLF(111−116) bind (Figure 10). This XRCC4 cavity seems

Figure 10. PPI structure (3sr2.pdb) of XLF homodimer (cyan and green, ribbons)-XRCC4 (gray, surfaced). Regions that are not on the PPI interface are omitted for clarification. XLF R64 (blue stick) interacts with XRCC4 E55 and D58 (light red). XLF L112 and L115 (magenta sticks) interacts with XRCC4 M61 and F106 (yellow).

Figure 11. Simplified HR pathway and molecules discussed for PPI inhibition. Black strands represent repaired DSBs, and blue strands are used as a homology template. Molecules that are not discussed in this chapter are not shown.

to be a primary PPI site that would be feasible to be targeted by a small molecule, which could be found by assays using XRCC4(1−157) protein and a XLF truncate protein probe containing XLF(61−67) and (111−116). Rational strategy (e.g., virtual screening in silico) could also be reasonable given that information for structure and mutational function are available. 53BP1. 53BP1 has been characterized as an NHEJ promoter/HR suppressor.169,170 Elimination of 53BP1 by siRNA promotes HR.171 53BP1 is inhibitory to HR in BRCA1-deficient cells, resulting in NHEJ activation.172 However, in a different cellular context, 53BP1 promoted HR.173 No matter whether 53BP1 suppresses or promotes HR, the therapeutic rationale for targeting 53BP1 could be justified given that expression of a dominant-negative form of 53BP1 enhances cytotoxicity of CPT,174 and genetic elimination of 53BP1 sensitizes cells to HU.171 The 53BP1 Tudor domain is responsible for recognizing DSB,175 but the 53BP1 tandem BRCT domain is dispensable for IR-induced DDR measured by γH2AX foci.176 The 53BP1 Tudor is also an epigenetic reader domain,177 and an inhibitor of the domain interaction to a dimethylated lysine peptide has been identified. The inhibitors showed inhibition for IgG class switching178 but were not characterized for HR/NHEJ modulation or chemotherapy sensitization.

genetically depleted, HR is suppressed and aNHEJ is activated.180 This raises a concern that suppressing HR under DSB generation may activate aNHEJ to rescue the cells. However, the fact that triple-negative breast cancers (BRCA1/ 2-null) are sensitive to a PARP inhibitor indicates that HR inhibition may sensitize HR-intact cells to the PARP inhibition. HR demands time-consuming processes for homology search and resolving Holliday junction (HJ resolution). An important step to initiating HR is extensive resection of the DSB ends to generate long single-stranded ends, which are required for the homology search to anneal to a homologous sequence in the sister chromatid. The exonucleases for this process could be a good target with which to selectively inhibit the HR initiation. The resection process is two steps, in which the first generates short (∼20 bp) single-stranded ends by MRN and CtIP. The second step of resection generates much longer single-stranded ends (up to 3.5 kbp181) by EXO1182 that could be specific for HR. The MRN complex is a scaffold for coordinating components for HR but also for NHEJ and, therefore, is not a specific target for HR inhibition. However, PPIs of the MRN complex with a component unique to HR could be a possible site for selective inhibition of HR (Figure 11). Targeting resolution of the HJ intermediate of HR is another considerable strategy. In human, a short HJ crossover can be resolved by BLM helicase and topoisomerase. A long HJ 9941



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possible that a phosphorylated CtIP sequence in the other region binds to NBS1 cooperatively with CtIP(22−45). EXO1. EXO1 is a 5′-end exonuclease that enables the strand invasion step forming a D-loop. In human cells, EXO1 is capable of pursuing end resection, and siRNA-induced elimination of EXO1 severely hampers HR activation (RPA and RAD51 chromatin foci formation) by IR.201 In yeast, Exo1 also facilitates resolution of double HJ by coordinating the Mlh1-Mlh3 complex (a MMR component) and Sgs1 (orthologue of BLM helicase).202 Thus, EXO1 is a promoter of the initial and final steps of HR. EXO1 interacts with several partners contributing to these processes (e.g., BLM,203 MLH1,204 and others205,206), which could be targeted. However, genetic elimination of EXO1 does not sensitize a mammalian cell line to either CPT or cisplatin,207 indicating that EXO1 is invalid as a therapeutic target. This may be due to its functional redundancy with DNA2 nuclease. Both EXO1 and DNA2 must be concurrently eliminated for effective chemotherapy sensitization,207,208 which may be difficult for a single PPI inhibitor to accomplish. RAD51-BRCA2. RAD51 has a major role, specific in HR, in the step of strand exchange. After the DSB ends are processed by the end resection, RAD51 is loaded on the single-stranded DNA (ssDNA) ends and forms filamentous structures on the DNA. A compound that covalently binds to RAD51 and inhibits its ssDNA interaction inhibits HR.209 BRCA2 is the main mediator of RAD51: it functions by directly interacting via a motif called BRC and promoting RAD51 filament formation on DNA. BRC is a short motif (36 amino acids210) repeated eight times on BRCA2 and is responsible for generating RAD51 filament.211 The isolated short BRC peptide is capable of inhibiting the PPI in vitro,212 RAD51 foci formation (when the BRC peptide was exogenously expressed in cells),213 and HR.214 Although the detailed mechanism of the interplay between BRCA2, RAD51, and ssDNA is not completely understood, these observations may suffice to justify targeting this PPI for HR inhibition. On this rationale, several attempts have been made to generate peptide RAD51 inhibitors (Figure 12)215,216 guided by the PPI structure.217 In another approach, the structure of RAD51 bound by BRC was modeled, and several RAD51 point mutants on the N-terminus domain were verified as having reduced BRC affinities.218 These observations could be served as a basis to rationally generate small-molecule inhibitors of this PPI. BLM-TopoIIIα. An inhibitor of BLM’s interaction with DNA (but not of BLM’s ATPase activity) has been characterized for aphidicolin sensitization (i.e., replication stalling) but not for DSB-inducing agent sensitization or HR suppression.219 BLM helicase (1417 AA) is structurally homologous to WRN helicase (1432AA)220 and interacts with several proteins.221 Functions of some BLM PPIs may be independent of the helicase activity that the BLM-DNA interaction inhibitor targets. Therefore, it is possible that a BLM PPI inhibitor has functional properties and a therapeutic benefit different from those of the BLM-DNA inhibitor. The BLM-TopoIIIα complex has been extensively characterized for topoisomerase activation and double HJ resolution.222−230 Cells expressing TopoIIIα but lacking BLM show more sensitivity to CPT than do their BLM-complimented counterpart,231 indicative of a functional rationale for targeting this PPI. Truncation analysis identified that BLM(1−212) can interact with TopoIIIα but BLM(1−142) can not,230 suggesting a potential site for the PPI. Unfortunately, no structural

crossover is nucleolytically resolved by MUS81-EME1 or SLX4SLX1. MUS81-EME1 and SLX1-SLX4 interact to form a multiprotein complex (shown later in Figure 17). In this complex, SLX1 cleaves one site of an HJ first (the rate-limiting step), and MUS81 next cleaves another site of the HJ. SLX4 is a 211 kDa nonenzymatic scaffold protein that has many PPI sites for coordinating nucleases (e.g., SLX1, XPF), which could each be a potential target for HR inhibition. However, elimination of these nucleolytic components sensitizes cells to agents inducing an ICL (e.g., MMC).183,184 This would be because those nuclease complexes are active also for cleaving a fork-structured DNA substrate and thus can resolve replication-stalling by an ICL. Therefore, they are discussed as targets for the ICL repair pathway in this article (see the ICL repair chapter). GEN1 is another endonuclease characterized for HJ resolution, but no regulatory PPIs for GEN1 have been reported yet. A functional assay for HR in mammalian cells was originally developed by Jasin et al. using an elegantly engineered GFP reporter (DR-GFP) containing an I-SceI site (that generates a DSB by exogenous I-SceI endonuclease) and a stop codon, which can be homologously recombined with downstream sequence encoding another truncated GFP to restore the GFP signal.185 This assay is robust, having been used in many studies and modified to assay NHEJ (described in the chapter for NHEJ) and ICL repair.186 HR activity has been validated also by RAD51 cofoci formation on chromatin with a DSB marker.187 γH2AX cofoci are commonly measured by using commercial kits for this purpose, but it is not really a reliable DSB marker because it is induced also by SSB,188 replication stress,189 and even in the absence of DNA damage.190 The phosphor-ATM191 and 53BP1192 are regarded as more-specific DSB markers than γH2AX is and should be used. CtIP(RBBP8)-NBS1. Among divergent PPIs of NBS1 (some overviewed in the MRN complex section in the former chapter for NHEJ), the CtIP PPI has been characterized as an event initiating the HR pathway. CtIP is required for DNA end resection exclusively in the S-G2 phase and promotes HR; knock-down of CtIP by siRNA sensitizes cells to DSB-inducing agents. 193 DSB end processing by CtIP requires its phosphorylation and PPI with NBS1 FHA domain in yeast. Schizosaccharomyces pombe expressing a mutant of the Nbs1 FHA domain are defective in Ctp1 (yeast CtIP homologue) localization and highly sensitive to DSB.194 In Xenopus, CtIP recruitment to DSBs requires both NBS1 and ATM activity.195 In mammalian cells, ATM and CDK hyperphosphorylate CtIP after DSB generation, and the NBS1 FHA is required for the CtIP phosphorylation.196 The phosphorylated CtIP stimulates MRE11 in the MRN for generating 3′-sticky ends from a DSB in vitro, and NBS1 is required for this activity.197 Interestingly, CtIP depletion from Xenopus egg extract suppresses Top2 protein liberation from supplemented DNA under etoposide, a TOP2 inhibitor.198 The phosphomimic CtIP mutant increases MRN endonucleolytic activity for a DNA substrate containing a DSB end blocked by biotin−avidin (i.e., a protein end), and NBS1 boosts this activity 10−15 times.199 These observations indicate that phosphor-CtIP-NBS1 PPI is important for removal of a topoisomerase−DNA adduct prior to the HR. The peptide Ctp1(62−83), in which T79 is phosphorylated, binds to an S. pombe Nbs1 truncate containing the FHA in ∼4 μM affinity.194 If this PPI mode is also common in humans, then it would be a good target for inhibition. However, CtIP(22−45), which is required for NBS1 PPI,200 does not contain any of the CDKs/ATM phosphorylation sites.196 It is 9942



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Figure 12. Rational generation of a potent peptide inhibitor. A BRC motif (cyan) binds to RAD51 (green) by multiregion interaction. A peptide (BRC4−24) derived from the BRC motif modestly inhibits the RAD51-DNA interaction (Ki = 10 μM). To improve the potency, His (BRCA2 H1525) was changed with a Tyr based on energy calculations, Ala (BRCA2 A1535) was changed with a Ser to allow interacting to RAD51 E213, and Leu (BRCA2 L1545) was changed with a Phe to enhance hydrophobic interaction with RAD51 F259. This generated a peptide inhibitor (Y-S2-F) with high potency (Ki = 0.75 μM).215 Figure 13. Simplified TLS pathway and molecules discussed for PPI inhibition. Star (magenta) represents a DNA lesion. The potential mechanism in which each steps occurs on different PCNA monomers is not shown for clarification. Majority of molecules that are not discussed in this chapter are not shown.

characterization of the PPI is available; therefore, nonrational strategy is needed to find the PPI inhibitor. To enable this, a minimal truncate of TopoIIIα proficient for the BLM(1−212) PPI should be identified and produced for the screening of the PPI inhibitors.

UbPCNA to preferentially interact with a TLS Pol for the first step of TLS. Polη(POLH, XPV) is a TLS Pol for CPD that is generated by UV. TLS over the CPD that is performed by Polη is exceptionally accurate for inserting correct GG sequences to prevent UV-mediated genetic mutations. Patients with XP group V (XP-V) are Polη-deficient and prone to skin cancer formation, indicating importance of Polη for CPD tolerance and making Polη a less plausible therapeutic target. The mechanism of transition from the first to second TLS step is not understood well, although studies indicate a possible scaffolding mechanism by REV1 to switch to Polζ, a major extender TLS Pol that is a REV3L-REV7 complex in mammals. REV1240 and REV3L241 have been characterized as being functionally important for acquiring resistance to chemotherapies and inducing mutations and therefore could be attractive therapeutic targets. REV1 and Polζ are indispensable for ICL repair and believed to be master TLS extenders on the damaged DNA strand containing an unhooked ICL remnant (next chapter). Conventional DNA damage assays (e.g., γH2AX analysis, Comet assay) cannot measure TLS activity because TLS is not DNA repair. What should be assayed for in studying TLS is DNA replication activity over a lesion that a TLS Pol can bypass. A classic in vitro example for this is primer extension using a oligonucleotide template containing a DNA crosslink.242 A functional cellular TLS/mutagenesis assay has been developed first in prokaryotes that uses a shuttle vector containing a site-specific lesion;243 in eukaryotes, the assay involves transfecting the plasmid into NER-deficient mammalian cells to allow TLS but not NER on the lesion.244,245 The TLS-induced mutations are identified by recovering and sequencing the plasmids, thereby assessing mutational risk for

9. TARGETING PPI TO INHIBIT TLS TLS bypasses a DNA damage by a DNA polymerase specialized for TLS232 (termed TLS Pol hereafter). TLS is an important mechanism of resistance of cells to DNA damage. An analogous damage bypass by RNA PolII during transcription (i.e., translesion RNA synthesis) is also a potential mechanism to avoid cell death.233 The therapeutic strategy to target TLS is justified by many observations that genetic elimination of a TLS Pol dramatically sensitizes the cells to DNA-damaging agents. Another important therapeutic aspect is that TLS induces mutations234 because the loss of original sequence by the damage disables inserting correct nucleotides. The mutagenesis can generate a carcinogenic neoplasm; therefore, inhibition of TLS is focused upon235 due to the benefit for preventing chemotherapy-induced secondary cancers (for an example of comprehensive studies, see ref 236) in addition to sensitizing cancer cells to the therapy. TLS has been characterized as a two-step process: first, nucleotides are inserted over the damaged template strand, and second, DNA is extended from the inserted nucleotides until undamaged template is reached and regular DNA replication is resumed237,238 (Figure 13). Because TLS is a process of DNA replication, TLS is active exclusively in S-phase, which is a desirable feature to selectively target proliferating cancer cells. TLS occurs on the DNA replication fork that is organized by PPI of PCNA and a TLS Pol. A key event to trigger this PPI is RAD18-mediated PCNA monoubiquitination on K164 (this species is referred to as UbPCNA hereafter).239 The majority of TLS Pols that interact with PCNA possess a module to interact with a ubiquitin moiety (e.g., UBZ, UBM). This allows an 9943



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a specific lesion and a TLS Pol. The assay uses LacZ′ expression (blue/white colony counting) as a marker of TLS events and is unfeasible for large-scale assays. For screening of TLS inhibitors, scalable variants of the plasmid assay would be feasible (e.g., using luciferase instead of LacZ′246). RAD18-RAD6(UBE2). RAD18 is the only known E3 ubiquitin ligase for PCNA K164-monoubiquitination and is a key upstream initiator of TLS. The biochemical and functional interactions of yeast Rad18-Rad6 have been characterized. In an early study, the domain of Rad18 used for Rad6 PPI was identified as Rad18(371−410), and a yeast strain expressing Rad18 lacking this PPI region is ∼50-fold more sensitive to UV than is the WT strain.247 However, in another study, functional characterization of Rad18 domains showed that Rad18(16− 366) can monoubiquitinate PCNA in vitro with Rad6 and E1 ligase although more weakly than full-length Rev18 does.248 It was later found that Rad6b is itself capable of PCNA polyubiquitination and Rad18 inhibits it by competition. A Rad18(339−366) peptide can suppress ubiquitin-chain formation by Rad6 in vitro,249 suggesting that a small molecule mimicking Rad18(339−366) could inhibit the Rad6 E2 enzyme. The NMR structure of the Rad18(339−366) complex with Rad6b249 revealed the structural feature and amino acid residues required for the PPI; some of them are conserved in human RAD18. An extensive analysis of truncated or pointmutated RAD18 identified that RAD18(1−341) lacking the RAD6A binding domain (340−395) cannot monoubiquitinate PCNA in vitro even if the truncate maintains other functional domains (RING, UBZ, SAP).250 The information could be useful to develop the PPI assay and design a compound by modeling based on the RAD6 PPI site of RAD18. Although DNA damage sensitivity of mammalian cells expressing RAD18(1−341) is not yet verified, cells expressing RAD18(C28F), another RAD6 PPI-deficient mutant, have increased sensitivity to UV, MMC, and MMS.251 REV1-Monoubiquitinated Proteins. REV1 was originally characterized as a TLS Pol because of deoxycytidyl transferase activity252 that exclusively inserts dC no matter what is on the template strand.253 However, many studies indicated that its major role for TLS is organizing other TLS components as a scaffold protein rather than inserting a dC at the lesion. REV1 has a BRCT domain and UBMs; each of them has been characterized for PPI with PCNA254,255 and UbPCNA,256,257 respectively. UbPCNA can activate TLS in yeast rev1 in vitro, but PCNA cannot.258 The rev1 UBM is required for the PPI: a point mutant in the UBM is defective for the UbPCNAmediated TLS.257 REV1 also recruits RAD18 on chromatin and promotes the PCNA monoubiquitination, possibly by interacting with monoubiquitinated RAD18: cells expressing a point mutant of REV1 UBMs are deficient in PCNA monoubiquitination.259 Altogether, these observations indicate that the PPIs of REV1 UBM to a monoubiquitinated protein are essential for TLS and, therefore, can be a therapeutic target. Human REV1 has two UBMs, neither of which are structurally characterized, but a structure of the ubiquitin complex of UBM with Polι, another TLS Pol homologous to REV1 UBM, has been characterized.260,261 This allowed homology modeling, and the REV1 UBM2 point mutants derived from the modeling promote hypersensitivity to UV and suppress UV-mediated mutagenesis,262 indicating that the UBM2 is the primary site for the PPI. The shallow cavity of the UBM binds to a hydrophobic patch of ubiquitin around L8 and V70 residues (Figure 14), which could be targeted by a PPI inhibitor. The conventional

Figure 14. PPI structure (2kwu.pdb) of Polι UBM2 (gray, surfaced)ubiquitin (light cyan, cartoon). A shallow cavity of UBM2 containing I685, V689, L693, V697, and L701 (yellow) interacts with two portions of a ubiquitin each containing L8 and V70 (magenta sticks). The C-terminus of ubiquitin (G76, orange) is exposed out from the interaction site.

approach using a peptide probe as a surrogate of the PPI (e.g., FP) is not applicable because the ubiquitin PPI is mediated not by a short peptide motif but by multipoint residues (L8 and V70). This limits the available options of screening protocols for this PPI. REV1-RIR Proteins. Another functionally important PPI of REV1 is mediated by its C-terminus region. An early study has identified that the REV1 C-terminus but not BRCT is required for the TLS function and chemoresistance.263,264 This region was later found to be capable of interacting with Polη265 and Polκ,266−268 indicating that the region is an organizer for switching these TLS Pols. XRCC1’s PPI with the REV1 Cterminus also has been characterized24 (see the BER chapter). As described in the section of XRCC1-REV1 for detail, these PPIs are mediated by a short RIR motif shared in these PPI partners of the REV1 C-terminus. Interestingly, the REV1 Cterminus can interact with the REV7 subunit of Polζ and a RIR peptide concurrently,267−269 suggesting REV7’s potential role for coordinating an RIR-containing TLS Pol to Polζ for transition from the first to the second step of TLS on REV1. The REV1 C-terminus also binds to Polδ3 RIR,270,271 which would terminate the TLS and resume the regular replication. These observations indicate that the REV1 C-terminus serves as a regulator for both proceeding and terminating the TLS, but it is unknown why or how so many and even opposite regulations can be managed on a single PPI site. No matter how these mechanisms are controlled, an inhibitor of the REV1 Cterminus PPIs could disrupt all of these processes. Because the RIR motif is a short peptide and the small REV1 C-terminus (∼100AA) should be readily produced, the inhibitor screening for the PPI can be easy and scalable. The knowledge of several of the structures for these PPI would also allow a rational strategy for creating inhibitors. Given the high similarity of the PPI structures among RIR motifs containing an FF motif (Figure 15), it is highly likely that a small molecule analogous to FF dipeptide binds to the REV1 CTD and inhibits the PPI. REV3L-REV7(MAD2L2). This is a PPI complex characterized as Polζ, a master extender in the second step of TLS, in 9944



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10. TARGETING PPI TO INHIBIT ICL REPAIR The ICL repair is a complex and time-consuming (∼4 h ex vivo281). In the simplest model (Figure 16), the first step of

Figure 16. Simplified ICL repair pathway under DNA replication. Star (red) represents an ICL.

ICL repair is double incision of both sides of ICL to unhook it, which is an NER. The structure of the ICL may influence this incision process, but XPF-ERCC1 is almost essential in this and later steps of ICL repair.97 Another mechanism of ICL unhooking is cleavage of the glycosyl bond of the cross-linked base, but this is not active for cisplatin ICL.282 The unhooked ICL remnant is next digested by a nuclease and bypassed by TLS to fill the patch in the incised strand. By using both biochemical281,283,284 and pharmacological285,286 approaches, REV1 and Polζ have been identified as essential for ICL repair.287−289 The mechanism of the ICL repair after the TLS step could be different among cell cycles when the ICL is detected and unhooked. If the ICL is on a DNA replication fork when detected (i.e., S-phase), the incision generates a DSB end of one of the forks, which needs to be resolved, presumably by HR. Several endonucleases for NER and HR are important for ICL repair (Figure 17).290 Less is known about the ICL repair mechanism when DNA replication is inactive and thus HR is impossible (i.e., G0-G1 phases). Several studies assayed reactivation of a plasmid containing an ICL in cells that cannot replicate it (e.g., plasmid lacking a replication origin), and

Figure 15. Upper: structure [pdb data courtesy of Robert London (NIEHS)] of REV1 C-terminus (gray, surfaced)-XRCC1 RIR peptide (light cyan, cartoon). Lower: structure (2n1g.pdb) of REV1 Cterminus (gray, surfaced)-POLD3 RIR peptide (light cyan, cartoon). Structure of XRCC1 RIR peptide (F209−F210, magenta sticks) and that of POLD3 RIR peptide (F238−F239, magenta sticks) on the REV1 C-terminus are very similar and both contain an FF motif.

which REV3L and REV7 respectively serve as the catalytic and noncatalytic subunit.272 Among 33 unique chicken DT40 cell strains from which unique DDR molecule(s) were eliminated, the one containing the REV3L deficiency exerted the highest sensitivity to cisplatin.273 Genetic elimination of REV3L from a tumor transplanted in mice dramatically sensitized them to cisplatin and suppressed the cisplatin-mediated mutation in the tumor.241 Coadministration of an siRNA targeting REV3L and REV1 with a cisplatin prodrug significantly suppress the tumor growth in mice.274 These observations validate REV3L as an attractive therapeutic target, but REV3L is a huge protein (3130 AA) and few studies have succeeded in purifying it for biochemical analysis.275 Thus, it is difficult to develop a strategy to directly target REV3L (i.e., REV3L polymerase inhibitor), but an alternative approach targeting the REV3L-REV7 PPI could be feasible. MEFs isolated from mice with a homozygous point mutation of REV7 (C70R) that abolishes the PPI with REV3L are ∼1.8-fold more sensitive to MMC than are normal MEFs, and their growth is arrested in S-phase by the MMC treatment,276 suggesting that inhibiting the REV7-REV3L blocks the Polζ activity. Deletion analysis of REV3L has identified a short sequence of REV3 responsible for the REV7 PPI,277 which was later narrowed down to REV3L(1873− 1885).278 The alanine scanning revealed that P1880 and P1885 of REV3L are especially important for the PPI, which was confirmed in the crystal structures of REV7 complex with this short REV3L peptide.267,269,279 An inhibitor of this PPI was identified by a screening using the REV3L peptide probe and shown to be inhibitory for ICL repair and sensitizing cells to cisplatin.280

Figure 17. Molecules for ICL repair discussed for PPI inhibition. Star (red) represents an ICL. Molecules that are not discussed in this chapter are not shown. 9945



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identified that several molecules of NER (XPA, XPF, XPG, CSB), TLS (Polκ, REV1, REV3), MMR (Msh2), and UbPCNA are required for the reactivation,291−295 suggesting that the TLS Pols can be recruited by UbPCNA to the ICL-unhooked patch without demanding the DNA replication. The molecules in each DDR pathway are reviewed in the respective chapter but not in this chapter. The FA pathway coordinates several steps in these processes and has been studied mainly for its role in the ICL repair. Several components of the FA pathway are shared with other DDR pathways as their alternative names indicate (e.g., XPF = FANCQ, BRCA2 = FANCD1). The first step of FA pathway activation is monoubiquitination of the FANCD2-FANCI complex (ID complex)296−298 on the replication fork stalled by an ICL. RAD18, the E3 ubiquitin ligase for PCNA, was shown to be responsible also for the ID monoubiquitination.299 The monoubiquitinated ID complex further assembles a multiprotein complex termed FA core, which is composed of numerous FA family proteins and others. The FA pathway is active after S-phase because recruitment and organization of the FA core occurs on a stalled DNA replication fork. The FA core seems to be required for initiating the ICL unhooking and subsequent TLS. There are numerous PPIs in the FA core, and a few of them are characterized. SLX4(FANCP) is a large multidomain scaffold protein that coordinates XPF-ERCC1, MUS81-EME1, and SLX1 by PPIs that coordinate for HJ resolution (also see the chapter of HR) and are required for ICL repair (Figure 17).300 FANCM interacts with FAAP24301 to mediate ICL-mediated RPA recruitment.302 Interestingly, FANCM is also capable of bypassing (traversing) an ICL without repairing it.303−305 However, contribution of the ICL tolerance by FANCM for the chemotherapeutic resistance may not be substantial because chemosensitization by genetic elimination of FANCM seemed much less significant306 than in those of the DDR molecules described above (e.g., XPF).285,286 Cellular toxicity to an ICL drug (e.g., cisplatin) is often used as a surrogate functional assay for ICL repair inhibition, but it is not reliable because no drugs are 100% specific to form ICLs (i.e., + intrastrand cross-link and monoalkylation) and resistance to a drug is often independent of DDR (e.g., drug excretion pump). A reactivation assay of a plasmid in which an ICL is site specifically incorporated is commonly used (as referenced in the second previous paragraph) and scalable for chemical library screening.307 To determine reduction of the ICL population generated by an ICL drug in nuclear DNA, modified cell nuclear comet tail assays were developed in which ICL reduces the tailing in alkaline conditions due to prohibition of the strand separation (Figure 18).307−309 Because the comet assay measures DNA damage based on the DNA integrity, it measures unhooking but not removal of an ICL. Tailing suppression does not tell whether unhooked ICL remnants are removed or still on the DNA and thus ICL repair is not completed. Direct measurement of ICL species in nuclear DNA is possible by LC-MS/MS310 but laborious and not scalable. For a scalable quantification of the ICL species, chemical probes that form an ICL and are capable of in situ labeling can be used.311−313 The mechanistic detail of an ICL repair inhibitor can be determined by using these multiple ICL repair assays. For instance, both an NER inhibitor and an HR inhibitor would inhibit the ICL plasmid reactivation, but only the former inhibits ICL unhooking in the comet assay.

Figure 18. ICL unhooking assay in cells (bar, DNA duplex; red star, ICLs; blue star, unhooked ICLs; yellow star, non-ICL lesions). Cell nuclei containing ICL and non-ICL (e.g., unhooked ICL) DNA damages are irradiated or treated with hydrogen peroxide to shred the DNA. By performing alkaline electrophoresis, DNA fragments that do not contain ICLs (i.e., those including unhooked ICL) are deannealed to single-stranded status and appear as a comet tail, indicative of ICL unhooking activity.

FANCD2-FANCI (ID Complex). This complex is the upstream initiator of the FA pathways triggered by their monoubiquitinations. The PPI is required to selectively bind to branched structures of DNA,314 which may be relevant to recognition of a DNA replication stalling on an ICL. Furthermore, a branched oligonucleotide duplex (mimicking a replication fork) stimulates FANCD2 monoubiquitination of the ID complex but an unbranched one does not,315 indicating selective activation of ID complex on a stalled replication fork. However, a study showed that the ID complex is recruited to ICL before the FANCD2 monoubiquitination, and genetic elimination of FANCD2 only is sufficient to sensitize cells to MMC,316 suggesting that the FANCD2 monoubiquitination is not required for the PPI and that nonubiquitinated FANCD2 is functionally druggable. Another study shows that FANCI but not FANCD2 is required for FA core complex formation upon MMC treatment,317 indicating that FANCD2 may be unnecessary for FA pathway activation. These conflicting observations hamper mechanistic justification for therapeutic PPI inhibition of the ID complex at this stage. However, a point mutant of chicken FANCD2 (L234R that corresponds to the L231R mutation identified in a human FA patient318) is defective in monoubiquitination and FANCI PPI, and DT40 cells expressing this mutant are >10-fold more sensitive to cisplatin than are those expressing WT,319 indicating functional justification for targeting this PPI. Structure of the ID complex includes the huge interface of the PPI.320 The FANCD2 L231 coordinates three helixes that may be required for proper folding for the PPI but is not located on the PPI interface. FANCD2 K559 and FANCI K522 were focused on as key structural elements of the PPI.320 Although it seems to be hard to disrupt the ID complex PPI due to the extensive interface, the sites in which these two lysine residues interact with could be potential hotspots to target (Figure 19, upper panel). Because the lysine-interacting sites of each FANCI and FANCD2 are structurally analogous (Figure 19, lower panel), 9946



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Figure 20. PPI structure (2zix.pdb) of MUS81 (gray, surfaced)-EME1 (light cyan, cartoon). A nodal structure of MUS81 adopts EME1 E454-A466 (magenta). F459 and W465 (sticks) of EME1 interact to the same side of the MUS81 crevasse, which could be blocked by a single small molecule.

Figure 19. Upper: structure of ID complex (3s4w.pdb), light green, FANCI; light blue, FANCD2. The PPI is extensive especially around FANCD2 K559 and FANCI K522 (magenta), which could be potential hotspots to target this PPI. Lower: superposition of the FANCD2 K559-interacting region of FANCI (light green) and FANCI K522-interacting region of FANCD2 (light blue). They are structurally similar and may be targeted by a single compound to dissociate the two lysine interactions at the same time.

The structure of the EME1(454−466) is somehow different among a couple of structures determined in the same study, indicating that this crevasse adopts divergent conformations of EME1(454−466), therefore, a structure-based strategy may not be efficient for this PPI. SLX4(FANCP)-XPF(ERCC4). SLX4 is a multidomain scaffold protein, organizes many important proteins (e.g., XPF-ERCC1) for ICL repair,328,329 and has been focused upon to elucidate the roles of each PPI. The SLX4 PPIs are stable enough to examine the endonuclease activities of SLX1, MUS81, and XPF ex vivo.330−332 XPF-ERCC1 unhooks an ICL by coordinating with SLX4. The in vitro ICL repair using Xenopus egg extract is abolished by XPF depletion but not by MUS81 or FAN1,333 indicative of the importance of the SLX4XPF PPI for ICL unhooking. A PPI complex of XPF-ERCC1 with a short SLX4 truncate is able to incise a Y-shaped DNA substrate containing an ICL at the crosspoint (i.e., mimicking a replication fork collision on an ICL).334 In the XPF-interacting region of SLX4 named MLR, 528−559 (in human) are especially well conserved in multiple species. Several point mutations of hydrophobic residues in this region (L530, F545, Y546, L550) are defective for the XPF PPI, and SLX4(L530Q) and SLX4(Y546C) cannot rescue SLX4-null MEF from MMC.335 Cells expressing SLX4 having a truncated XPFinteracting region are ∼10-fold more sensitive than are those expressing WT SLX4 to MMC but not CPT or a PARP inhibitor.329 These observations functionally justify targeting the SLX4-XPF PPI for ICL repair inhibition. The region(s) in XPF necessary for the SLX4 PPI is not identified yet; SLX4(528−559) would be used as a peptide probe to identify a XPF truncate that is capable of the PPI and, therefore, usable for the inhibitor screening. Given that the hydrophobic residues

they may be targeted by a single small molecule that could weaken the PPI and potentially dissociate the ID complex. MUS81-EME1. MUS81 is an endonuclease, forms a complex with noncatalytic EME1 protein,321 and can cleave a DNA fork in vitro (i.e., mimicking replication stalling). It can also cleave a HJ, but only when it contains a 12-bp322 and 26bp323 homologous core, which enables the branch migration.324 Therefore, MUS81 inhibition may be inhibitory for resolving a stalled DNA replication fork (e.g., ICL repair) rather than HJ specifically in HR (e.g., DSB repair). Indeed, MUS81-EME1 promotes converting an ICL to a DSB, and cells from MUS81knockout mice are ∼100-fold sensitive than are normal cells to ICL drugs (cisplatin, MMC, nitrogen mustard) but only faintly to IR,325,326 justifying their use as targets for inhibiting ICL repair. MUS81-EME1 and SLX4-SLX1 (next section) interact to form a higher-order complex mediated by MUS81-SLX4 PPI.184 MUS81-EME1 and SLX4-SLX1 complexes are each active as endonucleases, and they are functionally epistatic,183 therefore, this higher-order PPI seems not to be an absolute requirement. The sensitization effect observed by the single elimination of MUS81 or SLX4 suggests that they are not functionally redundant and that each is functionally druggable. The structure of the complex has been characterized well.327 The PPI interface is extensive and separated in several regions that may be difficult to efficiently target by using a single small molecule. However, MUS81 uses a characteristic node to adopt a short EME1(454−466) sequence (Figure 20), which could be a hotspot for targeting and be usable for inhibitor screening. 9947



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merely equal to simply increasing the chemotherapy dose. DDR pathways are the fundamental basis for maintenance of normal cells and, unfortunately, not unique to cancer. However, some genomic alterations of DDR molecules are known in cancers, and some of them have been proven to be effective and specific targets for certain cancer types (e.g., PARP for BRCA1/2mutated cancers). Targeting the epigenetic/chromatin dynamic pathways also offers an enormous unexplored opportunity to inhibit DDR, although few DDR molecules are known to be epigenetically upregulated selectively in cancers. Protein PTM is another important mechanism of DDR regulation because it enables changing the affinity of the PPI and even of the protein partners. Targeting PPIs of a DDR component with a PTM that is essential to activate the DDR (e.g., monoubiquitinated PCNA and ID complex) could specifically and effectively inhibit the DDR. Therefore, targeting the PPI of a DDR molecule with a cancer-specific PTM is a potential strategy to make chemotherapeutics selective to cancers. Some PPIs for PTM events in cancers have been successfully targeted (e.g., p53-MDM2). There should be numerous protein PTMs yet to be characterized for the DDR (e.g., SUMO357). By mapping PTM upregulation in cancers and analyzing their role in DDR pathways and their effect on the chemotherapeutic response, potential PPI targets for cancer-selective chemotherapeutic enhancement will be identified. As described in the Introduction, many DDR enzymes are unconventional and their enzymatic assays are often not easy ways to identify the inhibitors. Inhibition of enzymes’ PPI is a potential alternative strategy to inhibit functions of unconventional enzymes, as discussed in this article. This strategy will be especially useful for the DDR because several DDR enzymes also have functions as a PPI scaffold, which is often even more important than the enzymatic activity (e.g., REV1 recruits Polζ to DNA damage independently from its deoxycytidyl transferase function; see sections for REV1). Efforts to target PPIs have been hampered by inaccessibility to the assays and reagents. Nowadays, many novel methods became available for identifying compounds that directly bind a protein (e.g., WaterLOGSY NMR); some of these methods do not require purification of the protein and are even applicable for assaying the binding in cells (e.g., CETSA). These assays can be applied to virtually any PPI target regardless of the enzymatic activity. In addition, emerging gene editing techniques are available for validating the functional druggability of a specific PPI by generating cells expressing specific mutations in the PPI interface. These technological innovations and increased understanding of the specific role of PTM in cancers will fuel enthusiasm in many laboratories to identify PPI inhibitors of the DDR. Methods. All figures presenting PPI structures are produced by Pymol 1.3 (Schrodinger LLC). The superposition of protein structures in Figure 19 was calculated by SuperPose 1.0.358 The docking in Figure 7 was performed by Autodock Vina.359

of SLX4 are important, some chemical scaffolds designed or chosen for inhibiting hydrophobic PPIs26 could be preferentially screened. SLX4(FANCP)-SLX1. SLX4-SLX1 is another PPI for the endonucleolytic process of the ICL repair (partly described in the previous sections), especially for HJ resolution.330−332 This complex resolves a DNA substrate containing a mobile HJ in vitro.336 Human SLX1 is a 275-AA catalytic subunit and interacts with the C-terminus portion (1752−1811) of SLX4 named SBR.337 An analysis identified SLX1(95−275) as being required for the SLX4 PPI. This SLX1 region contains part of a nuclease catalytic core and PHD, but PHD only is not capable of the PPI,332 indicating multipoint interactions on SLX1’s interface. HEK293 cells in which SLX1 was eliminated by siRNA were ∼10-fold more sensitive than were those with control siRNA to cisplatin, CPT, and IR; the extent of the sensitivity was similar to that caused by SLX4 elimination,330 although SLX4 elimination caused greater sensitivity than did SLX1 elimination in HeLa cells.332 The structure of the PPI has been characterized for Candida glabrata338 and Schizosaccharomyces pombe.339 In the former structure, slx4(655−721) interacts with a large cavity composed of multidomains of slx1. Among the slx4 residues on the interface, Y679 and F681 are on a small helix each buried in a deep hydrophobic pocket, indicating a hotspot for inhibiting the PPI. In the latter structure, hydrophobic W373, L377, and Y379 residues on a small helix of slx4(349−417) interact with slx(177−246), which seems similar to the former structure. Thus, the PPI structure seems to be conserved in these species and is potentially druggable, although it is yet unknown whether such a PPI also exists in humans.

11. TARGETING PPI TO INHIBIT DPC REPAIR DNA−protein cross-linking (DPC) has received little attention as a therapeutic DNA damage, but cisplatin can generate DPC with a transcription factor or a histone,340 indicating that DPC also could be a driver of the chemotherapeutic effect of cisplatin. Topoisomerase 1 (TOP1) forms a covalent intermediate with the 3′ end of DNA, which is itself a DPC and stabilized by a TOP1 inhibitor. Such DPC can also be an intermediate of PARP inhibition (PARP trapping341−343). Although studies of DPC are too immature to define therapeutic value and solid rationale for targeting DPC repair at this moment, DPC repair could offer a novel opportunity for chemotherapeutic sensitization to these drugs. There are several recent studies identifying molecules required for DPC. During replication of a plasmid DNA containing a DPC in Xenopus egg extract, the DPC is degraded to a peptide-DNA cross-link that is next bypassed by Polζ,344 indicating that the DPC repair is a coordination of proteolysis and TLS extension. Spartan(DVC1, C1orf124) has been initially identified as a molecule interacting with UbPCNA to mediate TLS.345−351 Later, it was characterized as a protease facilitating replication of DPC-containing DNA,352−356 indicative of another link of proteolysis and UbPCNA-promoted TLS. Some molecules for NHEJ and HR (MRN, CtIP, BRCA1) are required to remove TOP2-DNA adducts.198 PPIs of these DDR molecules could also be targets for inhibiting the DPC repair.

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AUTHOR INFORMATION

Corresponding Author

*Phone: 901-595-5854. E-mail: [email protected]. ORCID

Naoaki Fujii: 0000-0002-2528-6944

12. FUTURE PERSPECTIVE An unresolved challenge in targeting the DDR is cancer specificity. A common criticism is that DDR inhibition is

Notes

The author declares no competing financial interest. 9948



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Biography

patch base excision repair in living cells. DNA Repair 2010, 9, 109− 119. (8) Gary, R.; Kim, K.; Cornelius, H. L.; Park, M. S.; Matsumoto, Y. Proliferating cell nuclear antigen facilitates excision in long-patch base excision repair. J. Biol. Chem. 1999, 274, 4354−4363. (9) Huehls, A. M.; Huntoon, C. J.; Joshi, P. M.; Baehr, C. A.; Wagner, J. M.; Wang, X.; Lee, M. Y.; Karnitz, L. M. Genomically incorporated 5-fluorouracil that escapes UNG-initiated base excision repair blocks DNA replication and activates homologous recombination. Mol. Pharmacol. 2016, 89, 53−62. (10) Seiple, L.; Jaruga, P.; Dizdaroglu, M.; Stivers, J. T. Linking uracil base excision repair and 5-fluorouracil toxicity in yeast. Nucleic Acids Res. 2006, 34, 140−151. (11) Klungland, A.; Rosewell, I.; Hollenbach, S.; Larsen, E.; Daly, G.; Epe, B.; Seeberg, E.; Lindahl, T.; Barnes, D. E. Accumulation of premutagenic DNA lesions in mice defective in removal of oxidative base damage. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 13300−13305. (12) London, R. E. The structural basis of XRCC1-mediated DNA repair. DNA Repair 2015, 30, 90−103. (13) Fan, J.; Wilson, P. F.; Wong, H. K.; Urbin, S. S.; Thompson, L. H.; Wilson, D. M., 3rd XRCC1 down-regulation in human cells leads to DNA-damaging agent hypersensitivity, elevated sister chromatid exchange, and reduced survival of BRCA2 mutant cells. Environ. Mol. Mutagen. 2007, 48, 491−500. (14) Saribasak, H.; Maul, R. W.; Cao, Z.; McClure, R. L.; Yang, W.; McNeill, D. R.; Wilson, D. M., 3rd; Gearhart, P. J. XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes. J. Exp. Med. 2011, 208, 2209−2216. (15) Gao, Y.; Katyal, S.; Lee, Y.; Zhao, J.; Rehg, J. E.; Russell, H. R.; McKinnon, P. J. DNA ligase III is critical for mtDNA integrity but not Xrcc1-mediated nuclear DNA repair. Nature 2011, 471, 240−244. (16) Vidal, A. E.; Boiteux, S.; Hickson, I. D.; Radicella, J. P. XRCC1 coordinates the initial and late stages of DNA abasic site repair through protein-protein interactions. EMBO J. 2001, 20, 6530−6539. (17) Yang, J.; Parsons, J.; Nicolay, N. H.; Caporali, S.; Harrington, C. F.; Singh, R.; Finch, D.; D’Atri, S.; Farmer, P. B.; Johnston, P. G.; McKenna, W. G.; Dianov, G.; Sharma, R. A. Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy. Oncogene 2010, 29, 463−468. (18) Trivedi, R. N.; Almeida, K. H.; Fornsaglio, J. L.; Schamus, S.; Sobol, R. W. The role of base excision repair in the sensitivity and resistance to Temozolomide-mediated cell death. Cancer Res. 2005, 65, 6394−6400. (19) Barakat, K. H.; Gajewski, M. M.; Tuszynski, J. A. DNA polymerase beta (pol beta) inhibitors: a comprehensive overview. Drug Discovery Today 2012, 17, 913−920. (20) Boudsocq, F.; Benaim, P.; Canitrot, Y.; Knibiehler, M.; Ausseil, F.; Capp, J. P.; Bieth, A.; Long, C.; David, B.; Shevelev, I.; FrierichHeinecken, E.; Hubscher, U.; Amalric, F.; Massiot, G.; Hoffmann, J. S.; Cazaux, C. Modulation of cellular response to cisplatin by a novel inhibitor of DNA polymerase beta. Mol. Pharmacol. 2005, 67, 1485− 1492. (21) Marintchev, A.; Gryk, M. R.; Mullen, G. P. Site-directed mutagenesis analysis of the structural interaction of the single-strandbreak repair protein, X-ray cross-complementing group 1, with DNA polymerase beta. Nucleic Acids Res. 2003, 31, 580−588. (22) Cuneo, M. J.; London, R. E. Oxidation state of the XRCC1 Nterminal domain regulates DNA polymerase beta binding affinity. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 6805−6810. (23) Gryk, M. R.; Marintchev, A.; Maciejewski, M. W.; Robertson, A.; Wilson, S. H.; Mullen, G. P. Mapping of the interaction interface of DNA polymerase beta with XRCC1. Structure 2002, 10, 1709−1720. (24) Gabel, S. A.; DeRose, E. F.; London, R. E. XRCC1 interaction with the REV1 C-terminal domain suggests a role in post replication repair. DNA Repair 2013, 12, 1105−1113. (25) Prasad, R.; Poltoratsky, V.; Hou, E. W.; Wilson, S. H. Rev1 is a base excision repair enzyme with 5′-deoxyribose phosphate lyase activity. Nucleic Acids Res. 2016, 44, 10824−10833.

Naoaki Fujii has practiced medicinal chemistry in Astellas Pharma Inc. and earned his Ph.D. by studying transition-metal catalyzed C−H bond carbocyclization reaction in Shinji Murai’s laboratory at Osaka University in Japan. At the University of California San Francisco, he designed the first nonpeptide PDZ domain PPI inhibitor in R. Kiplin Guy’s laboratory and validated it for lung cancer models in David M. Jablons’ laboratory. In 2006, he joined the Department of Chemical Biology and Therapeutics at St. Jude Children’s Research Hospital, where he is currently an Associate Member and focuses on identifying DDR inhibitors with novel MoAs.

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ACKNOWLEDGMENTS I thank Robert London (NIEHS) for the PDB data and permission to publish it in Figure 15, Akira Inoue for making comments for Chapters 4−11, and Cherise Guess for editing the manuscript. This work was supported by ALSAC.

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ABBREVIATIONS USED AA, amino acid; ADMET, absorption, distribution, metabolism, excretion, toxicity; ADP, adenosine diphosphate; AP, apurinic/ apyrimidinic/abasic; BER, base excision repair; BRCT, BRCA1 C-terminus; CETSA, cellular thermal shift assay; CPD, cyclobutane pyrimidine dimer; CPT, camptothecin; DDR, DNA damage response/repair; DPC, DNA protein cross-link; DSB, double-strand break; FA, Fanconi anemia; FP, fluorescence polarization; FRET, Förster resonance energy transfer; GFP, green fluorescent protein; HJ, Holliday junction; HR, homologous recombination; HSQC, heteronuclear single quantum coherence; ICL, interstrand cross-link; IP, immunoprecipitation; IR, ionic radiation; ITC, isothermal titration calorimetry; LOGSY, ligand observed via gradient spectroscopy; MEF, mouse embryonic fibroblast; MMC, mitomycin C; MMR, mismatch repair; MMS, methylmethanesulfonate; MoA, mechanism of action; NER, nucleotide excision repair; NHEJ, nonhomologous end joining; PH, Pleckstrin homology; PHD, plant homeodomain finger; PLA, proximity ligation assay; PoC, proof of concept; PPI, protein−protein interaction; PTM, posttranslational modification; SAR, structure−activity relationship; SPR, surface plasmon resonance; SSB, single-strand break; TAD, trans-activating domain; TLS, translesion synthesis; UBM, ubiquitin-binding motif; UBZ, ubiquitin-binding zinc finger; WT, wild-type

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REFERENCES

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