Discovery of DNA repair inhibitors by combinatorial library profiling

Abstract

Small molecule inhibitors of DNA repair are emerging as potent and selective anti-cancer therapies, but the sheer magnitude of the protein networks involved in DNA repair processes poses obstacles to discovery of effective candidate drugs. To address this challenge, we used a subtractive combinatorial selection approach to identify a panel of peptide ligands that bind DNA repair complexes. Supporting the concept that these ligands have therapeutic potential, we show that one selected peptide specifically binds and non-competitively inactivates DNA-PKcs, a protein kinase critical in double-strand DNA break repair. In doing so, this ligand sensitizes BRCA-deficient tumor cells to genotoxic therapy. Our findings establish a platform for large-scale parallel screening for ligand-directed DNA repair inhibitors, with immediate applicability to cancer therapy.

Introduction

Mammalian cells rely on vast interactive DNA repair networks to preserve genomic integrity in the face of constant genotoxic stress (1). Loss of DNA repair fidelity predisposes cells to mutation, genomic instability and carcinogenesis (2). Consequently, tumors often bear intrinsic DNA repair deficiencies absent in non-cancerous normal tissues, and these defects may represent tumor-specific therapeutic targets. The recent success of poly (ADP-ribose) polymerase (PARP) inhibitors against BRCA-deficient tumors has generated great interest in the pursuit of new tumor-specific drugs targeting DNA repair networks (3-5). However, both the magnitude and complexity of the DNA repair supra-molecular networks pose a serious obstacle to this goal and, currently, there is an underdeveloped repertoire of readily translatable molecular targets for this purpose.

Functional ligand-directed screening may provide an opportunity to accelerate the discovery of molecular targets within these pathways. This approach offers several advantages for mapping the DNA repair protein network: (i) its use of degenerate peptide sequences yields theoretically complete coverage of all possible binding sites, (ii) as it is non-biased, it has the potential to uncover targets within factors not known to be involved in DNA repair, and (iii) it selects for ligands binding accessible and active motifs, thus enriching for the recovery of functional small molecules potentially useful as DNA repair inhibitors. As such, we use here subtractive combinatorial ligand-directed mapping of DNA repair complexes to guide the selection of peptides potentially useful as DNA repair inhibitors.

Materials and Methods

Combinatorial screening

A phage random peptide library displaying the insert XXXXYXXXX (X, any residue; Y, tyrosine) was generated. pSP72 plasmid DNA (Promega) was linearized, biotinylated, and bound to magnetic streptavidin-coated beads (Dynabeads M-280, Invitrogen) at a DNA:bead molar ratio of 60,000:1. Nuclear extracts were prepared as described elsewhere (6). For negative counter-selection, 1 mg of DNA-coated magnetic beads was incubated with 10⁸ phage particles in PBS for 30 min at room temperature (RT). Unbound phage were collected, and positive selection was subsequently carried out on 1 mg of DNA-coated magnetic beads pre-bound to 100 μg of nuclear protein (37°C for 30 min) and incubated in PBS for 30 minutes at RT. This method of using DNA-coated beads to capture functional DNA repair proteins from nuclear abstracts was adapted from extensively validated published techniques (7-9). Bound phage were collected by the Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL) method (10). After three rounds of selection, the recovered phage library was introduced into host bacteria, individual clones were selected on LB agar plates, and the phage DNA corresponding to the peptide insert was sequenced via established primers using methods described previously (11).

Similarity analysis

For the first 500 peptides recovered from our screen, similarity analysis was performed using the NCBI human protein BLAST database search engine. To test for clustering of BLAST hits within functional annotated pathways, the top 10 hits for each peptide (5,000 hits total) were subjected to Ingenuity Pathway Analysis (12). To increase the stringency of the similarity analysis, BLAST results were filtered by the Entrez query “DNA damage repair”, and analysis limited to results with scores over 18.0 bits. These results were considered positive only when peptides resembled domains in DNA repair proteins known to be functionally-conserved (according to the Conserved Domain Database), or with protein sequences highly conserved amongst vertebrates. These results were organized using an online protein clustering algorithm (13), and a heatmap was generated to show quantitative results.

Peptide synthesis

Biotinylated and non-biotinylated synthetic custom peptides (GNFRYLAPP, RYPAGLPFL [scr], RQIKIWFQNRRMKWKK [pen], GNFRYLAPP-pen, and RYPAGLPFL-pen) were produced by PolyPeptide Labs and were reconstituted with sterile water (1 mM).

DNA-PKcs precipitation assay

Biotinylated peptides (10 μM) were incubated with 50 μg of pooled nuclear extract, with sonicated DNA (10 μg), ATP (1 μM), and/or excess free non-biotinylated GNFRYLAPP (100 μM) at 37°C for 15 min. 50 μg of streptavidin-bound beads was added to precipitate peptide-bound proteins. After three washes, recovered proteins were resolved on a polyacrylamide gel and analyzed via Coomassie staining and Western blotting.

Enzyme-linked immunosorbent assay (ELISA)

Streptavidin-bound 96-well plates (Roche) were coated with 100 ng of biotinylated linearized pSP72 or 10ng of biotin-GNFRYLAPP (vs. biotin-scrambled control). MaxiSorp 96-well plates (Fisher) were coated with 250 units of pure DNA-PK (Promega). After blocking, prey was added to each baited well and incubated at 37°C for 1 h, with 1 μM ATP, sonicated DNA (50 ng/μL), and a protease/phosphatase inhibitor cocktail (Sigma). To compete with GNFRYLAPP/DNA-PKcs interactions, we added non-biotinylated GNFRYLAPP or scrambled control peptide. To compete with DNA-PKcs/DNA interactions, we added either GNFRYLAPP or scrambled control peptide. We quantified DNA-PKcs immunoreactivity using fluorophore-conjugated antibodies detected via a fluorescence microplate reader. We quantified GNFRYLAPP reactivity using FITC-conjugated streptavidin (1:500, Jackson Immunoresearch # 016-010-084).

DNA-PKcs kinase assay

DNA-PKcs kinase activity assays were carried out with the SignaTect kit (Promega) according to the manufacturer's instructions, using pure DNA-PK (Promega) as the catalyst. Non-biotinylated GNFRYLAPP and wortmannin (WM, 10 μM) served as controls.

DNA-PKcs autophosphorylation assay

250 units of pure DNA-PK (Promega) was incubated for 7 minutes at 37°C in 1 mM HEPES with 1 μM γ-³²P-ATP and protease/phosphatase inhibitor cocktail (Sigma), with or without sonicated DNA (50 ng/μL), GNFRYLAPP (10 μM), DMNB (10 μM), or WM (10 μM). Reactions were resolved on a polyacrylamide gel and phosphorylation was detected by autoradiography. An identical experiment was run in parallel with cold ATP, and DNA-PKcs autophosphorylation was detected with a mouse anti-phospho T2609 DNA-PKcs antibody (Abcam # ab18356).

Electrophoretic mobility shift assay (EMSA)

Custom oligonucleotides (Sigma) were hybridized and end-labeled with α-³²P-dCTP by Klenow (New England Biolabs). Free radioactive nucleotide was removed with a PCR clean-up column (Qiagen). Labeled probe (4000 cpm) was incubated at 37°C for 10 min with 250 units of pure DNA-PK (Promega) in 1 mM HEPES buffer, 1 μM ATP, and protease/phosphatase inhibitor cocktail (Sigma). As indicated, GNFRYLAPP (10 μM) or a 10-fold molar excess of cold DNA probe was added. Reactions were resolved on a non-denaturing 4-20% PAGE gel (Bio-Rad), and mobility shifts were detected by autoradiography.

Protease protection assay

Standard techniques were used, as previously described (14). Briefly, pure recombinant DNA-PK (250 units, Promega) was incubated for 7 min at 37°C in 1 mM HEPES with 1 μM ATP and a phosphatase inhibitor cocktail, with or without sonicated DNA (50 ng/μL), GNFRYLAPP (10 μM), or scrambled control peptide (10 μM). Trypsin (Gibco) was then added (0.01%, wt:vol), and the mixture was incubated for 8 min at 37°C. Reactions were terminated by boiling, and resolved on a 5% SDS-PAGE gel (Bio-Rad). DNA-PKcs fragments were detected with a polyclonal antibody (1:1,000, Abcam # ab18356).

Cell culture

All cell lines were acquired from the American Type Culture Collection (ATCC) and cultured according to their recommendations, with the exception of the human KS1767 Kaposi sarcoma cell line (a gift from Dr. S. Leventon-Kriss). All peptides were added to standard growth media 72 hours prior to irradiation or mock-irradiation. Peptides were used at a concentration of 10 μM, as this was two-fold lower than the concentration needed to see any toxicity by MTT assay (not shown).

Irradiation

Cells were irradiated using a ¹³⁷Cs source (Shepherd Instruments) with a dose rate of 333 cGy per minute.

Immunocytochemistry

Cells were plated on coverslips. After irradiation, at the indicated times, cells were fixed for three minutes in a 1:1 mixture of ice-cold acetone:methanol. After washing, the cells were then blocked for one hour at room temperature with 5% goat serum. Primary antibodies, in 1% BSA, were then incubated overnight at 4°C. After washing, secondary antibodies were incubated for one hour at room temperature. Coverslips were mounted onto slides and imaged on an inverted fluorescence microscope.

Comet assay

One hundred fifty minutes following 6 Gy of radiation, 1000 cells were suspended in 0.5% low-melting temperature agarose and plated on microscope slides pre-coated with 1% agarose. Mounted cells were incubated at 4°C for 30 min in neutral lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris base, 1% sodium lauryl sarcosinate, and 1% Triton X-100, pH 7.6). The slides were next equilibrated in TAE (pH 7.6), followed by electrophoresis at 40 volts for 30 minutes. The slides were fixed in 70% ethanol for 10 min, DNA was stained with SybrGreen (Invitrogen), comets were imaged on an inverted fluorescence microscope, and unrepaired DNA damage was scored by CometScore (TriTek). One hundred fifty comets were analyzed per treatment and quantified as previously described (15).

Clonogenic assay

Cells were irradiated to the doses described after 72 h pre-incubation in the indicated conditions. Twenty-four hours later, the cells were replated at low density, under normoxic conditions, without peptide and/or reagent. Between 7 and 14 days later, colonies containing at least 50 cells were scored, and results were normalized to the plating efficiency observed for non-irradiated cells (16, 17).

Antibodies

Antibodies used were: anti-DNA-PKcs (1:1,000, Abcam # ab230), anti-53BP1 (1:1,000, BD # 612523), anti-53BP1 (1:1,000, Abcam # ab21083), anti-BRCA1 (1:1,000, Abcam # ab47429), anti-phospho-T2609 DNA-PKcs (1:1,000, Abcam # ab18356), anti-γH₂AX (1:1,000, Abcam # ab2893), anti-mouse IgG (1:10,000, Abcam # ab6789), anti-mouse IgG (1:5000, Abcam # ab6787), anti-rabbit IgG (1:5000, Abcam # ab6793), anti-rabbit IgG (1:1000, Abcam # ab6939), and anti-rabbit IgG (1:10,000, Jackson ImmunoResearch # 111-035-006).

Statistics

Tests for statistical significance between groups were carried out using either two-tailed Student's t-tests, Spearman rank, or log-ranked analyses, as indicated, with the level of significance defined as a P value less than 0.05.

Results

Construction and characterization of a DNA repair ligand library

A linear phage display library of 10⁸ unique insert sequences was produced with the general peptide arrangement XXXXYXXXX (X, any residue; Y, tyrosine) displayed on pIII. Peptide binding to DNA repair factors was enriched by three successive rounds of selection on immobilized DNA-nuclear protein complexes after an initial negative counter-selection on immobilized DNA alone (Fig. 1). Blunt-ended linear double-stranded DNA served as bait for nuclear proteins to focus the screen on double-strand DNA break (DSB) repair factors.

Figure 1.

Generation of a DNA repair-interacting combinatorial library. Linear, blunt, double-stranded plasmid DNA (blue) was used for an initial negative counter-selection of the phage library (grey), followed positive selection of recovered phage on identical DNA molecules pre-incubated with nuclear protein extract (red). Three rounds of selection were performed.

To assess for enrichment of DNA repair-interacting ligands, similarity analyses were performed for the first 500 peptides in the selected library versus an online database of human proteins (18). Functional pathway analyses (12) were performed on the top 10 BLAST hits for each (5,000 hits, total). This revealed significant enrichment of proteins clustering into known functional pathways, including DNA damage repair (Fig. 2A). To further increase the stringency of the analysis, BLAST hits were also separately reduced to only those wherein the queried peptide mimicked a conserved functional domain in the homologous protein. Amongst the peptides retained after this analysis, many resembled critical DNA repair factors whose absence causes recognized syndromes of genetic instability in humans, mapped to residues in these factors known to be mutated in human cancers, and overlapped with domains within these factors having defined roles in protein-protein interactions, protein-DNA interactions, or ATP binding (not shown). An interactivity map was created to display proteins with conserved domains mimicked by the recovered peptides, which shows extensive coverage of DSB repair pathways (Fig. 2B).

Figure 2.

Bioinformatic analysis of retrieved ligands. A, Retrieved peptide sequences were compared to human proteins using BLAST (18) in order to create a database of candidate mimicked proteins. This database was then subjected to functionality analysis using Ingenuity Pathway Analysis (12), which revealed a significant clustering of mimicked proteins in the DNA damage repair reactome, along with other annotated pathways. B, As a higher-stringency analysis, the database was filtered to include only proteins with conserved domains mimicked by a recovered peptide (see Materials and Methods). A fused interactivity-heat map is shown of such proteins, generated using the online STRING database (13). Proteins are colored according to a heat map reflecting the number of peptides recovered (0-5) mimicking conserved domains within that protein.

GNFRYLAPP is a non-competitive DNA-PKcs inhibitor

To explore whether mapped ligands might serve as DNA repair inhibitors, we produced one using solid-phase Merrified synthesis for functional analysis (GNFRYLAPP). GNFRYLAPP was chosen because of its similarity to Ku Autoantigen Related Peptide 1 (KARP-1), a primate-specific protein expressed as an in-frame N-terminal extension on Ku80 (Fig. 3A). Through undefined mechanisms, KARP-1 is known to modulate the activity of DNA-PKcs (19), a phosphatidylinositol 3-kinase-related kinase (PIKK) family member that plays a critical role in DSB repair (20, 21). GNFRYLAPP resembles an uncharacterized conserved motif in the N-terminal half of KARP-1 between its putative leucine zipper and nuclear localization domains (Fig. 3B), potentially responsible for its regulation of DNA-PKcs.

Figure 3.

KARP-1 is mimicked by a lead peptide. A, Shown are the structure of one retrieved ligand, GNFRYLAPP (bottom), and the aligned region of KARP-1 (top). B, Protein sequences for KARP-1 are shown, which is expressed as an amino-terminal extension on Ku80 in primates. Note that the mimicked structure (red) rests in a conserved region between putative leucine zipper and nuclear localization domains.

We first examined whether GNFRYLAPP and DNA-PKcs physically interact. The synthetic peptide GNFRYLAPP precipitated DNA-PKcs from nuclear extracts, and did so in a DNA- and ATP-dependent manner (Fig. 4A). This interaction was specific, as it was inhibited by excess free GNFRYLAPP, but not by scrambled peptide; also, purified recombinant DNA-PK was sufficient for attracting GNFRYLAPP (Fig. 4B).

Figure 4.

GNFRYLAPP binds and inhibits DNA-PKcs. A, GNFRYLAPP precipitated from nuclear extracts a high-molecular weight protein reacting with an anti-DNA-PKcs antibody by Western blot. The interaction depended on DNA and ATP, and was suppressed by excess free soluble GNFRYLAPP. A control scrambled peptide (Scr) demonstrated no such interaction. B, ELISA-based quantification of DNA-PKcs binding to immobilized GNFRYLAPP (black bars) or GNFRYLAPP binding to immobilized DNA-PKcs (blue bars), and the suppression of each by increasing concentrations of free GNFRYLAPP. Control scrambled peptide had no detectable interaction with DNA-PKcs. C, Increasing molar ratios of GNFRYLAPP to consensus DNA-PKcs peptide substrate decreased DNA-PKcs kinase activity. Wortmannin (WM) and scrambled peptide served as controls. GNFRYLAPP itself was not phosphorylated by DNA-PKcs (orange). D-E, GNFRYLAPP, DMNB, and WM each inhibited autophosphorylation of pure DNA-PKcs, visualized by (D) γ-³²P-ATP labeling and (E) phospho-specific antibody detection; scrambled peptide did not. F-G, GNFRYLAPP did not influence the DNA binding activity of DNA-PKcs by either (F) ELISA or (G) EMSA. An arrow marks the shifted band on EMSA. H, DNA-PKcs trypsin protease protection assay demonstrated partial protection of DNA-PKcs after activation by DNA binding, as detected by a residual immunoreactivity to a polyclonal DNA-PKcs antibody. Protease protection was lost in the presence of GNFRYLAPP, demonstrating that GNFRYLAPP blocked DNA-induced conformational changes in DNA-PKcs. Full-length DNA-PKcs is shown (left). All peptides and small molecule inhibitors were used at 10 μM, unless otherwise indicated.

To determine whether this interaction has functional consequences, we next examined the effects of GNFRYLAPP on DNA-PKcs activation and inactivation. When functioning normally, DNA-PKcs is activated by DNA binding, phosphorylates its downstream targets, participates in the recombination of DNA ends, and then deactivates through autophosphorylation and disengaging from DNA (20-22). Increasing molar ratios of GNFRYLAPP progressively inhibited the ability of DNA-PKcs to phosphorylate its p53-derived consensus substrate (Fig. 4C), suggesting the peptide induces an activation defect in its target. GNFRYLAPP inhibited DNA-induced DNA-PKcs autophosphorylation, as well (Fig. 4D-E), suggesting that it also affects target inactivation.

In considering how GNFRYLAPP causes multiple defects in DNA-PKcs function, we first hypothesized that the peptide might interfere with the recruitment of DNA-PK to DNA breaks. However, DNA precipitated DNA-PKcs from nuclear extracts equally well in the presence and absence of GNFRYLAPP (Fig. 4F). Moreover, GNFRYLAPP did not affect the electrophoretic mobility shift in DNA induced by incubation with purified recombinant DNA-PK (Fig. 4G).

We subsequently reasoned that GNFRYLAPP might function as an inhibitor of the conformational change induced in DNA-PKcs on binding to DNA, known to be required for its proper functioning (23, 24). To study its conformation, we examined patterns of protease protection in DNA-PKcs induced by its binding to DNA. In its open conformation, DNA-PKcs was digested by trypsin to an extent that no fragments remained detectable by immunoblot; by contrast, when bound to DNA, DNA-PKcs was partially protected from trypsin such that bands of approximately 80 and 130 kDa were detectable (Fig. 4H). GNFRYLAPP prevented the protection from trypsin digestion conferred on DNA-PKcs by DNA binding (Fig. 4H), suggesting GNFRYLAPP blocks the aforementioned conformational change in DNA-PKcs. Taken together, these data demonstrate that GNFRYLAPP functions as a non-competitive inhibitor of DNA-PKcs by blocking its DNA-induced conformational change and, thereby, suppressing its kinase activity.

GNFRYLAPP impairs DNA repair in homologous recombination-defective cells

To elucidate a biological role for GNFRYLAPP in mammalian cells, we synthesized a cell-internalizing version of the peptide by chemically fusing it in tandem to penetratin (pen), a 16-mer derived from the third helix of the homeodomain of the Drosophila melanogaster transcription factor Antennapedia. Added to the media of cells in tissue culture, penetratin-fused peptides undergo rapid cellular uptake and eventually distribute in various sub-cellular compartments, including the nucleus (25).

We first sought evidence for a physical and functional interaction between GNFRYLAPP and DNA-PKcs in cellulo. GNFRYLAPP-pen formed foci in the nuclei of irradiated tumor cells, and no such focus formation was seen in cells lacking DNA-PKcs (Fig. 5A, left). We observed delayed resolution of GNFRYLAPP-pen foci over time, with the majority of cells retaining nuclear foci as late as 12 hours following irradiation, potentially reflecting an acquired defect in DSB processing in the presence of GNFRYLAPP-pen (Fig. 5A, right). In support of this hypothesis, GNFRYLAPP-pen also suppressed DNA-PKcs autophosphorylation following irradiation of tumor cells (Fig. 5B), suggesting the peptide blocks activation of DNA-PKcs in cellulo. We next hypothesized that GNFRYLAPP-pen would impede DSB repair in cells and, thereby, might sensitize cells to DSB-inducing cytotoxic therapy, such as ionizing radiation. In light of the availability of alternative repair pathways in DNA-PKcs-deficient cells (26), we hypothesized that GNFRYLAPP-pen would show maximal inhibition of DNA repair in homologous recombination-deficient cells. In agreement with this hypothesis, GNFRYLAPP-pen both impaired DSB repair in cells lacking functional BRCA1 or BRCA2, and also sensitized these cells to ionizing radiation (Fig. 5C-E).

Figure 5.

GNFRYLAPP inhibits DNA-PKcs in cellulo. A, Biotinylated GNFRYLAPP-pen, detected with FITC-streptavidin, formed foci in DNA-PKcs +/+ cells (KS1767) 1 h after 3 Gy of radiation, but did not form foci in DNA-PK -/- cells (M059J) treated the same way (left). Quantification of GNFRYLAPP-pen foci in irradiated KS1767 cells (3 Gy) reveals slow resolution of these foci over time (right). B, Phosphorylated DNA-PKcs (T2609) was detected 30 minutes after 3 Gy of radiation in irradiated KS1767 cells treated with scrambled peptide, but no signal was detected in the presence of GNFRYLAPP-pen. C, Two BRCA-deficient cell lines (HCC1937, BRCA1-null; CAPAN1, BRCA2-null) showed increased residual double-strand breaks by the neutral comet assay (see Materials and Methods) at 3 and 6 hours after 6 Gy of radiation when treated with GNFRYLAPP-pen (red) or with a DNA-PKcs active site inhibitor (DMNB, orange). D-E, Both of these lines also exhibited increased radiosensitivity by clonogenic assay (see Materials and Methods) when exposed to GNFRYLAPP-pen (red) or DMNB (orange). All peptides and small molecule inhibitors were used at 10 μM.

Considering the potential therapeutic application of this peptide ligand, we next sought to further define the structure necessary for DNA-PKcs inhibition (Fig. 6A). Alanine scanning revealed GNFRY as the minimal required structure for DNA-PKcs inhibition. Tyrosine phosphorylation abolished the peptide's DNA-PKcs-inhibiting activity, as did mutating it back to GSFRYL, the consensus KARP-1 sequence. Serine phosphorylation of the KARP-1 sequence (GpSFRYL) reestablished DNA-PKcs inhibition, suggesting that GNFRYL serves as a mimic for the native active structure, GpSFRYL. A three-dimensional model of KARP-1 was generated (Fig. 6B) to view the mimicked domain from the perspective of its DNA-PKcs-binding site (19), which shows a high degree of accessibility of the aforementioned residues for protein binding.

Figure 6.

A, Alanine scanning of the synthetic peptide, GNFRYLAPP, revealed GNFRY as the minimal structure functionally inhibiting DNA-PKcs kinase activity (left). Phosphorylated mutant peptide derivatives of the parent compound demonstrate that GNFRYLAPP functionally mimics GpSFRYLAPP, accounting for the asparagine-serine mismatch between the peptide and KARP-1, and that tyrosine phosphorylation abolishes activity of both compounds (right). B, Superimposed on a predicted structural model of KARP-1 (green), the key DNA-PK-interacting residues (red) are shown from the perspective of the putative DNA-PKcs binding site. The asparagine mutation is shown in yellow.

Discussion

Although Phase I clinical trials have shown that DNA repair inhibitors may represent powerful and specific anti-cancer treatments, reactivating mutations in targeted pathways will likely lead to therapeutic resistance (3, 27-29), making the discovery of additional synthetically lethal interactions in DNA repair pathways critically important for the ultimate success of this emerging class of drugs. The platform described here provides an opportunity to accelerate the development of potential drug targets in the DNA repair network by allowing the simultaneous (i) discovery of targetable and functional motifs, (ii) prioritization of targets by functional interaction, and (iii) unbiased exploration for proteins with unrecognized roles in DNA repair.

To validate the utility of this approach, we selected GNFRYLAPP as a lead candidate over other promising peptides returned in the screen. The basis for this choice was first that the peptide mimics a small protein, increasing the odds that an oligopeptide might be sufficient to recapitulate a minimal structural and functional domain. Second, the region within KARP-1 mimicked by GNFRYLAPP is well-conserved amongst primates, and is nestled between two putative localization domains – a nuclear localization signal and a leucine zipper domain (19). The short interspersed domain is an ideal candidate, then, for carrying out the DNA-PKcs-regulating function of KARP-1.

As shown by our cellular data, GNFRYLAPP shows therapeutic potential as a DNA-PKcs inhibitor. Many DNA-PKcs inhibitors have shown promising anti-cancer activity in pre-clinical models, only to fail clinical development due to unacceptable toxicity levels. To date, there are no DNA-PKcs inhibitors available for clinical use. Potentially addressing an unmet clinical need, then, GNFRYLAPP may have immediate therapeutic relevance in oncology. Although toxicology aspects of its development are yet to mature, peptide-based drugs typically have favorable toxicity profiles, and it is likely that GNFRYLAPP will represent a deliverable alternative to existing DNA-PKcs inhibitors. Further work is needed to determine if this will be the case.

GNFRYLAPP may also provide added value to the repertoire of DNA-PKcs inhibitors by virtue of being the only available compound that functions through a non-competitive mechanism. As a result of its unique mechanism of action, GNFRYLAPP has several distinctive biochemical functions. First, though GNFRYLAPP inactivates the kinase activity of DNA-PKcs (Figs. 4B, 4C), DNA-PKcs must first be stimulated by a DSB before it is able to physically interact with GNFRYLAPP (Fig. 4A). The fact that DNA-PKcs must first engage a DSB prior to binding GNFRYLAPP is reinforced by the finding that the affinity of DNA-PKcs for DNA is unaffected by GNFRYLAPP (Figs. 4F, 4G). This behavior is reflected by the cellular data, which show GNFRYLAPP-pen focus formation only after DNA-PKcs is stimulated to bind to double-strand breaks following irradiation. All of these data point to the possibility of a “quasi”-activated state in DNA-PKcs, where binding to a DSB creates a biochemical environment suitable for further downstream protein-protein interaction (with KARP-1, for example), but during which time the conformational change required for kinase activation has not yet occurred. By preventing the conformational change induced in DNA-PKcs upon binding DNA, GNFRYLAPP blocks activation of the enzyme at the DSB. This distinguishes its mechanism of action from the active site inhibitors and, thus, may establish a novel class of DNA-PKcs inhibitors with immediate clinical applicability.

Even in the event that GNFRYLAPP does not demonstrate an improved therapeutic ratio compared to existing DNA-PKcs inhibitors, the true value of having pursued combinatorial drug selection for DSB inhibitors may lie in the sheer volume of candidate molecules uncovered. Many of the as yet uncharacterized peptides recovered in our initial screen may yield additional novel DNA repair inhibitors. Priority for further validation may be given to those mimicking domains in proteins for which structural data is available, as these data provide a significant advantage for structure-based rational drug design. Our ability to annotate these domains as likely being functionally involved in DSB repair ought to substantially increase the efficiency of such efforts. However, it should be noted that the screening method described here is not likely to have covered all DSB reactome protein domains comprehensively. The screen was designed to select for high-affinity reactions at the DSB end, and these represent a subset of all reactions relevant to DSB repair. We reasoned, though, that these high-affinity interactions may be the most therapeutically relevant and, therefore, a reasonable focus for initial efforts; our results with GNFRYLAPP suggest that this reasoning is sound.

In summary, we have used here subtractive combinatorial selection to guide the discovery of GNFRYLAPP as a non-competitive inhibitor of DNA-PKcs and a candidate anti-cancer therapy. This approach may establish a foundation for rapidly expanding the discovery and exploitation of ligand-based DNA repair inhibitors for cancer treatment.