CVR is often expressed as the ratio of cerebral blood flow change to CO2 change. We examine several factors affecting this measurement: blood pressure, stimulus pattern, response analysis and subject position.Cerebrovascular reactivity (CVR), measures the ability of the cerebrovasculature to respond to vasoactive stimuli such as CO2 were implemented in nine subjects, seated and supine. Middle cerebral artery blood flow velocity (MCAv), and mean arterial pressure (MAP) were determined breath-by-breath. Cerebrovascular conductance (MCAc) was estimated as MCAv/MAP. CVR was calculated from both the relative and absolute measures of MCAc and MCAv responses.Step and ramp increases in CO2 in some subjects so that relative CVR calculated from conductance responses were less than those calculated from CVR calculated from velocity responses. CVR measured from step responses were affected by the response dynamics, and were less than those calculated from CVR measured from ramp responses. Subject position did not affect CVR.MAP increased with CO2 and acts as a confounding factor for CVR measurement; (2) CVR depends on the stimulus pattern used; (3) CVR did not differ from the sitting versus supine in these experiments; (4) CVR calculated from absolute changes of MCAv was less than that calculated from relative changes.(1) MAP increases with CO We note that for these analyses, the CBF responses may be measured in absolute or relative values. We also introduce the possibility of using frequency domain analysis to calculate CVR with transfer function analysis (TFA), such as that used in analyzing dynamic pressure autoregulation  as the response.Our aim in these experiments was, therefore, to investigate how changes in MAP, and different body positions, stimulus patterns and analysis techniques affect the calculation of CVR. We used COThese studies conformed to the standards set by the latest revision of the Declaration of Helsinki. Nine (4 mol/L) healthy normotensive, nonsmoking subjects of mean (SD) age 27 (4) years participated in this study after approval from the Research Ethics Board at the Toronto General Hospital  and written informed consent from each of the subjects. We note that our subjects were considered healthy but were not subjected to examination of the neck vessels to exclude the presence of carotid steno-occlusive disease. They were not taking any medication other than oral contraceptives, and had no history or symptoms of cardiovascular, cerebrovascular, or respiratory diseases. In addition, they abstained from caffeinated or alcoholic beverages and vigorous exercise for at least 12 h before the study.2 and O2 , and recorded at 20 Hz. Each of these instruments saved a digital record for later analysis.Subjects were fitted with a face mask, connected to the breathing circuit via a mass flow sensor  to monitor ventilation. Beat-by-beat middle cerebral artery flow velocity was measured using bilateral transcranial Doppler  at 2 MHz and sampled at 125 Hz. Beat-by-beat MAP and HR were determined by finger plethysmography  sampled at 200 Hz. Tidal gas was sampled and analyzed for the partial pressures of CO2 tension (PetCO2) while maintaining O2 tensions at resting levels  circuit and a computer-driven gas blender ; a method described by  and PetCO2 so that the stimulus patterns applied are those of PaCO2  the slope of the linear regression of breath-by-breath values including all baseline breaths, but only the breaths from the final 2 min of the 5-min step increase in PetCO2 . In addition, the changes in blood pressure during the step changes of PetCO2 were measured and classified according to whether or not a threshold increase of 10 mmHg was exceeded. Finally, the speed of the MCAv response was determined by fitting an exponential rise, and classified as slow when the time constant exceeded 5 sec and fast when less.CVR was calculated from the step responses in two ways to take into account their dynamic aspects;  the slope of the linear regression of all breath-by-breath values of MCAv and MCAc from the start of baseline to the end of the 5-min step increase in PetCO2 of the ramp stimulus. CVR was calculated in two ways; (Lin) the slope of the linear regression, and (Sig) the slope of the sigmoid function, a + (b/(1 + exp(−(x−c)/d))), fitted to the data  at the baseline PetCO2. In addition, the changes in blood pressure during the ramp changes of PetCO2 were examined and fitted with two linear segments above and below a threshold determined as the best fit.The ramp responses included all breath-by-breath values of MCAv and MCAc between the minimum and maximum PFinally, we introduced the use of transfer function analysis (TFA) to calculate a TFA CVR from the gain function. Breath-by-breath step and ramp responses were selected from the recordings to obtain 500 sec samples . CVR values calculated from both MCAv and MCAc responses and were compared  between various measures with repeated measure analyses of variance (rmANOVA). Where factors were found to be significant, post hoc all pairwise multiple comparisons were made using the Holm–Sidak method. The correlations between step (SS) and ramp CVR (Lin) and TFA CVR values were examined with the Pearson product moment.etCO2, and MAP varies only slightly with PetCO2.All subjects completed all tests except for subject 7 where the supine ramp response was lost due to a technical failure. Figure etCO2 and 5.2 mmHg or less for PetO2. The differences in PetCO2 and PetO2 between adjacent breaths, expressed as the overall mean standard deviation, were also small; 1.0 mmHg or less and 2.2 mmHg or less, respectively. That there was little drift during the hypercapnic period of the step stimulus was indicated by the mean of the between breath differences, which was close to zero for both PetCO2 and PetO2. We concluded that stimulus drift and variation during the step tests was minimal and could not account for the response patterns observed.The step stimuli baseline and hypercapnic portions showed little variation, with the overall mean breath-by-breath variability, expressed as standard deviation, less that 1.6 mmHg for PP = 0.007; 1-way rmANOVA); higher in the supine position.Baseline values of MAP, MCAv, and MCAc Table  were tesTable P = 0.173 and 0.164 for All and SS, respectively), it was for CVR calculated from MCAc responses . CVR values calculated from MCAc were considerably lower when MAP increased (mean [SD] = 0.92 [1.14] vs. 2.75 [0.86] %/mmHg for the All determination and 0.93 [1.34] vs. 2.81 [0.9] %/mmHg for the SS determinations).We examined the relative CVR values to discover whether or not they differed when MAP increased using a 2-way ANOVA with factors response (MCAv vs. MCAc) and MAP increase (yes vs. no), treating all tests as independent. Table 2. We elaborate on this reasoning in the discussion. Consequently, further examinations were restricted to CVR values measured from MCAv responses during which MAP remained unchanged.With such a large decrease in CVR conductance (MCAc response) when MAP increased  and speed of response (fast vs. slow).P ≤ 0.001 and 0.002 for relative and absolute CVR, respectively), but did not differ for the fast responses , as illustrated in Figure The All and SS CVR values differed for slow responses  and position (sitting vs. supine). Table etCO2 as the Sig CVR. We examined the changes in MAP during the ramp increase in CO2 by fitting two linear segments above and below a threshold where the slope changed abruptly. Figure etCO2 where MAP began to increase. Ramp tests where the sub-threshold slope was > 0.6 mmHg/mmHg and the threshold < 46 mmHg were eliminated from further analysis as confounded by the concurrent increase in MAP. Table 2 where MAP was constant.The MCAv responses were fitted with either a straight line or a sigmoid as illustrated in Figure P = 0.153 and 0.198 for relative and absolute values, respectively). Furthermore, position was not a significant factor despite the finding of a decreased CVR in the sitting position . A 1-way rmANOVA comparison of the sitting vs. supine sigmoidal characteristics also found no difference between sitting and supine tests.These CVR measures were examined to determine if the linear and sigmoid fitting of the responses produced different CVR values, and to see if position was a significant factor using a 2-way rmANOVA with factors analysis (linear vs. sigmoid) and position (sitting vs. supine). Table We compared the CVR values obtained from the linear analysis of the ramp MCAv responses with their corresponding CVR values obtained from the SS analysis of the step MCAv responses (tests where MAP was not a factor) using a 2-way rmANOVA with factors test stimulus type (step vs. ramp) and relative  versus absolute  units. Table etCO2 at which the sigmoidal response slope was the highest (the midpoint parameter). A 1-way rmANOVA analysis showed that the midpoints of the sigmoid functions fitted to the relative MCAv responses (mean [SD] = 42.5 [2.6] mmHg) were not different from those of the absolute responses (mean [SD] = 41.7 [3] mmHg), but both were higher than the baseline PetCO2 of these selected tests (mean [SD] = 39.9 [0.8] mmHg). The baseline PetCO2 chosen for these experiments was, therefore, slightly lower than the PetCO2 where CVR is a maximum and the cerebrovasculature midway between its limits of vasoconstriction and vasodilation.Finally, the sigmoidal fitting of the responses also provided a measure of the PWe examined the ramp and step MCAv (cm/s/mmHg) responses where MAP was not a confounding factor with transfer function analysis. Figure We noted that the phase reflected the speed of the response; the mean (SD) phase of 6 step responses classified as slow or oscillatory was −0.27 (0.15) radians compared to 0.01 (0.05) radians for the fast responses.2, i.e. vascular diameter changes. It is this aspect that assumes clinical importance in defining the ability of a vascular bed to adjust its flow resistance to cope with challenges to its blood flow supply. Such cerebrovascular reactivity measurements to assess cerebrovascular health have important clinical applications. Impaired vascular reactivity has been linked to an increased risk of stroke  pressure autoregulation vasconstriction; (3) physical pressure vasodilation; (4) physical passive pressure flow increase. Pressure autoregulation vasoconstriction and CO2 vasodilation interact since both adjust vessel diameter, so that if autoregulation is not exhausted, then any increase in perfusion pressure invokes autoregulation adjustment of cerebrovascular conductance to regulate cerebral blood flow  CO2-induced vasodilation on cerebral blood flow. While MCAc in these experiments does provide an estimate of the conductance response to CO2 due to the various factors involved, it does not estimate the vasodilatory effect of CO2, and so using the MCAc response to measure CVR is misleading. We concluded that increases in MAP with CO2 remain a confounding factor when measuring CVR, and consequently responses where MAP increased were excluded from further analysis in this study.In none of these circumstances is it possible to separate the effects of perfusion pressure and CO2 that confound cerebral blood flow responses to CO2 are routinely disregarded in experiments such as breath-holding  and other subjects decreasing HR . The subjects with MAP increases with CO2 during ramp stimuli and showed no discernible concurrent changes in HR. Although these changes in heart rate may indicate changes in sympathetic and parasympathetic tone  and should be used when the stimulus range is large.The ramp stimulus range in these experiments, where PetCO2 produced by the step stimulus elicited responses that revealed their dynamic aspects; in these experiments, we were able to discern slow and fast responses as well as two oscillatory responses as previously observed  and the ramp and step stimuli. Therefore, the statistical test results between relative and absolute CVR measures did not differ. However, in cases where the baseline CBF values differ between conditions, the choice between relative and absolute CVR measures may affect the statistical test outcome, since changes in baseline CBF inversely affect the relative CVR.In these experiments, we introduced the use of transfer function analysis to calculate CVR, limiting the analysis to the ramp and step responses where MAP was constant. The TFA CVR values were similar to those calculated from the linear fits to the ramp responses and the steady-state step responses, and well correlated. Moreover, the phase values correlated with the speed of response determined from exponential fitting. We therefore concluded that TFA analysis deserves further investigation, and in particular we suggest that TFA analysis may be useful in BOLD MRI mapping of CVR, where the phase response could indicate areas of slowed responsiveness.2 as a stimulus, led to several conclusions bearing on the measurement of CVR. First, if the measurements of CVR are to be used as an indication of cerebrovascular vasodilatory reactivity, then MAP must be monitored, and CVR should be calculated from the change in MCAv while MAP remains constant to avoid the confounding action of changes in MAP with CO2. Second, when CVR is measured from the responses to a step stimulus pattern from baseline to hypercapnia, it is important to observe the dynamics of the response and use an analysis appropriate to the study aim. Third, if the ramp stimulus range is limited, then CVR values calculated from linear regression are equivalent to those calculated from fitting a sigmoidal function to the response, but the sigmoidal function parameters offer additional information. Fourth, transfer function analysis to calculate CVR appears to be a technique worthy of further investigation. Fifth, the subject position, sitting or supine did not alter the CVR. Finally, CVR calculated from relative responses differ from those calculated from absolute responses, but did not alter the statistical test results in these experiments.These experiments, using MCAv as a surrogate for CBF and CO


BRCA1, BRCA2 and PALB2. RAD52 also participates in the recovery of the stalled replication forks. Anticipating that ssDNA binding activity underlies the RAD52 cellular functions, we carried out a high throughput screening campaign to identify compounds that disrupt the RAD52-ssDNA interaction. Lead compounds were confirmed as RAD52 inhibitors in biochemical assays. Computational analysis predicted that these inhibitors bind within the ssDNA-binding groove of the RAD52 oligomeric ring. The nature of the inhibitor-RAD52 complex was validated through an in silico screening campaign, culminating in the discovery of an additional RAD52 inhibitor. Cellular studies with our inhibitors showed that the RAD52-ssDNA interaction enables its function at stalled replication forks, and that the inhibition of RAD52-ssDNA binding acts additively with BRCA2 or MUS81 depletion in cell killing.The DNA repair protein RAD52 is an emerging therapeutic target of high importance for BRCA-deficient tumors. Depletion of RAD52 is synthetically lethal with defects in tumor suppressors DOI:http://dx.doi.org/10.7554/eLife.14740.001 Cells are constantly in danger of losing or scrambling critical genetic information because of DNA damage. To cope with this stress, cells have numerous DNA repair systems. One of these systems – homology-directed DNA repair – involves the proteins BRCA1 and BRCA2, which are often missing or defective in breast and ovarian cancers. The BRCA-deficient cancer cells can still survive, but become “addicted” to other DNA repair proteins – among them a protein called RAD52. It might be possible to kill these cancer cells using drugs that stop RAD52 from working. Such treatments would have the benefit of not harming normal healthy cells, as these cells contain working BRCA proteins and can survive without RAD52.It is not currently known exactly how RAD52 allows the BRCA-deficient cells to survive, but this probably depends on RAD52’s ability to bind to single strands of DNA. Small molecules that block the interaction between the RAD52 protein and DNA might therefore help to kill cancer cells.Hengel et al. developed a high throughput biophysical method to search through a large collection of small molecules to find those that prevent RAD52 from binding to DNA. The best potential drug leads were then tested in laboratory-grown human cells and using biophysical and biochemical techniques. Computational approaches were also used to model how these molecules block the interaction between RAD52 and DNA at the atomistic level.Hengel et al. then used the information about how the small molecules bind to RAD52 to perform further computational screening. This identified a natural compound that competes with single-stranded DNA to bind to RAD52. The activity of this molecule was then validated using biophysical methods.The methods used by Hengel et al. provide the foundation for further searches for new anticancer drugs. Future studies that employ the small molecule drugs identified so far will also help to determine exactly how RAD52 works in human cells and how it helps cancer cells to survive.DOI:http://dx.doi.org/10.7554/eLife.14740.002 Understanding of synthetically lethal relationships between genome caretakers will help to define the molecular mechanisms underlying the maintenance of genomic integrity and may lead to the advancement of personalized cancer treatments. Depletion of the human DNA repair protein RAD52 is synthetically lethal with defects in tumor suppressors, BRCA1, BRCA2, or PALB2 . ImportaBRCA1 and BRCA2 are tumor suppressors that are commonly mutated or depleted in hereditary and sporadic breast cancers, and have important roles in homologous recombination (HR) , a templRAD52 has only a mild effect on recombination in vertebrates   and the ‘1’ ((−)−Epigallocatechin) and ‘6’  predicted to bind within the RAD52 ssDNA-binding site, inhibited the formation of the RAD52-dependent DSBs in hydroxyurea (HU)-stressed, checkpoint deficient cells to the same level as RAD52 depletion. Moreover, ‘1’ acts additively with the MUS81 depletion to kill cells treated with hydroxyurea (HU), which perturbs replication, and with checkpoint inhibitor UCN01. These data strongly suggest that the ssDNA binding activity of RAD52 is required for recovery of stalled replication forks in checkpoint deficient cells. We also show that ‘1’ selectively kills cells depleted of BRCA2, further supporting the importance of the RAD52-ssDNA interaction in BRCA deficient cells and the potential therapeutic value of RAD52 inhibition. Finally, in order to validate the strength of our hypotheses about the structural nature of the RAD52-inhibitor complex, we developed a validated in silico screening campaign, based on our HTS results, using a library of four thousand natural products. We describe the discovery of NP-004255, a macrocyclic compound, which we show by NMR WaterLOGSY and biophysical assays to be a completely novel and effective inhibitor of the RAD52-ssDNA interaction. The implication of these findings for the discovery of novel therapeutics that specifically inhibit the activity of RAD52 is discussed.Here, we report the development of a novel FRET-based high throughput screening (HTS) assay that led to the identification of compounds that disrupt the RAD52-ssDNA interaction. Initial HTS hits were biochemically validated in RAD52 functional assays and tested in two separate cellular assays. Two available high resolution crystal structures (PDB: 1H2I and 1KNO) of the conserved ssDNA-binding domain of RAD52 highlight the unique nature of this target . The ssD30-Cy5). When this ssDNA forms a stoichiometric complex with RAD52 (one 30-mer ssDNA molecule per one heptameric ring of RAD52), the two fluorophores are brought close to one another resulting in an increase in the FRET signal. The assay was successfully adapted to the 384-well plates HTS format. In each well, we recorded the fluorescence signal of the Cy3 dye, which was excited directly, and the signal of Cy5 dye, which was excited via the energy transfer from Cy3. The apparent FRET signal was then calculated as described in the Materials and methods. The separation between the positive control ) and the negative control (an unperturbed stoichiometric complex of RAD52 with Cy3-dT30-Cy5) initially resulted in a Z’ factor of 0.66 when calculated for the whole plate. Further optimization increased the Z’ factor calculated for the control rows in the screening experiments to 0.94, indicating excellent reliability of the assay (30-Cy5) of at least 5 S.D. The 96 preliminary hits were assembled into a 'cherry picked plate' and were tested in two more rounds of screening To identify compounds that disrupt the RAD52-ssDNA interaction we adapted a previously developed FRET-based assay  to the Hhe assay . Using t30-Cy5 complexes (1 nM Cy3-dT30-Cy5 and 8 nM RAD52). As the compounds bind and disrupt the ssDNA-RAD52 interaction we observed a decrease in FRET between the DNA-tethered Cy3 and Cy5 fluorophores. At each concentration of the compound, the FRET signal was adjusted for the change in Cy3 and Cy5 fluorescence in the presence of the compound, but in the absence of protein. From the hyperbolic inhibition curves we then calculated IC50 value for each compound under these conditions  selected for the in-depth follow-up characterization as disruptors of the RAD52-ssDNA interaction, we tested the ability of these compounds to interfere with the RAD52-dsDNA interaction, which involves a different site on the RAD52 ring  and the WaterLOGSY spectrum of compound ‘1’ in the presence of RAD52 (red) are compared, positive WaterLOGSY peaks are observed for the compound ‘1’, indicating the binding of ‘1’to RAD52 , an NMR technique, which is based on transfer of magnetization from bulk water to the protein-bound compound of interest , 2000. Ito RAD52 . Similarcompound . NotablySY peaks , thoughtSY peaks , while ‘GSY peak  nor inteGSY peak .1‘’ and 6’‘ can inhibit the RAD52 binding to and annealing of the RPA-coated ssDNA we added stoichiometric amounts of RPA (1 RPA per 30 nucleotides of ssDNA) to the FRET-based ssDNA binding/wrapping and ssDNA annealing experiments described above. RPA binds ssDNA with high affinity and extends the ssDNA to its contour length. In our assays such an extension manifests as a distinct FRET state of ~0.3, which is readily distinguished from a FRET state of ~0.48 of free Cy3-dT30-Cy5 ssDNA, as well as ~0.63 FRET of the stoichiometric ssDNA-RPA-RAD52 complex , the major eukaryotic ssDNA-binding protein essential for DNA replication, repair and recombination . The RPAplex see  for detatrations . Both, hhout RPA . Similarnnealing . Notablyproteins .In order to gain insight into the binding determinants of the various polyphenol hits obtained from the HTS screening, we undertook a computational investigation using the structure of the oligomeric ring formed by the conserved ssDNA-binding domain of RAD52 (PDB 1KNO). We utilized a layered approach involving docking and all atom-simulated annealing with explicit solvent, using a knowledge based force field . The lon‘6’, which resulted in a single pose scoring the highest in all three methods. To determine which methodology consistently provides the most accurate scoring, we employed a conservative approach, in which the 34 selected top scoring complexes were further subjected to all atom-simulated annealing studies, using explicit solvent and salt conditions.An all atom force field-based protocol was employed to distinguish binding affinities from a variety of docking poses that possessed various docking metrics. We compared the different scoring metrics, such as Triangle Matcher placement scores followed by rescoring with the affinity function versus the computationally expensive force field-based ligand refinement and subsequent MM/GBSA scoring. The use of all atom simulations (including explicit solvent models), when combined with docking, has been shown to significantly boost docking procedures' ability to predict and rank compound affinities. Therefore, we can compare the differences in free energy changes due to ligand binding for each of the poses, an ability that is not all within the realm of classical docking procedures . Thirty ‘1’ and ‘6’ yielded complexes with unique binding sub-pockets or 'hotspots' along the RAD52 binding groove, suggesting that they may have distinct biological activities and/or efficacies with regard to their ability to compete with ssDNA binding  Docking approach are listed in the  binding . The SAE binding  . We previously reported that concomitant depletion of RAD52 and MUS81 gives raise to the MUS81-independent DSBs . The ROC curve for '1'  for in vitro inhibition studies. The compound that was identified, NP-004255 is known as corilagin, and is a member of the class of secondary plant metabolites called ellagitannins. Corilagin is a macrocyclic ester consisting of three trihydroxylated phenolic moieties. NP-004255 binds to RAD52 in a similar manner as '1' and '6', in that it uses a buried interstitial water network, and is able to adopt a conformation that fits nicely into the ssDNA binding groove  in the narrow DNA binding groove. Indeed, it may be that this shape complementarity and the ability to utilize the resident waters dominates the binding determinants . Our iterative approach of compound discovery followed by the in silico screening was clearly successful in expanding the chemical space of our lead compounds, but more importantly provides a platform for strengthening the structure-activity relationship in an exceedingly challenging target pocket. Indeed, the discovery of the secondary plant metabolite, NP-004255, a macrocycle, as a means to effectively compete with a native substrate macromolecule (ssDNA and the ssDNA-RPA complex) may prove to be a new strategy in the field of disrupting protein-nucleic acid interactions. It has to be noted, however, that although our work provides a solid understanding of how shape complementarity and utilization of interstitial water networks drives complexation, there are limitations to these approaches. Specifically, the current experimental and computational studies do not address the underlying thermodynamics and kinetics that control ligand competition in the ssDNA binding groove. A fascinating aspect of the competition between small molecules and macromolecules for an extended and partially solvated binding pocket, as in RAD52, is the extent to which small molecules may exploit the potential enthalpy-entropy compensation of a large and floppy native DNA ligand. Additionally, residence times of lead compounds may be a critical factor in the successful design of therapeutic lead compounds. These factors will be addressed in the future studies that will be focused on the thermodynamics and kinetics of small molecule binding to the RAD52 protein, and in the studies that will confirm the ligand placement through high resolution structures.We chose to target the well characterized ssDNA binding activity of RAD52, which we expected to underlie RAD52 functions both in supporting replication and in promoting the survival of BRCA-deficient cells. The ssDNA-binding groove of RAD52 is an interesting target for small-molecule binding in that it spans the circumference of the RAD52 oligomeric ring and offers a repetitive pattern of potential binding pockets. This deep and circular groove surprisingly yields reasonable druggability scores, as described in the Results section. Nevertheless, it is a highly exotic cavity, and very distinct from enzyme and receptor pockets. The ssDNA binding groove consists of an alternating arrangement of hydrophobic and hydrophilic regions. Twelve compounds that inhibit RAD52-ssDNA interaction identified in this study  are pred1’ and 6-hydroxy-DL-dopa, suggests that disrupting the RAD52-ssDNA interaction or the integrity of the RAD52 oligomeric ring bears negative consequences for the RAD52 cellular functions. Considering that the efficient homology search and the DNA strand annealing requires the two complementary DNA strands (or the complementary ssDNA-RPA complexes) to be wrapped around the two different RAD52 oligomeric rings . Therefore, RAD52 inhibitors could be useful in combination with drugs which elicit replication stress. Tumors in which MUS81 is mutated or downregulated have been described , Target28Cy3 (T-28) (5′-ATAGTTATGGTGAGGACCC/iCy3/CTTTGTTTC-3′), Probe28Cy5 (P-28) (5′ GAAACAAAGGGGTCC/iCy5/ TCACCATAACTAT-3′) Oligo28-REV (5’-(Cy5)-GCAATTAAGCTCAAGCCATCCGCAACG-(Cy3)-3’, Cy3-Oligo28-Cy5 . All chemicals were reagent grade . All compounds were purchased from MicroSource Discovery systems, Inc  and Sigma-Aldrich. The NP-004255 was from AnalytiCon Discovery GmbH . Purity and structures of the purchased compounds were assessed from 1H NMR spectra collected on a Varian Unity Inova 600 MHz NMR spectrometer at 0.5 mM concentrations diluted into DMSO-d6.The HPLC purified ssDNA substrate -100. These were designated as negative controls as 10 nM poly(dT)-100 was sufficient to fully inhibit formation of the wrapped RAD52- Cy3-dT30-Cy5 complex under the selected experimental conditions (data not shown). Using a Multiflo dispenser (Biotek), 50 µL of the positive and negative controls were dispensed into their respective wells. Then 15 nM Cy3-dT30-Cy5 and 100 nM RAD52 in RAD52-HTS buffer were dispensed into each well. Next, 1 µL of each compound dissolved in DMSO at 833 µM concentration  was dispensed into the wells in columns 3 – 22 using a Microlab Star liquid handling robot (Hamilton) and mixed 3 times. Thus, 320 compounds were assayed per 384 well. The plate was incubated at 25°C for 30 min and the fluorescent signal of the Cy3 (λex(Cy3)= 530 nm; λem(Cy3)= 565 nm) and the Cy5 (λem(Cy5)= 660 nm) dyes were recorded using a Wallac, Envision Manager. The apparent FRET was calculated asHTS against the MicroSource Spectrum collection  was performed in nine 384 well plates. All measurements were carried out in the RAD52-HTS buffer containing 20 mM Hepes pH7.5, 1 mM DTT, and 0.1 mg/mL BSA. Each 384 well plate contained two columns of negative and positive controls as follow: Columns 1 and 24 were the positive controls, which contained 100 nM RAD52 (monomers) and 15 nM (molecules) Cy3-dTp and SDn are standard deviations, and μn and μp are means of the negative and positive control . NMR spectra were processed using NMR Pipe  NMR experiments , 2000. TNMR Pipe  and analNMR Pipe .FRET-based ssDNA binding, dsDNA binding, and annealing assays were carried out as previously described  using Ca30-Cy5 in the absence of RAD52. For each compound concentration we subtracted the difference in the FRET signal in the presence and absence of the compound from the respective FRET signal in the presence of RAD52. The FRET signal corrected for the compound fluorescence was calculated using the equation:To confirm that selected compounds inhibit RAD52 mediated binding and wrapping of ssDNA, compounds were titrated into stoichiometric complex containing 1 nM T30 and 8 nM RAD52. All experiments were performed in triplicates, and the data are shown as averages and standard deviations for three independent measurements. To remove possible experimental artifacts associated with chromogenic or fluorogenic compounds, as well as with the compounds that may quench or enhance Cy3 or Cy5 fluorescence, we also performed control titrations whereby we titrated each compound into 1 nM Cy3-dTas previously described  and plot0 is the initial FRET value of the RAD52-ssDNA complex in the absence of the compound and FRETmin is the FRET value at complete inhibition. FRET values were calculated as an average of three or more independent annealing reactions plotted against the concentration of the compound. Inhibition of the RAD52-dsDNA interaction was assayed in a similar experiments, except the stoichiometric complexes containing 1 nM molecules of Cy3-Oligo28-Cy5 duplex DNA and 10 nM RAD52.where FRETTo assess if selected compounds inhibit RPA-ssDNA mediated binding and wrapping by RAD52 compounds were titrated into stoichiometric complexes containing 1 nM T30, 1 nM RPA, and 10 nM RAD52. In all experiments, the FRET values for each data point were corrected for the effects of the compounds on the respective substrate in the absence of RAD52.app values were calculated as an average of three or more independent annealing reactions plotted against time (s). The average FRET values were fitted to a double exponential to calculate the final extent of annealing using Graphpad Prism4 software. The calculated annealing extent was plotted as a function of compound concentration and fitted to the same model as we used to determine IC50 values for the inhibition of DNA annealing.Annealing of complementary oligonucleotides by RAD52 was monitored under identical conditions as the binding assays described above. For each assay, the reaction master mixture containing 8 nM RAD52 protein in the presence and absence of the compounds at varying concentrations was prepared at room temperature and divided into two half reactions. Following baseline buffer and protein measurements, 0.5 nM of T-28 ssDNA substrate was added to the reaction cuvette and the signal was allowed to stabilize. The annealing reaction was initiated upon addition of the second half-reaction pre-incubated with 0.5 nM P-28 ssDNA substrate. The fluorescence of Cy3 and Cy5 were measured simultaneously over the reaction time course (500 s). FRETOur initial computational workflow employed a combination of classical docking, using the Triangle Matcher approach and scoring using the London dG scoring function  in MOE 2013.08 , followeThe all atom-simulated annealing energy minimization (here referred to SAEM for brevity), which is followed by a local docking protocol (as described below) is a customized protocol that was automated with a script using the Python-based Yanaconda scripting language, and use of the Yamber03 knowledge-based force field . Briefly‘1’ or ‘6’. Where indicated, the GM01604 cells were cells were transfected with siRNAs directed against GFP (Ctrl), or against RAD52 (Qiagen) 48 hr prior to induction of replication stress and/or inhibitor treatment. After that cells were subjected to neutral comet assay as described in Murfuni et al  according to the manufacturer’s instructions. Cell number was counted in randomly chosen fields and expressed as percent of dead cells  corrected for the cell loss observed in the population. For each time point, at least 200 cells were counted.GM01604 cells were transfected with siRNAs directed against GFP (Ctrl), or against MUS81 (Qiagen), BRCA2 (Sigma-Aldrich), and RAD52 (Qiagen) 48 hr prior to addition of 1 µM The AnalytiCon Discovery MEGx Natural Products Screen Library, which is the in silico version of an actual library of purified natural products from plant, fungal and microbial sources, which is available for purchase, was subjected to an in silico screening campaign. The campaign was designed based on the ability to optimally minimize false positives and false negatives, and to maximize true positives and true negatives. More specifically, the experimental hits identified in the HTS campaign described above, constitute the true positives, while specifically selected decoy compounds constitute the true negatives. Decoy compounds were generated using the Database of Useful Decoys – Enhanced (DUD–E) website . Decoys In order to validate the selected protocol , we emplA database containing the AnalytiCon Natural Products compound library, and a control selected from the initial HTS hits, were created and preprocessed for virtual docking (as described above). The top 30 final poses, generated using the Dock utility of MOE (as described above) were written to an output database. Poses of all compounds were ranked based on their scores. Compounds with the poses most favorable to binding, i.e. the poses with the lowest energy scores from the London dG scoring function were selected for further analysis. Those poses with better scores than the highest scoring pose of our control  were selected, and then subjected to a refining docking step involving force field-based energy minimization with the MMFF94x force field in MOE. Binding energies were ranked, and evaluated.The docking scores  were used for determining the ROC threshold values  The inhibition mechanism proposed is not convincing due to several issues in cell-based assays.2) Previous related studies reduce the impact of the current study is an important point concerning the significance of the work.3) The molecular modeling studies may be the main novelty compared to previous studies on Rad52 inhibitors by other researchers but do not add much to the manuscript in the absence of experimental validation.Reviewer 1:BRCA1, BRCA2, and PALB2-deficient cell survival. This work trails at least three other studies that have used similar HTS assays to identify RAD52 inhibitors . This manuscript needs to address how the compounds identified in this screen compare to the compounds that were previously identified as RAD52-ssDNA binding inhibitors. Is this largely a confirmation of what has been seen previously, or does the current work uncover new molecular scaffolds? In addition, this paper largely concludes that compounds '1' and '6' disrupt RAD52-ssDNA binding. However, an alternative hypothesis may be that these compounds affect the RAD52 superstructure, as suggested in Chandreamouly et al. This hypothesis can be easily tested here. Finally, the cell biology work requires multiple controls prior to publication in eLife.Hengel et al. identified novel small molecule inhibitors of RAD52, a protein required for Major Comments:1) Addition of RPA in 2) Compound '6' was dropped from The Discussion should also mention the RPA binding to '6'. Perhaps this is a reason for its high IC50 in cells.3) 4) 5) The authors could further test the method of action of their drugs by attempting to observe RAD52 foci in both stressed and unstressed cells. Presumably the drugs should reduce DNA damage-induced RAD52 foci.Reviewer #2:Hengel et al. present the results of a drug screen for compounds that inhibit the ability of RAD51 to bind ssDNA. A number of hit compounds are found and of these two characterized biochemically and in cell based assays. Evidence for direct binding of the two compounds was obtained by a method that I am unfamiliar with (WaterLOGSY) and computational analysis is used to identify potential binding sites. One of the lead compounds '1' is found to have inhibitory activity on DNA cleavage activity that depends on MUS81 and RAD52 in tissue culture cells. Analysis of the effects of the compound on cell viability indicates that the compound has modest toxicity in both the presence and absence of BRCA2 and MUS81. The compound also modestly enhances the toxicity of HU.eLife.Overall the experiments are appropriate and of good quality . However, there are two particularly disappointing aspects of the results presented, as described below. In addition, I do not feel that the impact of this work justifies publication in Problems with experiments or interpretations:First, although the molecular modeling studies seem promising, they are of little impact without experimental verification using mutant proteins altered at residues modeled to be critical for binding. Such mutants should be generated and tested. If an attempt to do this has been unsuccessful, that obviously raises serious concern regarding the validity of the computational work.Secondly, although the COMET assays do support the notion that compound '1' has inhibitory activity on RAD52 in cells, the cell viability assays do not connect the toxicity of the drug with replication stress or homologous recombination. Combined effects are essentially additive for BRCA2 depletion and for HU treatment. This is the expected result if the mechanism of toxicity of '1' and '6' is completely unrelated to any impact on replication stress or HR.One obvious missing control is a RAD52 knock-down in combination with '1" treatment. If the effect of '1' on viability is mediated by inhibition of RAD52, the combination should be no more toxic than the effect of siRNA RAD52 or "1" treatment alone (assuming siRNA treatment is effective). This control is provided in the COMET assay, but not the cell viability assays. Given that the authors discuss the importance of this control for the COMET assay data, it is not clear why it was omitted from the viability assays.eLife is the right place for it.Setting aside the shortcomings described above, the work does not provide significant biological or biochemical insight with respect to the mechanisms of replication stress tolerance or homologous recombination. Furthermore, if PARP inhibitors set a standard for what would constitute an influential approach to targeting BRCA2-defective tumor cells, the new compounds to not meet and certainly do not exceed that standard. Thus, while the work is nicely done and will certainly be of interest to some members of the replication and homologous recombination communities, I do not think Reviewer #3:Here the investigators have identified small molecule inhibitors of RAD52 with the goal of utilizing these in therapeutic treatment of cancers with defective homologous recombination genes. Using a FRET based assay for RAD52 DNA binding the investigators screened a small library of compounds and obtained several candidates. Two were characterized for effects on RAD52 promoted annealing of complementary strands, interaction with RPA, self-interaction, computational docking to the putative DNA binding groove in the RAD52 undecamer structure, and several cell based assays. The work is well-executed, the quality of the data is high, and the work is well written. The findings will be of interest to investigators in the field and there is potential for translational application.I have a few suggestions for the investigators to consider.1) Richard Pomerantz and colleagues recently reported identification of small molecule inhibitors of RAD52 using a different assay. That work should be cited..2) It would be confirmative to show the IC50s of the compounds on DNA binding by gel shift assay.3) 4) 5) 6) [Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]eLife. Your revised article has been favorably evaluated by Charles Sawyers (Senior editor), a Reviewing editor, and three reviewers.Thank you for resubmitting your work entitled "Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for survival of BRCA2 deficient cells" for further consideration at eLife, and while I concur with the other reviewers that your manuscript already has substantial amount of novel data and analysis that justify its publication in eLife, I request that you add a short paragraph to discuss limitations of your present study and what can be done in the future to further validate the approach.The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance. Reviewer 2 has concerns about the paper's suitability for Reviewer #1:eLife.This revised version had addressed my main concerns. While I agree with reviewer 2 that mutating multiple RAD52 residues would be a good way to validate the drug-interacting surface, this may also destabilize the protein or its multimers. I can see why the authors want to avoid going down that rabbit hole. Using the computational pipeline to discover another drug indicates that it has some predictive potential. This makes it a valuable tool in its own right. I am also satisfied by the additional cell biology results and a more thorough comparison with inhibitors that have been published by other groups. In sum, I think this paper is ready for publication in Reviewer #2:eLife.The revised manuscript addressed one of my concerns . However, two major issues prevent me from changing my mind with respect to the suitability of this paper for First, there is still no verification of the binding site by mutational analysis. The authors claim this is not possible because of the nature of the binding site. I agree that single point mutants may not be sufficient to detect an effect on binding, but this is not an impediment as it is just as easy to make multiple mutations, that lack several of the hypothesized contacts, as it is to make point mutants. A handful of double or triple mutants could have been designed (with the help of the in silico tools) and tested for effects on binding affinity.Second, as mentioned previously, the in vivo work reveals only modest additive effects and not the synergy required to make '1' a particularly attractive candidate to develop for treatment of BRCA2 patients.The new material added on in silico screening is interesting and has potential to be very important. However, again, there is no mutant validation. Furthermore, the compound that came out of the screen binds to RPA. Hence, it seems possible that NP-00425 is a relatively promiscuous binder and/or that the binding to RPA will result in problematic off pathway activity in vivo. There was no in vivo work done on the new compound to address this possibility. Furthermore, the binding to RPA raises the possibility that the compound binds a surface of RAD52 other than that predicted by the constraints of the in silico approach.eLife on the basis of this new method.I appreciate that the in silico method described COULD have high impact, but one will need to see if the success reported here is validated by demonstration that the hits are actually binding as expected. Even with mutational or other validation, one would want evidence that the approach is generally applicable to justify publication of the paper in eLife's standards as I understand them.I still feel it is unlikely that this paper is going to be "highly influential" relative to related studies that have been published in recent years. It is a very nice paper, but not up to Reviewer #3:This is a revised version of a manuscript that reported identification of inhibitors of Rad52 from a HTS. The issues that I had raised have been adequately addressed and from my perspective the manuscript has been tightened up and much improved. In addition, this revised version has an added new section on validation using in silico screening that led to discovery of novel lead compounds with different chemical spaces. While this approach is well beyond my area of expertise, it nonetheless seems clear that this is an authoritative analysis that adds a new level of innovation to the manuscript. I enjoyed reading the manuscript and find it to be a contribution that will be appreciated by investigators in the field. [Editors’ note: the author responses to the first round of peer review follow.]As you can see below, the reviewers appreciate the work and its potential importance but raised three important issues that preclude further consideration of the manuscript for eLife, at least in the present form. I can see that the issues may be addressed but the required experiments seem much beyond what can be done in two months, which is a requirement for considering a revision in eLife. However, should you be able to address all of the major concerns, we would be willing to consider a revised manuscript with no guarantee of acceptance. Of course, given the numerous changes specified by our reviewers, you may wish to submit this work elsewhere.We carefully considered the critiques and, I believe, we can fully address the identified concerns. The reviewers raised a number of excellent points, addressing these points allowed us to significantly strengthen the manuscript. I hope you will find the changes we implemented and our responses compelling.Major concerns:1) The inhibition mechanism proposed is not convincing due to several issues in cell-based assays.We have resolved the issues raised by the reviewers; see point by point responses below. Briefly, the revised manuscript now includes:1) Better quality Western blot images, which unambiguously show that depletion of one of the investigated proteins in no case affects the level of the others;2) Additional internal controls to exclude misinterpretation of data in the Comet and viability assays;3) Enhanced immunofluorescence images in the live/dead viability assays, to make it easier to evaluate the key phenotypes;4) Finally, Reviewer 1 inquired about the effect of the RAD52 inhibitor on the damage-induced and spontaneous RAD52 foci. In the point by point response below we show the results of the RAD52 immunofluorescence study that indicates that compound ‘1’ interferes with the recruitment of RAD52 at nuclear foci upon DNA damage. This result is provided for evaluation by reviewers but not included in the manuscript as it will dilute the main message of the work.2) Previous related studies reduce the impact of the current study is an important point concerning the significance of the work.eLife. While these papers do report the RAD52 inhibitors, they offer a limited insight into the mechanism of the identified small molecules. The third manuscript by Chandramouly et al.  identified a class of inhibitors that acts differently from the molecules we identified, as it interferes with the RAD52 oligomerization and the supramolecular assembly. We have included an extensive discussion on the complementarity of the two studies.We should note that the papers by Huang et al.  and Sullivan et al.  have been published while our manuscript was under consideration in The following statement has been added to the Discussion:Recently, Chandramouly and colleagues  identified a small-molecule RAD52 inhibitor, 6-hydroxy-DL-dopa, that acts differently from the molecules reported here. This inhibitor interferes with the RAD52 oligomerization and the supramolecular assembly by an unresolved mechanism. It may act by binding at the RAD52 monomer-monomer interface, or at a different site on the protein and acts allosterically. The existence of the distinct classes of RAD52 inhibitors, exemplified by ‘1’ and 6-hydroxy-DL-dopa, suggests that disrupting the RAD52-ssDNA interaction or the integrity of the RAD52 oligomeric ring bears negative consequences for the RAD52 cellular functions. Considering that the efficient homology search and the DNA strand annealing requires the two complementary DNA strands (or the complementary ssDNA-RPA complexes) to be wrapped around the two different RAD52 oligomeric rings , this is not surprising, and offers an exciting opportunity for development of more specific and potent agents for targeting recombination-deficient tumors.While this manuscript was under review, two studies reporting small-molecule inhibitors of RAD52 were published. In the first study, Huang et al.  carried out an HTS campaign to identify 17 compounds that inhibit RAD52-mediated annealing in vitro with IC50 values ranging between 1.7 and 17 µM, which physically bind RAD52, and selectively, albeit at high concentrations, inhibit the single-strand annealing pathway of DSB repair over homologous recombination. In another study, Sullivan et al.  reported an in silico docking screen of a library of drug-like compounds. Among 36 predicted small-molecules, 9 compounds inhibited RAD52-ssDNA interaction in vitro, and 1 in cells. As with all previous publications, the authors screened different libraries, resulting in compounds that represent a different chemical space from those that emerged from our campaign. In addition to the high lipophilicity and relatively poor ligand efficiency of inhibitors discovered in previous campaigns, our work is deeply linked with solid structure-activity relationships based on the RAD52-ligand complexes (i.e. the current work moves from phenomenology to a definitive workflow to exploit the ssDNA binding pocket). Additionally, the use of large scale natural product screening using an in silico approach is highly innovative, and has yielded a successfully hit, which provides an enormous opportunity utilize this pharmacophore in future campaigns.3) The molecular modeling studies may be the main novelty compared to previous studies on Rad52 inhibitors by other researchers but do not add much to the manuscript in the absence of experimental validation.We very much appreciate that the reviewers see the novelty and the potential insight offered by our computational studies. We agree that the experimental validation is needed to confirm the compound placement. However, the reason we did not carry out the analysis of a point mutant that would interfere with the compound binding is due to the nature of the compound-RAD52 complexes. The majority of the contacts are achieved through a shallow continuum of van der Waals interaction, and many are mediated by water molecules . Indeed, the very nature of the ssDNA binding groove presents unique challenges to direct attempts to parse the binding free energy contributions from the amino acid moieties. Our binding energy calculations suggest that none of the possible single mutants will have a significant effect on the compound binding, at least without destabilizing the protein. We have, however, another and more powerful validation of our compound placement: We applied our compound placement protocol  to virtually screen a larger library of natural products. The campaign yielded 9 promising compounds, all very distinct in structure from our original hits. Extraordinarily, the natural product compound NP-00425 that emerged from the in silico screen of ~ 5K compounds and was selected for in vitro testing, yielded a promising hit that binds to RAD52 and competes with ssDNA binding, as confirmed by both our biophysical assays as well as NMR WaterLOGSY experiments. The strength of our method is that the docking, scoring and selection protocol was designed to enrich the true positive hits  and decrease the false negatives and false positives. Importantly, this manuscript fully describes an integrated and highly complementary HTS/in silico screening approach that worked wonderfully in producing structure based lead compounds. In short, the initial HTS screen provided the critical de novo structure activity relationship, while the in silico method clearly elucidated the key metrics that describe complexation in the ssDNA binding groove. These studies will provide a strong foundation for the discovery of novel antineoplastic therapeutics. We believe that the success of this virtual screening campaign, not only validates the original compound placement, but goes a step further, and demonstrates the enormous success of such an approach in producing bone fide hits in this exceedingly difficult target class.The description of these studies was added as the following new sections:Results:In Silico Screening and Discovery of NP-00425: translating structure-activity relationships from HTS into novel inhibitorsNatural product NP-00425 physically interacts with RAD52 and RPA proteins, inhibits the RAD52 binding to ssDNA and the ssDNA-RPA complex, but does not affect RAD52-dsDNA interaction or the ssDNA binding by RPA.The relevant sections in the Introduction and Discussion were also updated.Reviewer #1:Hengel et al. identified novel small molecule inhibitors of RAD52, a protein required for BRCA1, BRCA2, and PALB2-deficient cell survival. This work trails at least three other studies that have used similar HTS assays to identify RAD52 inhibitors . This manuscript needs to address how the compounds identified in this screen compare to the compounds that were previously identified as RAD52-ssDNA binding inhibitors. Is this largely a confirmation of what has been seen previously, or does the current work uncover new molecular scaffolds?eLife. In the revised version we added a discussion of the compounds identified in both studies in terms of both scaffolds and potency (see comments above).As mentioned above, the studies by Huang et al. and Sullivan et al. were published while this manuscript was under consideration in Specifically, the high lipophilicity and relatively poor ligand efficiency of inhibitors discovered in previous campaigns suggests that novel scaffolds will be essential to take the RAD52 drug discovery to the next level. Our work is deeply linked with solid structure-activity relationships based on the RAD52-ligand complexes (i.e. the current work moves from phenomenology to a definitive workflow to exploit the ssDNA binding pocket). Additionally, the use of large scale natural product screening using an in silico approach rooted in the structure-activity relationships obtained in the analysis of the initial HTS hits is highly innovative, and has yielded a successfully hit with a completely novel scaffold. This methodology provides an enormous opportunity of utilizing this pharmacophore in future campaigns.In addition, this paper largely concludes that compounds '1' and '6' disrupt RAD52-ssDNA binding. However, an alternative hypothesis may be that these compounds affect the RAD52 superstructure, as suggested in Chandreamouly et al. This hypothesis can be easily tested here. Finally, the cell biology work requires multiple controls prior to publication in eLife.While all experimental evidence in our study points towards the inhibition of the RAD52-ssDNA interaction, we have considered the possibility that '1' and '6' (or any of our HTS hits) may also interfere with the oligomeric or supramolecular structure of RAD52. The dynamic light scattering analysis , howeverTo the original text “Dynamic light scattering experiments conducted in the presence of equimolar concentrations of each compound and RAD52 showed that the presence of these compounds neither breaks up the oligomeric ring of RAD52 nor causes protein aggregation .” We addMajor Comments:1) Addition of RPA in  in vivo effects of '6' See also . PerhapsThe positive WaterLOGSY peaks  indicate2) Compound '6' was dropped from The Discussion should also mention the RPA binding to '6'. Perhaps this is a reason for its high IC50 in cells.We agree with the reviewer’s conclusion and suggestion. The following statement in the Results section “Higher concentration of ‘6’ required to inhibit MUS81/EME1/RAD52-mediated DSBs  is likel3) It should be noted that decrease in the amount of BRCA2 in MUS81-depleted cells was only perceived because the amount of total protein in the siMUS81-depleted cells was lower than in the other lanes. Indeed, if normalized against LMNB1, the amount of BRCA2 was similar in the non-BRCA2 RNAi experimental points. To improve quality of the data, however, we repeated the viability assay and presented a better-quality Western blot  to show efficiency and specificity of the RNAi reagents used in the assay. This new Western blot clearly shows that MUS81 knock-down does not affect BRCA2 levels. Please, note that as the inhibitor does not affect the level of the investigated DNA repair proteins, for sake of clarity, we do not show in the revised Western blot image samples treated with RNAi and the RAD52 inhibitor.In the revised manuscript, we also included the analysis of cell death in double MUS81+BRCA2 knock-down cells. Interestingly, concomitant depletion of BRCA2 and MUS81 strongly increases cell death even in the absence of treatment. The lethality in the untreated cells co-depleted of MUS81 and BRCA2 is elevated compared to BRCA2-depleted cells with depleted or inhibited RAD52. This is not unexpected. Although we have previously shown that MUS81 and RAD52 can cooperate in supporting the replication fork recovery in the checkpoint-inhibited cells , MUS81 has multiple RAD52-independent functions, such as resolution of intermediates in M-phase. Loss of such MUS81 mitotic function may impact the viability of BRCA2-deficient cells. These new data may imply that BRCA2-deficent cells have to engage the MUS81-RAD52 route to deal with the demised replication forks, and, on the other hand, point to MUS81 as another potential target for small-molecule inhibitors to selectively kill BRCA2-deficent tumor cells.We now performed statistical analysis comparing each pair of experimental points, in the presence and absence of the inhibitor.Live/dead assay is analyzed blindly after acquisition of the images at a low magnification in order to collect a large population of the cells. So, it was not feasible to include high magnification images. However, we increased the contrast and brightness of all images to make easier identification of dead cells, which are red-stained, in the live population, which is green-stained. We also provide a more extended explanation in the methods that the percentage of cell death reported in the graph derives from calculation of the number of dead cells in the population but it is also normalized against the number of fields required to score the number of cells required in the assay. This normalization is performed in order to include in the number of dead cells also those that are floating in the well and are inevitably lost during the staining protocol. So, a very low number of green, live, cells, may end up in elevated percentage of cell death even in the presence of few red, dead, cells.4) As already stated for the revised 5) The authors could further test the method of action of their drugs by attempting to observe RAD52 foci in both stressed and unstressed cells. Presumably the drugs should reduce DNA damage-induced RAD52 foci.As for the reviewer’s suggestion, we analyzed the formation of RAD52 foci after DNA damage in the presence and in the absence of ‘1’. The figure summarizing the results is included below. We did not, however, include these data in the manuscript because this analysis goes beyond the scope of the current paper. The analysis of the RAD52 foci formation along with the effect of RAD52 inhibition on the HR, SSA, and other DNA repair pathways will comprise another detailed study, which will follow up this current manuscript and will require extensive work to verify the mechanism of the inhibitor action in the cells . These analyses will deviate too much from the scope and main message of the current work.As shown in hTert-immortalized primary human fibroblasts were transfected where indicated with RAD52 siRNAs, and 48h post-transfection treated with etoposide for 3h. After treatment cells were subjected to indirect immunofluorescence using anti-RAD52 primary antibody. Immunofluorescence was performed by in situ extraction to retain only chromatin-bound proteins. The number of RAD52-foci-positive nuclei was recorded, and the intensity of fluorescence evaluated using imageJ. Fluorescence intensity data are presented as the mean of the total number of positive nuclei expressed as arbitrary units after background normalization. Note that the inhibitor also qualitatively affects the RAD52 immunostaining with little persistence of well-defined foci in the nucleus.Reviewer #2:Hengel et al. present the results of a drug screen for compounds that inhibit the ability of RAD51 to bind ssDNA. A number of hit compounds are found and of these two characterized biochemically and in cell based assays. Evidence for direct binding of the two compounds was obtained by a method that I am unfamiliar with (WaterLOGSY) and computational analysis is used to identify potential binding sites. One of the lead compounds '1' is found to have inhibitory activity on DNA cleavage activity that depends on MUS81 and RAD52 in tissue culture cells. Analysis of the effects of the compound on cell viability indicates that the compound has modest toxicity in both the presence and absence of BRCA2 and MUS81. The compound also modestly enhances the toxicity of HU.Overall the experiments are appropriate and of good quality . However, there are two particularly disappointing aspects of the results presented, as described below. In addition, I do not feel that the impact of this work justifies publication in eLife.Problems with experiments or interpretations:First, although the molecular modeling studies seem promising, they are of little impact without experimental verification using mutant proteins altered at residues modeled to be critical for binding. Such mutants should be generated and tested. If an attempt to do this has been unsuccessful, that obviously raises serious concern regarding the validity of the computational work.We agree that the experimental validation is needed to confirm the compound placement. However, the reason we did not carry out the analysis of the point mutants that would interfere with the compound binding is due to the nature of the compound-RAD52 complexes. The majority of the contacts are achieved through a shallow continuum of van der Waals interaction, and many are mediated by water molecules . Indeed, the very nature of the ssDNA binding groove presents unique challenges to direct attempts to parse the binding free energy contributions from the amino acid moieties. Our binding energy calculations suggest that none of the possible single mutants will have a significant effect on the compound binding, at least without destabilizing the protein. We have, however, another and more powerful validation of our compound placement:This revised manuscript describes the implementation of a highly robust method for designing a hybrid in silico/in vitro screening, which successfully employed a library of natural products compounds. This campaign resulted in the discovery of macrocycle inhibitor of RAD52 (NP-00425). Extraordinarily, the natural product compound that emerged from the in silico screen (of ~ 5K compounds), and was selected for in vitro testing, yielded a promising hit that binds to RAD52 and competes with ssDNA binding, as confirmed by both our biophysical assays as well as NMR WaterLOGSY experiments. The strength of our method is that the docking, scoring and selection protocol was designed to enrich the true positive hits (the docked R-compounds) and decrease the false negatives and false positives. Importantly, this manuscript fully describes an integrated and highly complementary HTS/in silico screening approach that worked wonderfully in producing structure based lead compounds. In short, the initial HTS screen provided the critical de novo structure activity relationship, while the in silico method clearly elucidated the key metrics that describe complexation in the ssDNA binding groove. These studies will provide a strong foundation for the discovery of novel antineoplastic therapeutics. Additionally, we feel that the discovery of NP-00425 and its ability to compete with ssDNA in a ssDNA binding groove of RAD52 will generate significant interest in a broad range of disciplines, and strongly enhances the impact of the current study.Secondly, although the COMET assays do support the notion that compound '1' has inhibitory activity on RAD52 in cells, the cell viability assays do not connect the toxicity of the drug with replication stress or homologous recombination. Combined effects are essentially additive for BRCA2 depletion and for HU treatment. This is the expected result if the mechanism of toxicity of '1' and '6' is completely unrelated to any impact on replication stress or HR.The effect of RAD52 inhibition is additive with either HU-induced replication stress or with BRCA2 depletion, and these additive effects are then additive themselves. This phenomenon may be somehow related to the response to replication stress. One can consider the DSBs suppression and toxicity of the RAD52 inhibitor as two connected events. Indeed, as proposed by a seminal Genes&Dev paper from the Venkitaraman’s group, loss of BRCA2 results in the fork collapse because of accumulation of pathological replication intermediates. Recently, these results were corroborated by many groups, linking BRCA2-RAD51-FANCs to fork stabilization. The pathological replication intermediates left unprotected in the BRCA2 absence may be targeted by MUS81 in collaboration with RAD52, as may happen to those accumulating in BRCA2 wild-type cells experiencing replication stress induced by checkpoint inhibition or prolonged replication arrest . This control is provided in the COMET assay, but not the cell viability assays. Given that the authors discuss the importance of this control for the COMET assay data, it is not clear why it was omitted from the viability assays.We agree. The revised Setting aside the shortcomings described above, the work does not provide significant biological or biochemical insight with respect to the mechanisms of replication stress tolerance or homologous recombination. Furthermore, if PARP inhibitors set a standard for what would constitute an influential approach to targeting BRCA2-defective tumor cells, the new compounds to not meet and certainly do not exceed that standard. Thus, while the work is nicely done and will certainly be of interest to some members of the replication and homologous recombination communities, I do not think eLife is the right place for it.We believe that the addition of the new data, and in particular the discovery of a new RAD52 inhibitor based on the computational workflow, offers an important insight into the mechanisms of RAD52 inhibition. In order to validate the strength of our hypothesis about the structural nature of the ligand-RAD52 complex, we developed a validated in silico screening campaign, based on our HTS results. We describe the discovery of NP-004255, a macrocyclic compound, which we show by NMR WaterLOGSY and biophysical assays to be a completely novel and effective inhibitor of the RAD52-ssDNA interaction. The implication of these findings for the discovery of novel therapeutics that specifically inhibit the activity of RAD52 is discussed in the revised manuscript.Significance of our structural understanding of the RAD52 inhibitors is twofold. First, it has an important implication for the discovery and design of the novel therapeutics targeting the protein-DNA interactions; second, it confirms that the RAD52-ssDNA interaction plays a role in allowing the BRCA-deficient cells to survive. The latter was not obvious, as a coherent picture of what are the roles of human (vertebrate) RAD52 in genome maintenance is lacking. The inhibitors we identified, and in particular ‘1’, which appears selective for the ssDNA binding groove of the RAD52 ring, will be a valuable tool for dissecting the RAD52-dependent pathways.We agree that PARP inhibitors set a standard for therapeutics that can be used to treat the BRCA-deficient tumors since olaparib has been approved for treatment of some recurrent platinum-sensitive ovarian cancers. There exists, however, a need for the broader range of options for the precision treatment of the HR deficient tumors, and RAD52 is currently one of the most coveted targets. While we are not currently offering a solution ready for trials, we provide an avenue and the tools for development of the new drug-like compounds meeting the standards of efficacy like that of the PARP inhibitors, and for dissecting the RAD52-dependent cellular pathways, which may lead to the identification of the new anticancer targets.In addition to the use of these compounds to dissect RAD52-dependent cellular pathways, there may be significant utility in therapeutics developed from these studies. This is because, the cocktail therapies are the most effective ways of mitigating the evolution of drug resistance, especially in the antineoplastic therapies, and the RAD52-dependent pathways that allow BRCA-deficient cells to survive are likely overlapping with the pathway targeted by the PARP inhibitors.Reviewer #3:Here the investigators have identified small molecule inhibitors of RAD52 with the goal of utilizing these in therapeutic treatment of cancers with defective homologous recombination genes. Using a FRET based assay for RAD52 DNA binding the investigators screened a small library of compounds and obtained several candidates. Two were characterized for effects on RAD52 promoted annealing of complementary strands, interaction with RPA, self-interaction, computational docking to the putative DNA binding groove in the RAD52 undecamer structure, and several cell based assays. The work is well-executed, the quality of the data is high, and the work is well written. The findings will be of interest to investigators in the field and there is potential for translational application.I have a few suggestions for the investigators to consider.1) Richard Pomerantz and colleagues recently reported identification of small molecule inhibitors of RAD52 using a different assay. That work should be cited..We have included discussion of the differences between our compounds and the small molecule identified by Chandramouly and colleagues – please see our response to the editor above along with the discussion of the compounds identified in two studies that were published while this manuscript was under review. We believe that the results of our studies are complementary and provide broader opportunities for the successful drug discovery.2) It would be confirmative to show the IC50s of the compounds on DNA binding by gel shift assay.Our previous studies  have unambiguously established the equivalency of the FRET-based assay and the EMSA, with an advantage of FRET-based assay being an equilibrium binding assay. Due to the nature of the dynamic RAD52-ssDNA complex, the gel shift in the EMSA assays can be achieved only upon crosslinking the protein to DNA. We believe, therefore, that the cross-linking will undermine the validity of the competition experiment. Moreover, the presence of the secondary DNA binding site on the top of the RAD52 ring may further convolute the data interpretation in EMSA experiments.3) This is a really good point. Under our experimental conditions and on the reaction time scale, spontaneous annealing is not observed ; there is no change in the spontaneous annealing rate in the presence of inhibitors, so the inhibitor only signal has not been subtracted. The same applies to the annealing of the RPA-ssDNA complexes.We have added the spontaneous annealing data to the 4) This is a valid control, which we have previously performed. In the  manuscript we demonstrated that RAD51 depletion does not affect formation of the MUS81-dependent DSBs. We therefore consider of little relevance to analyze the effect of RAD52 inhibition in RAD51-depleted cells.5) In the revised Materials and methods section we have included a more detailed description of the assay. Briefly, the assay evaluates the number of dead cells, red, in the population of live cells, visualized as green. That is the reason why results are presented as percentage of cell death.6) We apologize for the oversight. We now amended the legend title and included the analysis of cell death in cells depleted of RAD52, alone and combined with the inhibitor. Evaluation of cell death in siBRCA2 cells was present in the original figure.[Editors’ note: the author responses to the re-review follow.]The manuscript has been improved but there are some remaining issues that need to be addressed before acceptance. Reviewer 2 has concerns about the paper's suitability for eLife, and while I concur with the other reviewers that your manuscript already has substantial amount of novel data and analysis that justify its publication in eLife, I request that you add a short paragraph to discuss limitations of your present study and what can be done in the future to further validate the approach.As you requested, the following paragraph has been added to the Discussion section of the revised manuscript:“It has to be noted, however, that although our work provides a solid understanding of how shape complementarity and utilization of interstitial water networks drives complexation, there are limitations to these approaches. Specifically, the current experimental and computational studies do not address the underlying thermodynamics and kinetics that control ligand competition in the ssDNA binding groove. A fascinating aspect of the competition between small molecules and macromolecules for an extended and partially solvated binding pocket, as in RAD52, is the extent to which small molecules may exploit the potential enthalpy-entropy compensation of a large and floppy native DNA ligand. Additionally, residence times of lead compounds may be a critical factor in the successful design of therapeutic lead compounds. These factors will be addressed in the future studies that will be focused on the thermodynamics and kinetics of small molecule binding to the RAD52 protein, and in the studies that will confirm the ligand placement through high resolution structures.”


We examined the real-world effectiveness of ART as an HIV prevention tool among HIV serodiscordant couples in a programmatic setting in a low-income country.We enrolled individuals from HIV serodiscordant couples aged ≥18 years of age in Jinja, Uganda from June 2009 – June 2011. In one group of couples the HIV positive partner was receiving ART as they met clinical eligibility criteria (a CD4 cell count ≤250 cells/ μL or WHO Stage III/IV disease). In the second group the infected partner was not yet ART-eligible. We measured HIV incidence by testing the uninfected partner every three months. We conducted genetic linkage studies to determine the source of new infections in seroconverting participants.A total of 586 couples were enrolled of which 249 (42%) of the HIV positive participants were receiving ART at enrollment, and an additional 99 (17%) initiated ART during the study. The median duration of follow-up was 1.5 years. We found 9 new infections among partners of participants who had been receiving ART for at least three months and 8 new infections in partners of participants who had not received ART or received it for less than three months, for incidence rates of 2.09 per 100 person-years (PYRs) and 2.30 per 100 PYRs, respectively. The incidence rate ratio for ART-use was 0.91 . The hazard ratio for HIV seroconversion associated with ART-use by the positive partner was 1.07 (95% CI 0.41-2.80). A total of 5/7 (71%) of the transmissions on ART and 6/7 (86%) of those not on ART were genetically linked.Overall HIV incidence was low in comparison to previous studies of serodiscordant couples. However, ART-use was not associated with a reduced risk of HIV transmission in this study. Antiretroviral therapy (ART) has been shown to dramatically reduce plasma viral load (VL) in HIV-infected individuals. Studies conducted prior to the widespread introduction of ART in Uganda demonstrated that the HIV VL of the infected partner is the primary determinant of HIV transmission among serodiscordant couples  This andHowever, the context of highly-resourced randomized trials and ecologic studies from industrialized countries is very different from that of ART programs in low-income countries, where the majority of HIV-infected individuals accessing treatment reside. In such programs support for adherence to therapy, transportation, nutrition and other ancillary services are often lacking. As well, human resources , 8 and cWe conducted an observational study to examine the effectiveness of ART as prevention in a large HIV care and treatment program in rural Uganda in order to determine the real-world effectiveness of HIV treatment as prevention among serodiscordant couples.We conducted a prospective cohort study of co-habiting HIV serodiscordant couples of individuals aged ≥18 years of age known as the Highly Active Antiretroviral therapy as Prevention (HAARP) study. All HIV-infected participants were clients of The AIDS Support Organization (TASO), in Jinja, Uganda. Participants were eligible if they reported at least two episodes of sexual intercourse in the previous three months. Prior to enrollment, the HIV negative partner must have undergone an HIV test and received the results. Both the positive and negative participants were required to have already disclosed their HIV serostatus to their partner. The TASO clinic in Jinja provides care to over 8000 HIV infected individuals with over 5000 receiving ART. The ART program began in 2004 and is financed through the US Presidents Emergency Fund for AIDS Relief.Study participants provided informed written consent to their participation. Prior to study enrollment a research assistant read aloud the consent form in the preferred, of one of three local languages and responded to all questions raised by potential recruits. If the individual agreed to study participation, they then signed the form . The study, including the consent forms and data collection tools, received scientific and ethics approval from the Institutional Review Boards of the Uganda Virus Research Institute and the Uganda National Council for Science and Technology in Uganda and the University of British Columbia in Vancouver, Canada.In one group of couples the HIV positive partner was receiving ART because of meeting the clinical or laboratory eligibility requirements in effect at the time of the study; a CD4 cell count ≤250 cells/ μL or WHO Stage III or IV disease. In the HIV incidence in the two groups was measured by testing the uninfected partner every three months using two different point of care tests, applied in a serial algorithm . In the event of discordant results between the first and second tests, a third test  was used to make the final diagnosis. All new HIV diagnoses were also subsequently confirmed by a laboratory-based ELISA test. The study was powered to detect a 75% reduction in HIV incidence associated with ART-use assuming that HIV incidence in the couples with untreated HIV would be 4 per 100 person years with an α of 0.05 and a power of 0.8, using a two-sided test.For individuals who initiated ART near enrollment or during the study, observation time was considered to be not on ART until three months after the individual first started receiving ART. Once these individuals had received ART for three months, all subsequent observation time was considered to be while receiving ART. We collected sociodemographic and behavioural data through questionnaires administered by research assistants at enrollment and every six months thereafter. We also conducted clinical assessments at the same intervals and collected vaginal swabs for bacterial vaginosis and performed rapid diagnostic tests for syphilis , every six months. Treatment for other sexually transmitted infections was based on syndromic management in accordance with local guidelines.We collected and stored plasma and serum samples every six months for viral load testing ) and herpes simplex type 2 (HSV2) antibody testing  at the end of the study. In couples where HIV seroconversion occurred, we conducted VL testing on all samples from the HIV positive participant during the study and for non-seroconverting couples, we tested only the sample drawn at the last study visit. We conducted HSV2 serology on all study participants using the enrollment sample and again on those individuals who were HSV2 negative at enrollment on the last samples drawn. HIV sequencing was conducted on plasma samples drawn from the HIV positive participant and their partners who seroconverted where the VL measured was ≥300 copies/ mL using an in house method. For seroconverting couples where we could not obtain sufficient virus in plasma samples for genotyping, we drew additional whole blood samples to extract proviral DNA from intact CD4 cells.pol gene generated a 1341 base-pair fragment. We analyzed Protease (PR aa 1–99) and Reverse Transcriptase (RT aa 1–320) regions of the pol gene (fragment length 1341 base-pairs). This fragment was purified, sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit , and analysed on an ABI Prism 3130 Genetic Analyser . All resulting sequences were genotyped using SCUEAL [pol sequences with dates were downloaded from the Los Alamos National Laboratory Database . A sequence similarity search (ViroBlast) [Briefly, RT-PCR followed by nested PCR of the 5’ region of the g SCUEAL . In orderoBlast) was perfoWe conducted Kruskal Wallis and Chi-squared tests to compare couples where the HIV positive participant did not receive ART, with those where the positive participant was receiving ART from enrollment and couples where the positive participant began ART during the study. We also conducted bivariate analyses comparing couples where seroconversion occurred during follow-up with those where seroconversion did not occur. We calculated the incidence rate ratio for HIV seroconversion using exact Poisson regression and used univariate Cox proportional hazards modeling to examine the hazard associated with time-to-seroconversion in the HIV negative participants, with ART exposure of the HIV positive participant used as a time-updated variable. Given the small number of transmission events we observed, we were unable to develop multivariate models, rather we conducted several stratified Cox proportional hazard analyses to accouA total of 586 couples were enrolled between June 1, 2009 and June 20, 2011. In 249(42.5%) of these couples, the HIV-infected participant was receiving ART at enrollment and in 99 (17%) couples the HIV positive participant initiated ART during the study. In the remaining 238 (40.6%) couples the HIV-infected participant did not receive ART during the study. The median duration of ART-use for those on ART at enrollment was 2.3 years. Couples where the positive participant was receiving ART at enrollment were more likely to report always using condoms in the past three months in comparison to those who initiated during the study or were never on ART . They also reported longer relationships with their primary sexual partner than non-ART couples , but not longer than couples who started ART during the study (median 12 years). There were no differences between the three groups in terms of condom use at last sex, pregnancy intentions, alcohol use, number of non-spousal sexual partners or other behavioural characteristics.There were also differences in clinical characteristics between the three groups . The HIVpol gene sequence from both partner viruses in 14 of the 17 seroconverter couples. Of these 79% (11/14) were found to be genetically linked; 5/7(71%) in the ART arm and 6/ 7(86%) in the non-ART arm. Linked infections were always supported by posterior probabilities of 1. Mean pairwise genetic distance among couples was 0.02 nucleotide substitutions per site, and median 0.015.Follow-up of study participants continued until December 14, 2011 when the median duration of study participation was 1.5 years. A total of 42 couples did not return for the first follow-up visit, such that 544 couples were able to be assessed for an outcome . We founThe bivariate analysis of factors associated with seroconversion is shown in Univariate Cox proportional hazards modeling found that no association with time-updated ART use . Similarly none of our stratified Cox analyese demonstrated associations between ART use and seroconversion. The HR for ART-use when stratified by male circumcision status was 1.01 (95% CI 0.39–2.67); when stratified by HSV2 serostatus of the HIV positive participant it was 0.94 (95% CI 0.35–2.55); when stratified by the HSV2 serostatus of the HIV negative participant it was 0.90 (95% CI 0.31–2.60); when stratified by the gender of the HIV positive participant it was 1.02 (95% CI 0.39–2.68) and when stratified by the CD4 cell count nadir of the HIV positive ≤200 cells/μL, it was 0.581 (95% CI 0.10–3.27).In this study, ART use by the HIV positive partner was not associated with a reduced risk of HIV transmission among HIV serodiscordant couples. This result contrasts with many previous clinical studies of ART as prevention that have found effectiveness varying from 26%- 96% , 15–19, The HPTN 052 study found a 96% reduction in HIV transmission associated with the early use of ART among 1763 serodiscordant couples . HoweverThe relatively low rate of HIV incidence in the non-ART couples (2.30 per 100 PYRs) may be due to the high prevalence of condom-use in this cohort, but may more accurately reflect the background HIV transmission risk of serodiscordant couples where the positive partner is already engaged in HIV care and receiving prevention counselling. In the systematic review, above, the median incidence rate for this group was 7.5 per 100 PYRs. ReporteIt is possible that the higher proportion of uncircumcised men and HSV2 seroprevalence in the couples where ART was used may have diminished the effectiveness of ART in preventing transmission. However, these differences in distribution were quite small  and seem unlikely to completely counteract the expected efficacy of ART in preventing HIV transmission. Furthermore, we did not find any effect on ART-use by the HIV positive partner in analyses stratified on the basis of circumcision status, gender or HSV2 seroprevalence. Nevertheless, we cannot rule out interactions in the differences between these important co-factors of HIV transmission. However, it is also worth noting that some of the differences in variables known to be associated with HIV transmission, such as condom-use and relationship duration, would have biased our study towards having a reduced risk of HIV transmission among the ART couples, irrespective of the effect of ART. Such factors have been previously identified as biases in observational studies examining this issue.Our study has a number of limitations. Firstly, our study was underpowered to detect the moderately sized decreases in HIV transmission (75%) associated with ART-use because the incidence in the couples where the positive partner did not receive treatment was lower than we had anticipated. However, a post-hoc power calculation did find that we had sufficient statistical power to detect a 95% reduction in transmission associated with ART-use. Our study, however, cannot rule out an effect of ART which was less than this. Secondly, the VL measurements we obtained could have been taken up to six months prior to the date of seroconversion and therefore may have missed episodes of high viremia which may have caused transmission. The small number of transmission events also meant that we were unable to adjust our analyses to control for all potential confounders. Finally, as with all observational studies, confounding by other factors which we did not measure may also have contributed to the lack of effect we observed.In summary, we did not demonstrate a benefit of the use of ART in preventing HIV transmission among serodiscordant couples in Uganda. A possible explanation for this observation is that couples were already using condoms and had participated in some risk reduction counseling, thus limiting the possibility to show further benefits from ART. Our results highlight the need for conducting operational research in similar program settings to determine the real-world impact of interventions which have been proven in clinical trials.