RPA/replication protein A

HAMNO is a novel protein interaction inhibitor of replication protein A (RPA). The ATR/Chk1 pathway includes RPA. In both HNSCC cell lines, HAMNO inhibits colony formation at low micromolar concentrations. Colony formation is significantly inhibited by HAMNO and etoposide when compared to HAMNO alone. A dose-dependent increase in pan-nuclear -H2AX staining happens after UMSCC38 cells are exposed to HAMNO. Both the cancer cell line UMSCC11B and cancer cell line UMSCC38 exhibit strong -H2AX staining, especially following incubation with 20 M HAMNO. Following the addition of HAMNO, both UMSCC38 and OKF4 cells exhibit increased -H2AX staining, with S-phase showing the greatest increase in signal[1].

---

HAMNO is selective for DBD-F [1]
HAMNO (Fig. 1A) was first identified as a RPA DBD-F inhibitor in a high-throughput screen that determined the ability of a small molecule to dissociate a Rad9-GST fusion protein from a RPA-ssDNA complex, an interaction that requires DBD-F. Binding of HAMNO to DBD-F was further investigated through in silico methods (Fig. 1B). These studies utilized a crystal structure of DBD-F that was earlier optimized for binding to the in vitro DBD-F inhibitor, fumaropimaric acid (FPA). The site of highest predicted affinity was to a position immediately adjacent to R43 on DBD-F (Fig. 1B: right panel), where the compound would predictively act to hinder protein-protein interaction, as this residue is essential for DBD-F-protein binding.

To confirm an interaction of HAMNO with DBD-F in vitro, we took advantage of the ability of DBD-F to weakly bind a labeled oligonucleotide, and looked for changes in mobility and intensity of the complex in the presence of HAMNO using a EMSA (Fig. 1C). In the absence of DBD-F, HAMNO does not bind labeled oligonucleotide (Fig. 1C: left panel). In the presence of both DNA and DBD-F, the addition of HAMNO results in the formation of a band between the free ssDNA and protein-bound ssDNA bands. (Fig. 1C: right panel; denoted by an asterisk). As HAMNO is uncharged at neutral pH, it is unlikely that binding of the compound to the protein-DNA complex alone would result in this newly formed band detected on the gel. Rather, this stimulation of DBD-F binding to DNA by HAMNO suggests a direct interaction of the small molecule with DBD-F, resulting in a conformational shift that alters DNA binding.

We then wanted to confirm that HAMNO is selective for inhibiting only DBD-F, and not DBDs A-E which are important in RPA binding to ssDNA. This selectivity would ensure that HAMNO would affect protein recruitment more adversely than ssDNA binding. To determine this, we took advantage of a DNA unwinding activity by RPA that is dependent on the DBD-F, but does not require DBD-F for stable binding of the resultant ssDNA that is formed. To do this, we utilized EMSAs with full length RPA using ssDNA and dsDNA probes. HAMNO did not affect RPA binding to ssDNA at concentrations up to 200 μM, but prevented dsDNA unwinding and subsequent ssDNA binding at 100 μM (Fig. 1D). This inhibition of dsDNA unwinding by HAMNO is nearly equivalent to that by our previously identified in vitro inhibitor, FPA, whose subsequent dissociation constant for DBD-F was determined to be 9.0 μM. Together these data show a preference of HAMNO for selectively inhibiting DBD-F at micromolar levels, an ability that would predictively target the replication stress response in replication-stressed cancer cells over normal cells.

---

HAMNO induces γ-H2AX staining in a cell-cycle specific manner [1]
DBD-F binds ATRIP, Rad9, Rad17 and Nbs1 thereby recruiting and stabilizing the proteins involved in ATR activation. Inhibition of DBD-F- interactions with these proteins would likely short-circuit ATR signaling, leading to increased replication stress that can be monitored by pan-nuclear phosphorylation of H2AX in S-phase. This type of phosphorylation has been shown to be S-phase specific as previously demonstrated with Chk1 inhibitors and differs from the punctate foci that occur in response to double-strand breaks. We evaluated whether HAMNO induces replication stress as assessed through increases in pan-nuclear γ-H2AX staining (Fig. 2A). After UMSCC38 cells were exposed to HAMNO, increased pan-nuclear γ-H2AX staining occurred in a dose dependent manner (Fig. 2A,B). When H2AX phosphorylation was assessed via western blot, cancer derived UMSCC38 cells, as well as another cancer cell line, UMSCC11B, had prominent γ-H2AX staining, particularly after incubation with 20 μM HAMNO. In contrast, the telomerase-immortalized keratinocyte cell line, OKF4, did not show enhanced γ-H2AX staining at this concentration, suggesting that HAMNO is more effective in inducing H2AX phosphorylation in cancer cell lines that are potentiated for oncogene-induced stress. To further validate that pan-nuclear γ-H2AX represents replication stress and the differences in replication stress between HNSCC cells and OKF4 cells is S-phase specific we employed flow cytometry. To measure cell cycle specific γ-H2AX staining we assayed cells in the absence of HAMNO as a negative control which determined the threshold between γ-H2AX positive and γ-H2AX negative cells for each cell cycle phase (Fig. 2D). Both UMSCC38 and OKF4 cells presented increased γ-H2AX staining after addition of HAMNO, with the greatest increase in signal occurring in S-phase (Fig. 2D). In further comparison of the two cell lines, the ratio of γ-H2AX positive to γ-H2AX negative cells in S-phase was six- to eight-fold greater in UMSCC38 cells than OKF4 cells at 20 μM and 50 μM, respectively (Fig. 2E). These data suggest that HAMNO selectively increased γ-H2AX staining in S-phase, indicative of increased replicative stress. These data also indicate that cells predicted to have high levels of oncogene-induced stress, such as mutant p53 squamous cell carcinoma cells, are selectively potentiated for increased replication stress by HAMNO.

---

HAMNO affects RPA and ATR phosphorylation [1]
Inhibition of DBD-F by HAMNO is expected to inhibit RPA32 phosphorylation directly by inhibiting DBD-F interactions with replication stress response proteins involved in activating ATR. We tested this hypothesis under conditions that enhance RPA32 Ser33 phosphorylation (Fig. 3A). Ser33 of RPA32, an ATR substrate, is highly phosphorylated after two hours of treatment with 20 μM of etoposide, which was reduced with the addition of 2 μM HAMNO, and was nearly absent at higher concentrations, demonstrating an in vivo effect of HAMNO as an inhibitor of RPA32 phosphorylation by ATR.

The increase in γ-H2AX staining and decrease in RPA phosphorylation in HAMNO treated cells suggests a deregulation of ATR signaling. To further describe this loss of competent DNA damage response (DDR) signaling, we assessed the autophosphorylation of ATR at T1989, a marker for ATR activity (Fig. 3C). As expected, etoposide strongly induced ATR autophosphorylation, while treatment with HAMNO alone showed a slight increase in T1989 phosphorylation. Interestingly, the addition of etoposide and HAMNO together resulted in a decrease in ATR autophosphorylation as compared to etoposide alone, suggesting that ATR activity has been compromised. Taken together, these data show that HAMNO inhibits RPA32 phosphorylation and decreases ATR autophosphorylation, indicative of HAMNOs ability to affect ATR signaling.

---

HAMNO enhances etoposide toxicity [1]
The ability of HAMNO to inhibit ATR signaling suggested that the compound may be cytotoxic, and sensitizes cells to DNA damaging agents. HAMNO alone inhibited colony formation in both HNSCC cell lines in the low micromolar range (Fig. 4A). Next, we compared the colony formation of UMSCC38 cells in the presence of increasing concentrations of HAMNO with or without an initial 20 μM/2 h exposure to etoposide (Supplementary Fig. S1). When these two conditions are normalized to the 0 μM HAMNO treatment, HAMNO combined with etoposide significantly inhibited colony formation to a greater degree than HAMNO alone (Fig. 4B).

HAMNO slows the growth of UMSCC11B tumors in mice. After two hours of treatment with 20 M etoposide, Ser33 of RPA32, an ATR substrate, is heavily phosphorylated. This phosphorylation is reduced with the addition of 2 M HAMNO and is almost absent at higher concentrations, demonstrating an in vivo effect of HAMNO as an inhibitor of RPA32 phosphorylation by ATR[1].

---

To further examine the potential of HAMNO as an anticancer agent, we tested HAMNO in a mouse xenograft model. In mice, HAMNO slowed the progression of UMSCC11B tumors (Fig.4C). This inhibition was also seen in mice cotreated with sub-lethal etoposide as well as in similarly treated UMSCC38 cells (Fig. 4D,E) These results raise the exciting possibility that a DBD-F inhibitor alone can reduce tumors or can sensitize tumor cells to other chemotherapy agents [1].

Cell cycle assessment and γ-H2AX staining are monitored in UMSCC38 and OKF4 cells after 2 h incubation with HAMNO (2, 20, 50 μM) and fixed in 70% ethanol overnight. Cells are washed with PBS and incubated overnight in PBS containing 1% BSA, 10% goat serum and PS139-H2AX antibodies, washed and incubated in goat anti-mouse Alexa Fluor 647 antibody for 30 min at room temperature. Cells are incubated in 50 μg/mL propidium iodide and 100 μg/mL RNase A for 30 min, and 10,000 cells per sample are analyzed[1].

---

Cells and clonogenic assays [1]
The squamous cell carcinoma cell lines UMSCC38 and UMSCC11B were propagated in DMEM with 10% fetal bovine serum (FBS). The immortalized primary oral keratinocyte cell line, OKF4 was propagated in fortified KBM-2 media with 10% fetal bovine serum. For clonogenic assays, cells were trypsinized and diluted in media to 1000 cells/mL, then dispersed into 60 mm dishes (3 mL) overnight. After addition of HAMNO, cells were grown for 9 d, then fixed in PBS containing 6% glutaraldehyde for 30 min, and then dyed in 0.5% crystal violet for 30 min and rinsed. Colonies containing over 50 cells were counted. For studies requiring etoposide, HAMNO was added one h before addition of 2 μM etoposide. After 2 h of etoposide exposure, media was removed and rinsed with PBS, before adding back media containing HAMNO. Data were analyzed using an unpaired 2-tailed Student t test to determine statistical significance.

---

Flow cytometry [1]
Cell cycle assessment and γ-H2AX staining were monitored in UMSCC38 and OKF4 cells after 2 h incubation with HAMNO and fixed in 70% ethanol overnight. Cells were washed with PBS and incubated overnight in PBS containing 1% BSA, 10% goat serum and PS139-H2AX antibodies, washed and incubated in goat anti-mouse Alexa Fluor 647 antibody for 30 min at RT. Cells were incubated in 50 μg/mL propidium iodide and 100 μg/mL RNase A for 30 min, and 10,000 cells per sample were analyzed on a BD FACSarray using 532 and 635 nm excitations and collecting fluorescent emissions with filters at 585/42 nm and 661/16 nm (yellow and red parameters, respectively). BD FACSarray and WinList™ software were used for data collection and analysis, respectively.

In this study, naked thymic mice were used. The tumor cells (2 to 6105 cells in 100 mL of sterile PBS) are subcutaneously injected into 6-week-old female mice to implant UMSCC38 and UMSCC11B cells. Tumor volume [tumor volume=1/2(lengthwidth2)] and vernier calipers are used every day to measure the growth rates of tumors. Etoposide (10 mg/kg mouse) and HAMNO (2 mg/kg) are injected intraperitoneally every day for three days once the tumor has grown to a size of 50 mm3. The volume of the tumor is compared between all experimental groups, and tumor size is tracked daily. Every group includes a minimum of three mice[1].

[1]. RPA inhibition increases replication stress and suppresses tumor growth. Cancer Res. 2014 Sep 15;74(18):5165-72.

The ATR/Chk1 pathway is a critical surveillance network that maintains genomic integrity during DNA replication by stabilizing the replication forks during normal replication to avoid replication stress. One of the many differences between normal cells and cancer cells is the amount of replication stress that occurs during replication. Cancer cells with activated oncogenes generate increased levels of replication stress. This creates an increased dependency on the ATR/Chk1 pathway in cancer cells and opens up an opportunity to preferentially kill cancer cells by inhibiting this pathway. In support of this idea, we have identified a small molecule termed HAMNO ((1Z)-1-[(2-hydroxyanilino)methylidene]naphthalen-2-one), a novel protein interaction inhibitor of replication protein A (RPA), a protein involved in the ATR/Chk1 pathway. HAMNO selectively binds the N-terminal domain of RPA70, effectively inhibiting critical RPA protein interactions that rely on this domain. HAMNO inhibits both ATR autophosphorylation and phosphorylation of RPA32 Ser33 by ATR. By itself, HAMNO treatment creates DNA replication stress in cancer cells that are already experiencing replication stress, but not in normal cells, and it acts synergistically with etoposide to kill cancer cells in vitro and slow tumor growth in vivo. Thus, HAMNO illustrates how RPA inhibitors represent candidate therapeutics for cancer treatment, providing disease selectivity in cancer cells by targeting their differential response to replication stress. Cancer Res; 74(18); 5165-72. [1]

---

The ability of HAMNO to work effectively with etoposide attests to the effective strategy of inducing replication stress and reducing the replication stress response to increase cell death selectively in cancer cells that have constitutive DNA replication stress. This approach would be beneficial in the clinic, as the therapeutic efficacy would increase with the addition of a RPA inhibitor and reduce unwanted side effects. HAMNO also has the potential to be used as a stand-alone agent due to its ability to selectively increase cytotoxicity in cancer cells that already have oncogene-induced replicative stress.
This is the first in vivo study showing the potential of a RPA DBD-F inhibitor as a cancer chemotherapeutic agent. The ability of HAMNO to induce cytotoxicity alone and kill cells synergistically with etoposide suggests that HAMNO could be used alone or in combination with other chemotherapeutic agents. HAMNO is tolerated in mice at doses that affect tumor growth, attesting to the clinical potential of this compound. The structure of HAMNO allows for the addition and substitution of many possible moieties that could result in increased affinity to the DBD-F. We are currently testing several HAMNO derivatives for increased DBD-F inhibition.[1]

C17H13NO2

263.3

263.095

C, 77.55; H, 4.98; N, 5.32; O, 12.15

138736-73-9

138736-73-9

85895

Solid powder

3.514

2

3

2

20

344

0

OC1C=CC2C=CC=CC=2C=1/C=N/C1C=CC=CC=1O

NADCEWZYITXLKJ-KAMYIIQDSA-N

InChI=1S/C17H13NO2/c19-16-10-9-12-5-1-2-6-13(12)14(16)11-18-15-7-3-4-8-17(15)20/h1-11,18,20H/b14-11-

HAMNO; CID 6335338; NSC-111847; HAMNO; 138736-73-9; 894-93-9; 2(1H)-Naphthalenone, 1-[[(2-hydroxyphenyl)amino]methylene]-; NSC111,847; 137320-35-5; 1-(((2-Hydroxyphenyl)imino)methyl)-2-naphthol; 1-(((2-Hydroxyphenyl)amino)methylene)naphthalen-2(1H)-one; NSC111847; NSC111847; MLS000737724;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: 2.5 mg/mL (9.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

 (Please use freshly prepared in vivo formulations for optimal results.)