WO2013148114A1 - P300/cbp inhibitors and methods of use - Google Patents

Abstract

The subject invention pertains to a class of compounds that inhibit the function of p300/CBP, GCN5 and PCAF. P300/CBP inhibition was noted to result in inhibition of tumor cell growth and killing of tumor cells. P300/CBP inhibition is, thus, useful for treatment of various types of cancers, including acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immunoblastic lymphoma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, central nervous system (CNS) cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer and breast cancer. The compounds disclosed herein can be used alone or in combination with other cancer treatment regimens (e.g., radiation therapy and/or other chemotherapeutic agents that are administered to a subject having a tumor, cancer or neoplasia).

Description

DESCRIPTION

P300/CBP INHIBITORS AND METHODS OF USE CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Serial Nos. 61/617,728, filed March 30, 2012 and 61/702,896, filed September 19, 2012, the disclosures of which are hereby incorporated by reference in their entirety, including all figures, tables and amino acid or nucleic acid sequences.

BACKGROUND OF THE INVENTION

Breast cancer (BC) is a heterogeneous disease in terms of cancer biology and therapeutic options. Based on mRNA expression microarray profiling data, BC could be broadly classified into four main subtypes: normal-like, HER2 overexpressing, luminal A and B, and basal-like BC (1). Characteristic markers for each subtype have been described. Normal-like BC cells exhibit gene expression pattern resembling basal epithelial cells with low expression of genes characteristic of luminal epithelial cells. Erb-B-2/HER2 -positive BC cells display high levels of expression of HER2. The luminal tumors show heightened expression of luminal cytokeratins and genes specific to the luminal epithelial cells. The luminal BC is subdivided into A and B types with the A type being low-grade tumors and estrogen receptor (ER) positive. Luminal B tumors are generally of high-grade and ER positive. The basal-like BC is characterized by the lack of expression of ER, progesterone receptor (PgR), HER2 overexpression and upregulation of genes associated with basal myoepithelial cells.

Triple-negative BC (TNBC) is defined by the lack of expression of ER and PgR as well as the absence of HER2 gene amplification or overexpression with distinct molecular, histological and clinical characteristics. Among the four BC subtypes defined by gene expression profiling, TNBC shares many of the molecular features of basal-like BC (2). Clinically, TNBC exhibits an invasive ductal phenotype and high histological tumor grade. Patients with TNBC appear to have a much worse prognosis than other BC subtypes, accounting for a disproportionately large number of deaths due to BC. TNBC represents about a quarter of advanced BC at diagnosis, and African American women appear to be overrepresented in this population. With respect to therapy, patients with TNBC seem highly responsive to conventional chemotherapeutic regimens initially, but drug resistant tumors recur rapidly with the majority of deaths occurring in the first five years (2).

Although mechanisms underlying TNBC disease relapse and unfavorable prognosis remain to be established, it has been hypothesized that a subpopulation of TNBC cells display cancer-stem cell (CSC) properties such as ability of self-renewal, slow growth and resistance to chemo- and radiotherapies (3, 4). Recent studies have shown that basal-like BC and TNBC cells contain a relatively high percentage of CSCs with CD44^h7CD24^lo/" phenotypes (5, 6). These features in conjunction of ER, PgR and HER2 negativity provide a reasonable explanation for the lack of benefits from the currently available targeted therapies and conventional chemotherapies. Although EGFR is generally expressed in TNBC, agents targeting EGFR (e.g. cetuximab) alone did not seem to provide obvious benefits against TNBC (2). Therefore, new therapeutics and targeted therapies against TNBC need to be developed in order to improve survival of patients with TNBC.

p300 (also known as EP300 and KAT3B; see SEQ ID NO: 1) was originally identified as a binding protein of adenovirus El A protein (7). It is a large protein with multiple domains that bind to diverse proteins including many DNA-binding transcription factors (8). The cyclic AMP-responsive element-binding protein (CREB) binding protein (CBP, also known as KAT3A) is a cellular paralog of p300. p300 and CBP share extensive sequence identity and functional similarity. As such, they are often referred to as p300/CBP in the scientific literature. p300/CBP are lysine acetyltransferases that catalyze the attachment of an acetyl group to a lysine side chain of histones and other protein substrates. As large proteins, p300/CBP were proposed to activate transcription in part by bridging DNA-binding transcription factors to RNA polymerase machinery or by helping assemble the transcriptional pre-initiation complex (PIC). Importantly, p300/CBP-catalyzed acetylation of histones and other proteins is pivotal to gene activation (8). Heightened p300 expression and activities have been observed in advanced human cancers such as prostate (9, 10) and liver (11, 12) cancer and appear to be associated with poor prognosis of these cancer types. Compared to normal tissue counterparts, the expression levels of p300 are higher in human primary breast cancer specimens and in mouse mammary carcinomas induced by poiyomavirus middle-T oncogene (13). These observations suggest that p300 might be a potential therapeutic target for treating diverse types of human cancer (14).

Importantly, p300/CBP act as a critical coactivator of several oncogenic transcription factors such as STAT3, NF-κΒ and HIF-Ι . Genes regulated by these transcription factors are involved in cytokine or hypoxia-induced cancer cell survival and sustained proliferation. Not only does p300/CBP serve as their coactivators, STAT3 and NF-κΒ are also substrates of p300/CBP-mediated acetylation (15, 16). Recent studies revealed that p300-mediated acetylation of multiple lysine residues of STAT3 is a prerequisite for its phosphorylation at Y705 by JAK kinases (17). Interestingly, Marotta et al. reported recently that IL- 6/JAK2/STAT3 pathway is preferentially active in CD44⁺/CD24^~ breast CSCs and is required for their growth. Inhibition of this pathway by JAK2 inhibitor is effective to kill CSCs and cause regression of xenografted tumors (18). Additionally, CD44 has been shown to undergo nuclear translocation. In the nucleus, CD44 mediates the interaction between STAT3 and p300/CBP to acetylate STAT3 and its activation (19). STAT3 can further strengthen oncogenic signaling through activating NF-κΒ directly or indirectly (19, 20). Aside from acetylation of substrates that are directly involved in transcriptional regulation, p300/CBP acetylates cellular proteins that impact metabolism (21), autophagy (22) and motility (23). For example, p300/CBP might contribute to metastasis through protein acetylation such as chaperone protein Hsp90 (23). These observations provide a strong rationale for targeting p300/CBP as an anticancer therapeutic strategy. Targeting p300/CBP in cancer therapy may offer additional advantages. Because multiple growth factor receptors converge to activate STAT3, NF-KB and HIF-Ι and other transcription factors that recruit p300/CBP to their target genes, inhibition of p300/CBP may be more effective than suppression of receptor tyrosine kinases, as inhibition of one kinase often leads to the activation of an alternative pathway that still permits cancer cell survival and tumor progression. Furthermore, chemical inhibition of p300/CBP that possesses intrinsic acetyltransferase enzymatic activity is more feasible than blocking transcription factors with small molecules, as discovery of chemical inhibitors of transcription factors has proven extremely challenging.

High-throughput screening (HTS) was conducted to identify chemical inhibitors of p300/CBP that selectively kill TNBC cells. The TNBC cell line MDA-MB-231 was used in the primary HTS assay coupled with a counterscreening against human normal marry epithelial cells (HMEC) in order to discover compounds that are only toxic to TNBC cells but not to HMEC. MDA-MB-231 is one of the most widely studied breast cancer cell lines since its establishment in 1973 from pleural effusions of metastatic mammary carcinoma patients (24) and is generally resistant to a wide array of chemotherapeutic agents. p300 is expressed in MDA-MB-231 and appears to play a critical role in driving the invasive growth of this cell line (25). Hit compounds emerging from the primary HTS were tested for inhibiting p300 enzymatic activity in vitro.

BRIEF SUMMARY OF THE INVENTION

Applicant has identified a class of compounds that inhibit the function of p300 and/or

CBP, PCAF or GCN5. P300/CBP inhibition was noted to result in inhibition of tumor cell growth and killing of tumor cells. P300/CBP inhibition, alone or in combination with PCAF and/or GCN5 inhibition is, thus, useful for treatment of various types of cancers, including acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyeloeytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immunoblascie lymphoma, plasmacytoma, multiple myeiorna, Hodgkin's lymphoma, non- Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, central nervous system (CNS) cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer and breast cancer. The compounds disclosed herein can be used alone or in combination with other cancer treatment regimens {e.g., radiation therapy and/or other chemotherapeutic agents that are administered to a subject having a tumor, cancer or neoplasia). The compounds also can be used to treat a variety of other diseases and disorders, such as HIV and other infectious diseases, heart disease, diabetes mellitus, inflammation and airway inflammation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, with color drawing(s), will be provided by the Office upon request and payment of the necessary fee.

Fig. 1. Summary of the uHTS campaign for identifying compounds that are selectively toxic to TNBC cell line and also inhibitors of p300 enzymatic activity. A library of 622,079 compounds was screened in a primary assay based on cytotoxicity in MDA-MB-231. HMECs were used in a counterscreen. The compounds that are toxic to MDA-MB-231 cells but not to HMECs were further tested for inhibiting p300 catalytic activity in an in vitro assay.

Figs. 2A-2B. HTS identification of L002 as a p300 inhibitor. (Figure 2A) L002 chemical structure is shown (top). The middle panel shows growth inhibition of MDA-MB- 231 cells by L002 alone (red; circles) or in combination with SAHA (1.5 μΜ) (black; triangles), and HMEC (green; squares). Bottom panel shows inhibition curve of purified p300 in vitro by L002 using a fluorescence-based assay (black circles; IC50, 1.98 μΜ). The compound did not quench fluorescence (green triangles). (Figure 2B) MDA-MB-231 cells were untreated (control, lane 1), treated with hit #1, HDAC inhibitor TSA (lane 3), hit #1 plus TSA (lane 4), L002 (hit #2, lane 5) and L002 plus TSA (lane 6). The hit compounds were added to 30 μΜ 2 h followed by 1 h treatment with TSA (0.2 μΜ) before cell harvest. Histones were isolated by acid extraction method and separated by acid-urea gel electrophoresis. Western blotting was done with an anti-acetylated histone H4 antibody (H4K5/8/12/16ac). Total histones were separated by SDS-PAGE and detected with colloidal blue staining (bottom).

Figs. 3A-3B. L002 is highly toxic to TNBC cells. (Figure 3A) MDA-MB-231 (red line; squares) and MCF7 cells (blue line; diamonds) were seeded in 96-well plate, and exposed to vehicle or the indicated doses of L002. Viable cells were assayed 96h after drug addition. (Figure 3B) Colony formation assay. MDA-MB-231 and MCF7 cells were exposed to DMSO or the indicated doses of L002. Two days later, cells were split and reseeded. Medium without (solvent control) or with the compound were changed every four days. The plates were stained with methylene blue two weeks after initial drug exposure.

Fig. 4. L002 induces cell cycle arrest and apoptosis in MDA-MB-231 cells. Cells were exposed to DMSO (blue/lighter gray column; left column in each column pair) or L002 (red/darker column; right column in each column pair; 10 μΜ). At 24h after the agent addition, cells were processed for flow cytometry analysis. Percentage of cells in different phases of the cell cycle as well as cells with sub-Gl DNA content is plotted. Shown are average values of two independent experiments along with standard deviation.

Figs. 5A-5B. Relative potency of p300 inhibition by L002 analogs. (Figure 5A) The chemical structures of six compounds structurally related to L002. (Figure 5B) Relative IC50 values of the six compounds against p300 determined by radioactivity-based filter- binding assays.

Figs. 6A-6C. Molecular docking of L002 to the acetyl-CoA-binding pocket in the p300 catalytic domain. The chemical structure of L002 was fitted to the acetyl-CoA-binding pocket of a crystal structure of the p300 catalytic domain using in silico molecular docking. Potential hydrogen bonds between the ligand and the indicated residues in the p300 catalytic domain are depicted (dotted yellow lines). The residues of interest are labeled in yellow. (Figure 6 A) Cartoon representation of a model of the ligand-p300 interaction. (Figure 6B) The space-filled representation of p300 catalytic domain (green) in complex with the ligand. (Figure 6C) The ligand-p300 complex in a space-filled model. The ligand and the p300 catalytic domain are shown in red and green respectively.

Figs, 7A-B. Identification of L002 and its analogs as p300 inhibitors. (A) Dose- response curves of p300 inhibition by L0Q2 (black dots; left), and anacardic acid (AA, right). Purified recombinant p300 catalytic domain was incubated in a solution containing 50 μΜ of acetyl-CoA and a histone H3 N-terminal peptide in the presence of L002 or AA at a specified concentration. Co ASH, a product of the acetyl atkm reaction, was detected and quantified through a fluorescence-readout, in separate assays, L002 was added after the completion of the acetylation reaction and the fluorescence detection to assess whether LQ02 would quench fluorescence. No quench was observed (gray triangles, left panel). (B) The chemical structures of L002 and its analogs are shown. L001, L002, L004, L007 and LOOS were distinct hits identified in HTS assays. L003, L004 and L006 were identified based on their structural similarity to L002. Their inhibitor}' activities to acetyltransferases were tested in vitro (see Table 1 ).

Figs. 8A-C. Inhibition of p300-dependent functions in cells. (A) MDA-MB-468 ceils were untreated (NT, lane 1), treated with DMSO (lane 2), TSA (lane 3), L002 (lane 4), and L002 plus TSA (lane 5). The cells cultured in a complete medium with 10% bovine calf serum were exposed to DMSO or L002 for 7 h. TSA was added to 0.2 uM at 1 h before iysing cells for western blotting. A blot was probed with an antibody against acetyiated lysine (top panel) or acetyiated histone H4 (H4ac, middle panel). The blot was reprobed with an anti-PCNA antibody as a loading control. In a separate gel, an equal amount of the samples was loaded and stained with colloidal blue (bottom panel). (B) HCT116 cells were exposed to DMSO or L002 as indicated for 7 h. Etoposide (Etop) was added 1 h after the addition of L002. Cells were lysed for western blotting with the indicated antibodies. (C) MIA PaCa-2 cells were exposed to the indicated doses of L002 or L004 for 6 h before harvesting cells for western blotting using the indicated antibodies.

Figs. 9A-C. In vitro cell growth suppression by L002. (A) MDA-MB-231 and MCF7 cells were cultured and exposed to DMSO (0 μΜ) or the indicated doses of L002. Viable cells were assayed. Sho wn are the a verage values of three assays along with SD, (B ) Colony formation assay. The cells were exposed to DMSO or the indicated doses of L002. Surviving colonies were stained with methylene blue. (C) Reversibility of L002-mediated cytotoxicity. MDA-MB-231 or HCT116 cells were exposed to the indicated doses of L002. At 24h of exposure, L002 was washed out in one set of the experiments as indicated . Ceil viability was determined at 96h after L002 addition.

Figs, 1ΘΑ-Β. L002 induces cell cycle arrest and apoptosis. (A) MDA-MB-231 cells were exposed to DMSO, or 10 μΜ of L002 or AA for 24 h. Cells were then processed for flow cytometry analysis. Percentage of cells in different phases of the ceil cycle as well as cells with sub-Gl DMA content (apoptosis) is plotted. (B) HCT116 cells were exposed to DMSO or the indicated doses of L002 for 24h. Percent thymidine incorporation is plotted against L002 concentration.

Figs. 11A-C. In vivo anticancer efficacy of L002. Mice bearing MDA-MB-468 xenografts were untreated or treated with the vehicle (DMSO) or L002. Each treatment groups consisted of five mice (n=5). The vehicle or L002 (0.5 mg per injection) was injected intraperitoneally twice weekly for three weeks. Tumor volumes (A) and the percent change of body weights (B) of tumor-bearing mice along with the standard error of the mean (SEM) are plotted. The arrow in the left graph denotes treatment endpoint. (C) Tumor sections of mice treated with DMSO or L002 were subjected to H&E staining or immunohistochemicai analysis with an anti-H4ac antibody. Three different magnifications of the tumor sections stained with the anti-H4ac antibody are shown.

Figs. 12A-B. Effects of L002 and other hits on histone modifications. (A) MDA-MB- 231 ceils were treated with the indicated hit compounds (final concentration of 30 J.tM). Histone proteins were extracted from the treated cells and analyzed by SDS-PAGE and western blotting with the indicated antibodies (anti-H2BK12ac, Epitomics 1755-1 ; anti- H3K18ac, Abeam, abl l 91; anti-H3K4me3, Upstate, 07-473). (B) Similarly, HCT116 cells were treated with the indicated agents and analyzed as in (A). Lower panels: the extracted histones were analyzed with SDS-PAGE and stained with colloidal Coomassie blue. The ChemBridge catalog numbers are 6743374 (hit#l ), 6625948 (hit#2), 5473210 (hit#3), and 5861253 (hit#4).

Figs. 13A-C. L002 does not inhibit HDACs. (A) Purified full-length HDAC1 (15.4 nM) was incubated with L002 or SAHA at a specific concentration as indicated in the HDAC-Glo Ι/ΊΙ buffer (Promega) at room temperature for 30 rain. An equal volume of a developer solution was then added to the reaction mixture. Luminescence signals were read with a BMG plate reader. Similar assays were done for purified full-length HDAC6 (11 nM) in the presence of different concentrations of L002. Shown are average HDAC activities along with standard deviations from three assays. (B) HDAC11 (0.9 μΜ) was incubated with L002 or trichostatin A (TSA) at an indicated concentration, A fluorogenic acetylated peptide from p53 residues 379-382 (RHK Ac, 50 μΜ) was added to the HDAC 11 -inhibitor solution. Fluorescence signal was detected after adding a developer solution and quantified. The relative HDAC activity was plotted against the inhibitor concentration.

Fig, 14. L002 does not inhibit HMTs. A purified human histone methyltransferase (HMT) was mixed with a histone substrate (the core hisione, oligonucieosomes, histone 3 or H4) and L002 at an indicated concentration. The radioactive S-adenosyi-L-[methyl— H] methionine (1 μΜ) was then added to initiate the methyl transfer reaction. The reactio solution was spotted on a filter, which was washed and dried. The radioactivity was detected and quantified. The relative HMT activity was plotted against L002 concentration. In the control reactions, the solvent DMSO was incubated with an HMT.

Fig. 15. L002 suppresses migration of triple-negative breast cancer ceils. A confluent monolayer of TNBC MDA-MB-231 cells was scratched. DMSO (solvent control) or L002 at two different concentrations were immediately added to the scratched cell cultures. Images of the scratched areas were acquired immediately after scratch (0 hours) and at 8 and 20 hours after scratch. (A) Representative images are shown. The open areas devoid of cells were quantified, and the relative unfilled areas were plotted. (B) Shown are the average values of at least 10 images across the scratch along with standard deviation. The data are representative of three independent experiments with similar results. The data show that L002 inhibited TNBC' cell migration, suggesting that it may suppress cancer metastasis. Thus, it may have therapeutic application for treating metastatic cancer.

DETAILED DISCLOSURE OF THE INVENTION

In one aspect of the invention, Applicants have identified a class of compounds that inhibit the function of p300/CBP, PCAF and/or GCN5. P300/CBP inhibition was noted to result in inhibition of tumor cell growth and killing of tumor cells. P300/CBP inhibition is, thus, useful for treatment of various types of cancers, including acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytic leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large ceil imm noblastic lymphoma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, central nervous system (CNS) cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer, and breast cancer. As indicated in the claims, the p300/CBP inhibitors disclosed herein can be used alone, or in combination with radiation therapy and/or chemotherapeutic agents for the treatment of cancers, tumors or neoplasias. The compounds also can be used to treat a variety of other diseases and disorders, such as HIV and other infectious diseases, heart disease, diabetes mellitus, inflammation and airway inflammation.

Accordingly, one aspect of the invention provides methods for treating cancer in a subject by inhibiting the activity of p300/CBP by administration of a compound of Formula I or Formula II. In various embodiments of this aspect of the invention, cancer can be treated by administering an effective amount of a p300/CBP inhibitor, alone or in combination with a chemotherapeutic agent and/or radiation therapy, to a subject in need treatment. In embodiments of this aspect of the invention, the inhibitor is a compound of Formula I, Formula II, L001, L002, L003, L004, L005, L006a, L006b, L007 or L008or an analog or derivative thereof.

Another embodiment of this aspect of the invention provides for combination therapies for cancer that include the administration of an effective amount of a p300/CBP inhibitor selected from a compound of Formula I, Formula II, L001, L002, L003, L004, L005, L006a, L006b, L007 or L008 in combination with radiation treatment and/or at least one additional chemotherapeutic agent. The radiation therapy can be any X-ray therapy or radiopharmaceutical therapy that is well-known in the art. The chemotherapeutic agent is any agent for treating cancer that is well-known in the art.

In embodiments, the cancer is acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocyte leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immurioblastic lymphoma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, CNS cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer, or breast cancer. In some embodiments, the cancer is brain cancer, lung cancer, or melanoma.

In another aspect, methods for inhibiting the growth, proliferation, or survival of a neoplastic or cancer cell by contacting the cell with an effective amount of a p300/CBP inhibitor are provided. In embodiments of this aspect of the invention, the inhibitor is a compound of Formula I, Formula II, L001, L002, L003, L004, L005, L006a, L006b, L007 or L008or an analog or derivative thereof. Yet another aspect of the invention provides in vitro methods of inhibiting p300/CBP activity assays (e.g., serving as a control for screening other p300/CBP inhibitors). In various embodiments of this aspect of the invention, samples or biological samples containing p300/CBP are contacted with an amount of a p300/CBP inhibitor disclosed herein effective to inhibit p300/CBP activity within the sample or biological sample. Exemplary in vitro methods for performing such inhibition studies are provided in the examples, below.

Yet another aspect of the invention provides methods of treating diseases or disorders associated with excessive p300/CRB, PCAF and/or GCN5 activity comprising the administration of the compounds disclosed herein. Yet another aspect of the invention provides methods of treating HIV and other infectious diseases, heart disease, and diabetes mellitus, inflammation and airway inflammation comprising administration of a compound of the invention to a subject having HIV and other infectious diseases, heart disease, and diabetes mellitus, inflammation and airway inflammation.

One aspect of the invention provides for compounds of Formula I and Formula II,

wherein:

R¹, R², R³ (if present, e.g., in Formula I), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl. In certain embodiments, R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), lower alkyl-, lower alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or lower perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

Yet other embodiments provide for compounds of Formula II in which:

i) R¹, R⁴, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are -H, R² and R⁵ are lower alkyl, R³ is an oxo (=0) group and n is 0-4;

ii) R¹, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group and n is 0-4;

iii) R¹, R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group and n is 0-4;

iv) R¹, R⁴, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group and n is 0-4; or

v) R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R¹ is a lower alkyl group and n is 0-4. In certain specific embodiments for (i)-(v) as set forth above, n is 0.

Certain specific embodiments provide for compounds that are identified herein as compounds LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008:

Certain other aspects of the invention provide pharmaceutical compositions comprising one or more of the compounds (compounds of Formula I, Formula II or a compound identified as LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008), disclosed herein in combination with chemotherapeutic agent and a pharmaceutically acceptable carrier and/or excipient. Pharmaceutical compositions, as disclosed herein, can be formulated in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York) known by a person skilled in the art. Pharmaceutical composition according to the invention may also be formulated to release active agents (e.g., a p300/CBP inhibitor as disclosed herein alone or in combination with a chemotherapeutic agent) substantially immediately upon administration or at any predetermined time or time period after administration.

Compositions for parenteral administration are generally physiologically compatible sterile solutions or suspensions which can optionally be prepared immediately before use from solid or lyophilized form. Adjuvants such as a local anesthetic, preservative and buffering agents can be dissolved in the vehicle and a surfactant or wetting agent can be included in the composition to facilitate uniform distribution of the active ingredient.

For oral administration, the composition can be formulated into conventional oral dosage forms such as tablets, capsules, powders, granules and liquid preparations such as syrups, elixirs, and concentrated drops. Non toxic solid carriers or diluents may be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, magnesium, carbonate, and the like. For compressed tablets, binders, which are agents which impart cohesive qualities to powdered materials are also necessary. For example, starch, gelatine, sugars such as lactose or dextrose, and natural or synthetic gums can be used as binders. Disintegrants are also necessary in the tablets to facilitate break-up of the tablet. Disintegrants include starches, clays, celluloses, algins, gums and crosslinked polymers. Moreover, lubricants and glidants are also included in the tablets to prevent adhesion to the tablet material to surfaces in the manufacturing process and to improve the flow characteristics of the powder material during manufacture. Colloidal silicon dioxide is most commonly used as a glidant and compounds such as talc or stearic acids are most commonly used as lubricants.

For transdermal administration, the composition can be formulated into ointment, cream or gel form and appropriate penetrants or detergents could be used to facilitate permeation, such as dimethyl sulfoxide, dimethyl acetamide and dimethylformamide.

For transmucosal administration, nasal sprays, rectal or vaginal suppositories can be used. The active compound can be incorporated into any of the known suppository bases by methods known in the art. Examples of such bases include cocoa butter, polyethylene glycols (carbowaxes), polyethylene sorbitan monostearate, and mixtures of these with other compatible materials to modify the melting point or dissolution rate. A number of terms and phrases are defined below.

The singular forms "a", "an", and "the" include plural forms unless the context clearly dictates otherwise. Additionally, as used herein, the terms "comprises," "comprising," "containing," "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean "includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals, in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Nonlimiting examples of such cancers include squamous cell cancer, small- cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, lung cancer, leukemia, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma (i.e., brain cancer), CNS cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney/renal cancer, liver cancer, melanoma, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocyte leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immunoblastic lymphoma., plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and various types of head and neck cancer. In certain embodiments, methods of treating triple negative breast cancer (TNBC) are provided.

The terms "proliferative disorder" and "proliferative disease" refer to disorders associated with abnormal cell proliferation, such as cancer or dysplasia.

The term "tumor" as used herein refers to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including precancerous lesions.

The term "neoplastic" refers to those cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. A neoplastic disease state may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness.

The term "inhibit tumor growth" and its grammatical equivalents refer to any mechanism by which tumor cell growth can be inhibited. In certain embodiments, tumor cell growth is inhibited by slowing proliferation of tumor cells. In certain embodiments, tumor cell growth is inhibited by halting proliferation of tumor cells. In certain embodiments, tumor cell growth is inhibited by killing tumor cells. In certain embodiments, tumor cell growth is inhibited by inducing apoptosis of tumor cells. In certain embodiments, tumor cell growth is inhibited by preventing migration of tumor cells. In certain embodiments, tumor cell growth is inhibited by preventing invasion of tumor cells.

The term "p300/CBP inhibitor" refers to a compound of Formula I, Formula II or compounds L001, L002, L003, L004, L005, L006a, L006b, L007 or L008that inhibits the histone acetyltransferase (HAT) activity of as measured by an inhibition assay that is well- known in the art, including the coupled spectrophotometric assay, the direct radioactive assay, and the HAT assays described herein.

The term "radiation therapy", "radiotherapeutic treatment" or "radiotherapy" is a term commonly used in the art to refer to multiple types of radiation therapy including internal and external radiation therapy, radioimmunotherapy, and the use of various types of radiation including X-rays, gamma rays, alpha particles, beta particles, photons, electrons, neutrons, radioisotopes, and other forms of ionizing radiation. Preferably, the radiotherapy involves the use of X-rays.

The methods and pharmaceutical composition of the invention can further utilize a chemotherapeutic agent suitable for the treatment of cancers, tumors and/or neoplasias. The "chemotherapeutic agent" may be selected from the group consisting of anthracyclines, platinum-based chemotherapy drugs, pyrimidine analogues, kinase inhibitors and alkylating agents, and combinations thereof. Anthracyclines may include, but are not limited to, doxorubicin, epirubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, carminomycin and detorubicin. Platinum-based chemotherapy drugs may include, but are not limited to, carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate and satraplatin. Pyrimidine analogues may include, but are not limited to, 5- Fluorouracil (5-FU), cytarabine and floxuridine. Alkylating agents may include, but are not limited to, nitrogen mustards such as cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine; nitrosourea compounds such as carmustine, lomustine, semustine and streptozotocin; busulfan; dacarbazine; procarbazine; altretamine; mitozolomide; and temozolomide. Kinase inhibitors that can be used in this aspect of the invention include, and are not limited to, sorafenib, sunitinib and imatinib.

Terms such as "treating," "treatment," "to treat," "alleviating," and "to alleviate" refer to both 1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or proliferative disorder, and 2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or proliferative disorder. Thus, those in need of treatment include those already with the proliferative disorder; those prone to having the proliferative disorder; and those in whom the proliferative disorder is to be prevented. A subject is successfully "treated" according to the methods of the present invention if the patient shows one or more of the following: a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs, including the spread of cancer into soft tissue and bone; inhibition of or an absence of tumor metastasis; inhibition or an absence of tumor growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; and improvement in quality of life. By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease or a symptom thereof.

The term "administering" is defined herein as a means of providing an agent or a composition containing the agent to a subject in a manner that results in the agent being inside the subject's body. Such an administration can be by any route including, without limitation, oral, subcutaneous, intradermal, intravenous, intra-arterial, intratumoral, intraperitoneal, and intramuscular.

The term "sample" is defined herein as blood, blood product, biopsy tissue, serum, and any other type of fluid or tissue that can be extracted from a subject or a mammal or which can contain p300/CBP, PCAF or GCN5 (e.g., a sample from a cell that recombinantly produces p300/CBP). The terms "sample" and "biological sample" may be used interchangeably in this application.

The term "subject" or "patient" refers to an animal which is the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a bovine, equine, canine, ovine, murine or feline. In certain embodiments, the treatment of humans is contemplated by this invention.

The term "effective amount" means the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient or to produce some desired therapeutic effect. The effective amount of active compound(s) used to practice the present invention for prevention or treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen.

The term "analog" means a molecule that is not identical, but has analogous functional or structural features. For example, an amide, ester, carbamate, carbonate, ureide, or phosphate analog of a compound is a molecule that either: 1) does not destroy the biological activity of the compound and confers upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is itself biologically inactive but is converted in vivo to a biologically active compound.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. "About" can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The term "alkyl" refers to a straight or branched or cyclic chain hydrocarbon radical with only single carbon-carbon bonds. Representative examples include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, and cyclohexyl, all of which may be optionally substituted. Alkyl groups are C₁-C₁₂ and include alkyl groups that are Ci-Cg in some embodiments or C₁-C5 in other embodiments, each of which can be optionally substituted.

The term "optionally substituted" or "substituted" includes groups substituted by one to six substituents, independently selected from lower alkyl, lower aryl (substituted or unsubstituted), lower aralkyl, lower cyclic alkyl, lower heterocycloalkyl, hydroxy, lower alkoxy, lower aryloxy, perhaloalkoxy, aralkoxy, lower heteroaryl (substituted or unsubstituted), lower heteroaryloxy, lower heteroarylalkyl, lower heteroaralkoxy, azido, amino, halo, lower alkylthio, oxo, lower acylalkyl, lower carboxy esters, carboxyl, -carboxamido, nitro, lower acyloxy, lower aminoalkyl, lower alkylaminoaryl, lower alkylaryl, lower alkylaminoalkyl, lower alkoxyaryl, lower arylamino, lower aralkylamino, sulfonyl, lower -carboxamidoalkylaryl, lower -carboxamidoaryl, lower hydroxyalkyl, lower haloalkyl, lower alkylaminoalkylcarboxy-, lower aminocarboxamidoalkyl-, cyano, lower alkoxyalkyl, lower perhaloalkyl, and lower arylalkyloxy alkyl.

The term "aryl" refers to aromatic groups which have 5-14 ring atoms and at least one ring having a conjugated pi electron system and includes carbocyclic aryl, heterocyclic aryl and biaryl groups, all of which may be optionally substituted.

Carbocyclic aryl groups are groups which have, in various embodiments, 6-10 or 6-14 ring atoms wherein the ring atoms on the aromatic ring are carbon atoms. Carbocyclic aryl groups include monocyclic carbocyclic aryl groups and polycyclic or fused compounds such as optionally substituted naphthyl groups.

Heterocyclic aryl or heteroaryl groups are groups which have, in various embodiments, 5-10 or 5-14 ring atoms wherein 1 to 4 heteroatoms are ring atoms in the aromatic ring and the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include oxygen, sulfur, nitrogen, and selenium. Suitable heteroaryl groups include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolyl, pyridyl-N-oxide, pyrimidyl, pyrazinyl, imidazolyl, and the like, all optionally substituted.

"Substituted aryl" and "substituted heteroaryl" refers to aryl and heteroaryl groups substituted with 1-3 substituents. These substituents are selected from the group consisting of lower alkyl, lower alkoxy, lower perhaloalkyl, halo, hydroxy, and amino.

The term "-aralkyl" refers to an alkylene group substituted with an aryl group.

Suitable aralkyl groups include benzyl, picolyl, and the like, and may be optionally substituted. "Heteroarylalkyl" refers to an alkylene group substituted with a heteroaryl group.

The term "alkylaryl-" refers to an aryl group substituted with an alkyl group. "Lower alkylaryl-" refers to such groups where alkyl is lower alkyl.

The term "lower" referred to herein in connection with organic radicals or compounds respectively defines such as with up to and including 10, in one aspect up to and including 6, and in another aspect one to four carbon atoms. Such groups may be straight chain, branched, or cyclic.

The term "cyclic alkyl" or "cycloalkyl" refers to alkyl groups that are cyclic of 3 to 10 carbon atoms, and in one aspect are 3 to 6 or 3 to 8 carbon atoms. Suitable cyclic groups include norbornyl and cyclopropyl. Such groups may be substituted. The term "heterocyclic", "heterocyclic alkyl" or "heterocycloalkyl" refer to cyclic groups of 3 to 10 atoms, and in one aspect are 3 to 6 atoms, containing at least one heteroatom, in a further aspect are 1 to 3 heteroatoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen. Heterocyclic groups may be attached through a nitrogen or through a carbon atom in the ring. The heterocyclic alkyl groups include unsaturated cyclic, fused cyclic and spirocyclic groups. Suitable heterocyclic groups include pyrrolidinyl, morpholino, morpholinoethyl, and pyridyl.

The terms "arylamino" (a), and "aralkylamino" (b), respectively, refer to the group -NR ' wherein respectively, (a) R is aryl and R' is hydrogen, alkyl, aralkyl, heterocycloalkyl, or aryl, and (b) R is aralkyl and R' is hydrogen, aralkyl, aryl, alkyl or heterocycloalkyl.

The term "arylalkyloxyalkyl" refers to aryl-alk-O-alk- group where "alk" is an alkylene group.

The term "acyl" refers to -C(0)R where R is alkyl, heterocycloalkyl, or aryl.

The term "carboxy esters" refers to -C(0)OR where R is alkyl, aryl, aralkyl, cyclic alkyl, or heterocycloalkyl, all optionally substituted.

The term "carboxyl" refers to -C(0)OH.

The term "oxo" refers to =0 in an alkyl, carbocyclic, aryl or heterocycloalkyl group.

The term "amino" refers to -NRR' where R and R' are independently selected from hydrogen, alkyl, aryl, aralkyl and heterocycloalkyl, all except H are optionally substituted; and R and R' can form a cyclic ring system.

The term "-carboxylamido" refers to -CONR₂ where each R is independently hydrogen or alkyl.

The term "-sulphonylamido" or "-sulfonylamido" refers to -S(=0)₂NR₂ where each R is independently hydrogen or alkyl.

The term "halogen" or "halo" refers to -F, -CI, -Br and -I.

The term "alkylaminoalkylcarboxy" refers to the group alkyl-NR-alk-C(0)-0- where "alk" is an alkylene group, and R is a H or lower alkyl.

The term "sulphonyl" or "sulfonyl" refers to -S0₂R, where R is H, alkyl, aryl, aralkyl, or heterocycloalkyl.

The term "sulphonate" or "sulfonate" refers to -S0₂OR, where R is -H, alkyl, aryl, aralkyl, or heterocycloalkyl.

The term "alkenyl" refers to unsaturated groups which have 2 to 12 atoms and contain at least one carbon-carbon double bond and includes straight-chain, branched-chain and cyclic groups. Alkenyl groups may be optionally substituted. Suitable alkenyl groups include allyl. "1-alkenyl" refers to alkenyl groups where the double bond is between the first and second carbon atom. If the 1-alkenyl group is attached to another group, e.g., it is a W substituent attached to the cyclic phosphonate, it is attached at the first carbon.

The term "alkynyl" refers to unsaturated groups which have 2 to 12 atoms and contain at least one carbon-carbon triple bond and includes straight-chain, branched-chain and cyclic groups. Alkynyl groups may be optionally substituted. Suitable alkynyl groups include ethynyl. "1 -alkynyl" refers to alkynyl groups where the triple bond is between the first and second carbon atom. If the 1 -alkynyl group is attached to another group, e.g., it is a W substituent attached to the cyclic phosphonate, it is attached at the first carbon.

The term "alkylene" refers to a divalent straight chain, branched chain or cyclic saturated aliphatic group. In one aspect the alkylene group contains up to and including 10 atoms. In another aspect the alkylene chain contains up to and including 6 atoms. In a further aspect the alkylene groups contains up to and including 4 atoms. The alkylene group can be either straight, branched or cyclic.

The term "acyloxy" refers to the ester group -0-C(0)R, where R is H, alkyl, alkenyl, alkynyl, aryl, aralkyl, or heterocycloalkyl.

The term "aminoalkyl-" refers to the group NR₂-alk- wherein "alk" is an alkylene group and R is selected from -H, alkyl, aryl, aralkyl, and heterocycloalkyl.

The term "alkylaminoalkyl-" refers to the group alkyl-NR-alk- wherein each "alk" is an independently selected alkylene, and R is H or lower alkyl. "Lower alkylaminoalkyl-" refers to groups where the alkyl and the alkylene group are lower alkyl and alkylene, respectively.

The term "arylaminoalkyl-" refers to the group aryl-NR-alk- wherein "alk" is an alkylene group and R is -H, alkyl, aryl, aralkyl, or heterocycloalkyl. In "lower arylaminoalkyl-", the alkylene group is lower alkylene.

The term "alkylaminoaryl-" refers to the group alkyl-NR-aryl- wherein "aryl" is a divalent group and R is -H, alkyl, aralkyl, or heterocycloalkyl. In "lower alkylaminoaryl-", the alkyl group is lower alkyl.

The term "alkoxyaryl-" refers to an aryl group substituted with an alkyloxy group. In

"lower alkyloxyaryl-", the alkyl group is lower alkyl.

The term "aryloxyalkyl-" refers to an alkyl group substituted with an aryloxy group. The term "aralkyloxyalkyl-" refers to the group aryl-alk-O-alk- wherein "alk" is an alkylene group. "Lower aralkyloxyalkyl-" refers to such groups where the alkylene groups are lower alkylene.

The term "alkoxy-" or "alkyloxy-" refers to the group alkyl-O-.

The term "aryloxy-" refers to the group aryl-O-.

The term "arylalkoxy-" refers to the group aryl-alkyl-O-.

The term "alkoxyalkyl-" or "alkyloxyalkyl-" refer to the group alkyl-O-alk- wherein "alk" is an alkylene group. In "lower alkoxyalkyl-", each alkyl and alkylene is lower alkyl and alkylene, respectively.

The terms "alkylthio-" and "alkylthio-" refer to the group alkyl-S-.

The term "alkylthioalkyl-" refers to the group alkyl-S-alk- wherein "alk" is an alkylene group. In "lower alkylthioalkyl-" each alkyl and alkylene is lower alkyl and alkylene, respectively.

The term "alkoxycarbonyloxy-" refers to alkyl-0-C(0)-0-.

The term "aryloxycarbonyloxy-" refers to aryl-0-C(0)-0-.

The term "alkylthiocarbonyloxy-" refers to alkyl-S-C(0)-0-.

The term "amido" refers to the NR₂ group next to an acyl or sulfonyl group as in NR₂-C(0)-, RQC -NR¹-, NR₂-S(=0)₂- and RS(=0)₂-NR¹-, where R and R¹ include -H, alkyl, aryl, aralkyl, and heterocycloalkyl.

The term "carboxamido" refer to NR₂-C(0)- and RC(0)-NR¹-, where R and R¹ include -H, alkyl, aryl, aralkyl, and heterocycloalkyl. The term does not include urea, -NR-C(0)-NR-.

The terms "sulphonamido" or "sulfonamido" refer to NR₂-S(=0)₂- and RS(=0)₂-NR¹-, where R and R¹ include -H, alkyl, aryl, aralkyl, and heterocycloalkyl. The term does not include sulfonylurea, -NR-S(=0)₂-NR-.

The term "carboxamidoalkylaryl" and "carboxamidoaryl" refers to an aryl-alk-NR¹-C(0)-, and ar-NR¹-C(0)-alk-, respectively where "ar" is aryl, "alk" is alkylene, R¹ and R include H, alkyl, aryl, aralkyl, and heterocycloalkyl.

The term "sulfonamidoalkylaryl" and "sulfonamidoaryl" refers to an aryl-alk-NR¹-S(=0)₂-, and ar-NR¹-S(=0)₂-, respectively where ' 'ar" is aryl, "alk" is alkylene, R¹ and R include -H, alkyl, aryl, aralkyl, and heterocycloalkyl.

The term "hydroxyalkyl" refers to an alkyl group substituted with one -OH.

The term "haloalkyl" refers to an alkyl group substituted with one halo. The term "cyano" refers to -C≡N.

The term "nitro" refers to -N0₂.

The term "acylalkyl" refers to an alkyl-C(0)-alk-, where "alk" is alkylene.

The term "aminocarboxamidoalkyl-" refers to the group NR₂-C(0)-N(R)-alk- wherein R is an alkyl group or H and "alk" is an alkylene group. "Lower aminocarboxamidoalkyl-" refers to such groups wherein "alk" is lower alkylene.

The term "heteroarylalkyl" refers to an alkylene group substituted with a heteroaryl group.

The term "heteroaryloxy" refers to heteroaryl-O-.

The term "heteroaralkoxy" refers to the group heteroaryl-alkyl-O-.

The term "perhalo" refers to groups wherein every C-H bond has been replaced with a C-halo bond on an aliphatic or aryl group. Suitable perhaloalkyl groups include -CF3 and

-CFCI2.

The term "carboxylic acid moiety" refers to a compound having a carboxylic acid group (-COOH), and salts thereof, a carboxylic acid ester, or a carboxylic acid surrogate. Suitable carboxylic acid surrogates include a tetrazole group, a hydroxamic acid group, a thiazolidinedione group, an acylsulfonamide group, and a 6-azauracil. (see, e.g., The Practice ofMedicinal Chemistry; Wemuth, C.G., Ed.; Academic Press: New York, 1996; p. 203).

The term "pharmaceutically acceptable salt" includes salts of compounds of Formula I or II (including L001, L002, L003, L004, L005, L006a, L006b, L007 or L008) and an organic or inorganic acid or base. Suitable acids include acetic acid, adipic acid, benzenesulfonic acid, (+)-7,7-dimethyl-2-oxobicyclo[2.2.1]heptane-l-methanesulfonic acid, citric acid, 1 ,2-ethanedisulfonic acid, dodecyl sulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glucuronic acid, hippuric acid, hydrochloride hemiethanolic acid, HBr, HC1, HI, 2-hydroxyethanesulfonic acid, lactic acid, lactobionic acid, maleic acid, methanesulfonic acid, methylbromide acid, methyl sulfuric acid, 2-naphthalenesulfonic acid, nitric acid, oleic acid, 4,4'-methylenebis [3-hydroxy-2-naphthalenecarboxylic acid], phosphoric acid, polygalacturonic acid, stearic acid, succinic acid, sulfuric acid, sulfosalicylic acid, tannic acid, tartaric acid, terphthalic acid, and /?-toluenesulfonic acid.

Following is an example which illustrates procedures for practicing the invention.

These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted. EXAMPLE 1

MATERIALS AND METHODS

Cell culture

The human breast carcinoma cell lines MCF7, MDA-MB-231, MDA-MB-436, and

MDA-MB-468 were cultured with Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% v/v fetal calf serum, 10 units/ml penicillin, and 10 μg/ml streptomycin sulfate. Human Mammary Epithelial Cells (HMEC) were purchased from Invitrogen (A10565) and cultured in HuMEC Ready Medium (Invitrogen, 12752010) supplemented with 1% penicillin- streptomycin-neomycin antibiotic mix (Invitrogen, 15640) and incubated at 37°C in an atmosphere of 5% C02 and 95% relative humidity.

HTS compound library

The uHTS screening campaign to identify p300 acetyltransferase inhibitors (HATi) was run against the Scripps Drug Discovery library. This comprises 622,079 unique and drug-like compounds originating from both internal medicinal chemistry/drug discovery efforts and commercial sources. The library contains several sub-libraries such as GPCR, Kinase, "Rule of 5" natural products, and transcription factor and "Click" chemistry collections. Additionally, the library contains collections of clinically relevant compounds and approved drugs. In particular, it includes a collection of 1280 drugs that have reached clinical trial stages in the USA or that are marketed in Europe and/or Asia, a collection of over 1,000 commercial bioactive compounds identified from the MDL® Comprehensive Medicinal Chemistry database or DrugBank database, a LOPAC collection of 1280 pharmacologically active high purity compounds, as well as other active compounds such as nuclear receptor signaling ligands (Androgen (AR), Estrogen (ER), Glucocorticoid (GR), Mineralocorticoid (MR), Peroxisome proliferator-activated (PPARa, PPARx or PPAR5), Progesterone (PR), Liver X (LXR), Retinoic acid (RAR), Retinoid X (RXR), and Thyroid hormone), off-patent small molecules from Prestwick Chemicals and unique and diverse bioactive compounds from Tocris. All compounds have been tested for purity and structural confirmation via liquid chromatography and mass spectrometry (LC-MS) and/or nuclear magnetic resonance (NMR). Relevant compounds with high purity for cell-based assays including those for cell viability, colony formation, inhibition of histone acetylation using western blotting analysis, and radioactivity-based in vitro inhibition of acetyltransferases were purchased from ChemBridge.

Cell-based ultra HTS (uHTS) assay

All assays were adapted to 1536-well format using uHTS platform. For the cell-based assays, the 96-well format protocols were adapted to 1536-well format first by reducing dispensed volumes. Cell density was optimized to give the best balance between assay performance and practical considerations related to reagent costs, cell scale-up and handling. In the final assay format, 500 cells (MDA-MB-231 or HMEC) were seeded per well in 3 of medium and cultured overnight. Library compound dissolved in DMSO was then added to the final concentration of 6.2 μΜ (final DMSO concentration was 0.6%) with final assay volume of 4 μΐ, and the cells were incubated for 72 hr. To assay cell survival, 4 μΐ, of CellTiter-Glo reagent (Promega; Madison, WI) was added per well and the plates were allowed to develop for 15 min in dark at room temperature. Luminescence was quantified with the PerkinElmer Viewlux. The effect of treatment on cell growth was calculated for each compound using the equation: % Growth inhibition = [l-(RFU_Test_Compound - Median RFU High Control) / (Median RFU Low Control - Median RFU High Control )]* 100, where RFU Test Compound is the luminescence signal intensity of well containing a test compound; RFU High Control is defined as the luminescence intensity of well containing 50 μΜ SAHA, and RFU Low Control is defined as the luminescence intensity of wells with DMSO. The average percent inhibition of each compound and standard deviation were calculated. Any compound that exhibited an average percent inhibition greater than the hit cutoff calculated for the primary screen was selected for subsequent assays. Fluorescence-based p300 acetyltransferase activity assay

An enzyme mix consisting of 0.39X Assay Buffer (final concentration) and 50 μΜ acetyl-CoA (final concentration) with or without purified p300 catalytic domain (amino acids 965-1810; ^g/ml, final concentration), was dispensed in 1 μΐ_^ per well to 1536-well plates. Thirty nL of a test compound or DMSO was then added and the plates were incubated at room temperature for 15 min, followed by the addition of 1 μί of 101.5 μΜ of a histone H3 peptide solution as the acetylation substrate to each well to the final concentration of 50 μΜ. The plates were allowed to incubate for 30 min at room temperature. The acetylation reaction was stopped with the addition of 1.25 μΐ, of stop solution to each well. To generate a fluorescent product, 2.5 μΐ, of the 28.9 μΜ developing solution was added to each well. Plates were incubated for 15 min in the dark at room temperature and fluorescence was detected with the Tecan Saffire instrument with excitation at 360-390 nm and emission at 450-470 nm. To detect potential fluorescence quenchers among the library compounds, a test compound was added after the completion of the acetylation and fluorescence adduct formation, so that compounds that suppress fluorescence readout independently of p300 inhibition could be identified. All relevant reagents for the in vitro acetyltransferase assay were obtained from Active Motif (Carlsbad, CA). Radioactivity-based filter-binding (HotSpot) HAT assays, in which [³H]-acetyl-CoA and histone H3 were used as substrates, were done by Reaction Biology Cooperation (Malvern, PA).

Compound titration assays

The 640 compounds selected for titration assays based on the results of the cell-based primary and counterscreen uHTS were prepared as 10-point, 1 :3 serial dilutions, starting at a 15.5 μΜ final test concentration. Each titrated compound was tested in triplicate in all assays. For each test compound, percent inhibition was plotted against compound concentration. Relevant CC50/IC50 values were generated using the Assay Explorer software.

CC₅₀/IC₅₀ determination

Data analysis was performed using GraphPad Prism v.5 software (San Diego, CA).

For each test compound, percent inhibition was plotted against compound concentration. A four-parameter equation describing a sigmoidal dose-response curve was then fitted with adjustable baseline. The reported CC50/IC50 values were generated from fitted curves by solving for the X-intercept value at the 50% inhibition level of the Y-intercept value. In cases where the highest concentration tested (15.5 μΜ) did not result in > 50% inhibition, the CC50/IC50 was reported as > 15.5 μΜ. Compounds with CC50/IC50 values of greater than 10 μΜ were considered inactive, whereas compounds with CC50/IC50 values equal to or less than 10 μΜ are considered active. Structure-activity response clustering and Promiscuity profiling

In addition to the biological results, in silico data were generated for the 640 compounds tested in dose-response titration assays to facilitate compound prioritization for further studies. First, compounds were grouped (clustered) by structure with a 0.85 Tanimoto similarity threshold cutoff using Accelrys Pipeline Pilot 8.0 (Accelrys, San Diego, CA), followed by manual inspection to merge clusters sharing a common scaffold. Compounds were grouped into 81 individual scaffolds, each encompassing from 2 to 48 members. Three hundred-fifteen compounds that did not exhibit similarity to any other were classified as singletons. "Promiscuous" compounds are found active across a wide range of assays either by interfering with the assay readout (e.g. fluorescent properties) or because of their physicochemical properties (e.g. aggregation). To this end, each titrated compound was compared to its past performance in primary HTS assays executed at Scripps Florida in previous drug discovery efforts. Promiscuity results were expressed as the number of times a compound was found active over how many primary HTS assays in which it was tested.

Cell viability and colony formation assays

Cells (10,000 per well) were seeded in 96-well plate in 100 μΐ of medium. After culturing overnight, cells were exposed to solvent (DMSO) or L002 in triplicate. The plates were then incubated for 96 h and cell viability was assayed with CellTiter-Glo reagent kit. Luminescence was detected with a BMG POLARstar OMEGA multiplate reader. Luminescence readout in each well was normalized against that from solvent-treated wells. Data were fitted with non-linear semi-log dose response curve for CC50 calculation using Prism 5 software. In colony formation assay, cells were seeded in 6-well plates and exposed to various doses of L002 for 48 h. The treated cells were trypsinized and 3,500 cells per well were reseeded in a 12-well plate in triplicate. Cells were cultured with medium containing L002 at the doses as in the initial treatment. Medium with a proper dose of L002 was changed very four days until colonies appeared in about 2 weeks. Colonies were stained with 1% methylene blue.

Cell cycle analysis

MDA-MB-231 cells were seeded in 12-well plates. At 24 li after seeding, cells were exposed to exposed to DMSO or .10 μ L002 for 24 h. Ceils were then trypsinized, and processed for flow cytometry using a FACSort instrument (BD Biosciences) essentially as described previously (26).

Histone isolation and western blotting

A near confluent monolayer culture of MDA-MB-231 cells or other cell lines was exposed to DMSO or 30 μΜ of L002 (final concentration) 2 h before cell harvest. One hour before cell harvest, cells were mock-treated or exposed to trichostatin A (TSA, 0.2 μΜ final concentration). The cells were trypsinized and histones were isolated using acid extraction method as described previously (27). The isolated histones were separated with conventional SDS-PAGE or acid-urea gel electrophoresis as described (27). Histones acetylated at specific lysine residues were detected with a proper antibody (H4ac, Upstate 06-866; H3K9ac, Upstate 07-352). To detect total histones, the isolated histones were subjected to SDS-PAGE and the gels were then stained with colloidal blue staining kit (Invitrogen). In silico molecular docking

L002 was docked to a crystal structure of the catalytic domain of p300 (RCSB Protein Data Bank Accession Number: 3BIY) (28). All heteroatoms were removed from the .pdb file of the structure and the MSROLL program was used to generate a molecular surface for the protein. Spheres representing possible binding sites were then generated from the surface using the SPHGEN program. The spheres corresponding to the docking site of interest were manually isolated, creating a cluster of 31 spheres. A three-dimensional rectangle was formed around the selected spheres using SHOWBOX, leaving a 20-angstrom buffer on all sides of the spheres. A .mol2 file suitable for docking was generated from the .pdb file of 3BIY using Chimera. The energy environment of the area contained in the box was calculated using the GRID program. The .mol2 file of L002 was obtained from the ZINC database and DOCK6.3 was used to evaluate and score possible docking orientations based both on electrostatic interactions as well as van der Waals forces, allowing for energy minimization of the ligand. Similarly, L002 was also docked to the acetyl— CoA— binding pockets of PCAF (KAT2B, PDB accession #: 1CM0) (57), Tip60 (KAT5, PDB accession #: 20U2), and MYST1 (KAT8, PDB accession #: 3TOB) (58). All programs are part of the DOCK suite developed by UCSF.

RESULTS

uHTS campaign for identifying p300 inhibitors that selectively kills TNBC cells

The purpose of this screen was to identify novel compounds that selectively promote cell death of TNBC cells through inhibiting acetyltransferase p300. The primary screen was to identify compounds that are toxic to TNBC cell line MDA-MB-231 but not to HMEC. Compounds that satisfied this criterion were then tested to inhibit p300 acetyltransferase activity in an in vitro biochemical assay. The original hypothesis was that inhibition of p300 might potentiate HDACi-mediated cytotoxicity based on the initial observation that shR A- mediated knockdown of p300 and CBP sensitized HDACi to kill MDA-MB-231 cells (data not shown). Thus, the primary screen was conducted in the presence of 1.5 μΜ of SAHA (vorinostat), which induced 12% growth inhibition for MDA-MB-231 cells (CC₁₂), in order to discover HDACi "potentiators". Each compound in the Scripps Drug Discovery library (n = 622,079 compounds) was screened as a single point, single dose at 6.2 μΜ in the MDA- MB-231 cytotoxicity assay with the presence of 1.5 μΜ of SAHA. The primary screen exhibited a Z' score of 0.82. The hit-cutoff, established using an average plus three-fold standard deviation calculation method, was 30.01%. This cutoff yielded a total of 6,471 primary hits at a 1.04% hit-rate. From these hits, 6,400 compounds exhibiting the highest inhibition values were selected for secondary screening. Eight compounds were not available; therefore 6,392 were included in further testing. These compounds were retested in the "potentiator" confirmation assay in the presence of 1.5 μΜ of SAHA against MDA-MB-231 in triplicate at a single dose (6.2 μΜ). They were also tested in counterscreens against MDA- MB-231 and HMEC cells in the absence of SAHA. The vast majority of compounds exhibited similar inhibitory activities in assays in the presence or absence of SAHA, suggesting that few "potentiators" could be uncovered in the screen.

Of the 6392 compounds emerging from the primary/confirmation screens, 640 compounds were chosen based on selectivity against MDA-MB-231 cells in comparison to activity vs. HMEC for dose-response assays. These compounds were tested in 10- concentration titration assays in triplicate. Furthermore, the 640 compounds were also tested for inhibiting p300 enzymatic activity in vitro using a fluorescence-based assay (see Materials and Methods section). The CC₅₀ cytotoxicity values of the 640 compounds vs. MDA-MB-231 in the presence or absence of SAHA and HMEC as well as IC₅₀ of p300 inhibition were determined. Of these 640 compounds, ten exhibited selectivity against MDA- MB-231 (CC₅₀ < 10 μΜ vs. MDA-MB-231 and > 10 μΜ vs. HMEC), while inhibited p300 in vitro with IC₅₀ < 10 μΜ (Figure 1). Effects of hit compounds on histone acetylation in cell-based assays

The final hit compounds were tested for their effects on histone acetylation in cell- based assays. MDA-MB-231 cells were exposed to a test compound at 30 μΜ for 2 h. Cells were also treated with TSA one hour after the addition of a test compound to inhibit deacetylation. Data presented in Figure 2 show that hit #2 (L002, ChemBridge ID 6625948 whose chemical structure is shown in Fig. 2A) inhibited acetylation of histone H4 with much smaller effects on acetylation of histone H3 at lysine 9 (H3K9; Figure 2B, lane 6). In contract, hit #1 (ChemBridge ID 6743374) had no effects on H4 or H3K9 acetylation (Fig. 2B lane 4), despite the observation that both compounds exhibited potent in vitro inhibition of p300 with ICso of 1.98 μΜ for L002 and 0.78 μΜ for hit #1 (Fig. 2A and data not shown). Additionally, L002 but not hit#l inhibited acetylation of H3K18 and H2BK12 in MDA-MB- 231 cells. Furthermore, the effects of several hit compounds on histone acetylation in HCT116 cells were tested. L002 inhibited acetylation of H4, H3K18 and H2BK12, whereas hit #1 and two additional hits, #3 (Chembridge ID 5473210) and #4 (Chembridge ID 5861253), did not obviously affected histone acetylation at these sites (data not shown). Since p300 has been shown to mediate the specific acetylation examined in these experiments including H2BK12ac, H3K18ac and H4ac (acetylation at K5/K8/K12/K16) (29), these cell- based assays provide a validation for inhibitory effects of L002 on p300. Interestingly, anacardic acid (AA), a known inhibitor of p300 and other histone acetyltransferases (30), moderately reduced H2BK12ac, but seemed to have no obvious effects on H3K18ac and H4ac. These results suggest that L002 might be a more potent acetyltransferase inhibitor than AA. L002 selectively induces cytotoxicity to TNBC cells

Compared to HMEC, TNBC MDA-MB-231 cells were highly sensitive to L002 (Fig. 2A). To further validate the selectivity of L002 against TNBC cell lines, the sensitivity of several cancer cell lines including three TNBC cell lines (MDA-MB-231 , MDA-MB-436 and MDA-MB-468) to L002 was tested. As shown in Table 1 and Figure 3, TNBC cell lines were much more susceptible to treatments with L002 compared to normal mammary epithelial cells (HMEC) and luminal subtype BC cell line MCF7. The CC₅₀ of L002 against TNBC cell lines were approximately 8 to 20-fold lower than those vs. MCF7 (Table 1). Additionally, colon cancer cell line HCT116 and prostate cancer cell line DU145 were also more resistant to L002 than TNBC cell lines.

To further assess the differential sensitivity of TNBC cell line MDA-MB-231 and

MCF7 to L002, the impact of L002 treatment on clonal growth of these two cell lines was examined. As shown in Figure 3, in the presence of 1 μΜ of L002, the clonal growth of MDA-MB-231 cells were completely suppressed, whereas colonies of MCF7 cells were not affected, compared to the control (DMSO-treated cells). For MCF7 cells, significant number of colonies was survived at 5 μΜ of L002, although no colonies were detected at 10 μΜ (Fig. 3B).

The effects of L002 treatment on cell cycle profiles of MDA-MB-231 and MCF7 cells were also assessed. As shown in Figure 4, compared to treatment with DMSO, exposure of MDA-MB-231 to L002 increased cell numbers in the Gl phase of the cell cycle with concomitant reduction of the percentage of cells in the S phase. The proportion of cells with sub-Gl DNA content, indicative of cells undergoing apoptosis, was higher in MDA-MB-231 cells exposed to L002 compared to those treated with DMSO. In contrast, L002 had little or no effects on the cell cycle profile of MCF7 compared to those exposed to DMSO (Fig. 4). Taken together, these results indicated that L002 appears to induce Gl arrest and apoptosis in TNBC cells as a potential mechanism of action.

Initial structure-activity relationship analysis

Six analogs of L002 were identified in the ChemBridge online catalog (Fig. 5). These structurally related compounds were tested for inhibiting p300 in vitro using the HotSpot HAT assays, in which radiolabeled acetyl-CoA and histone H3 were used as substrates. Among these compounds, L001, L005 and L006 were more potent, whereas L003 and L004 were about 2-fold less potent, compared to L002 (Fig. 5). L002, L003 and L004 all contain the methoxyphenyl group, while the other three more potent compounds lack the methoxyl moiety or have a methyl group at the corresponding position, suggesting that the absence of the methoxyl moiety might enhance p300 inhibitory potency. Nonetheless, these compounds exhibited similar potency in suppressing the survival of TNBC and other cancer cell lines (data not shown), possibly reflecting their largely similar IC₅₀ (within one order of magnitude) in inhibiting p300's enzymatic activity. These data provide further validation of the activity of L002 and its analogs in inhibiting p300. Importantly, the common structural scaffold of these compounds may serve as a starting point for further improvement of the inhibitory potency against p300. In silico docking of L002 to the crystal structure of the catalytic domain of p300

To assess whether L002 could dock to the active site of the catalytic domain of p300, the chemical structure of L002 was fitted to a crystal structure of p300 catalytic domain (28). In an energy-minimized model of L002-p300 complex, an oxygen atom in the sulfonyi moiety of L002 potentially forms two hydrogen bonds with the side chain of R1410 in the helix a3 of the p300 catalytic domain (Fig, 6). R1410 of p300 was shown to form hydrogen bonds with the synthetic bisubstrate p300 inhibitor Lys-CoA in its co-crystal structure with the p300 catalytic domain (28), and was also proposed to form similar hydrogen bonds with the recently identified p300 mhibitor C646 (31), Additionally, a possible hydrogen bond might also form between the oxygen linked to the nitrogen atom of the imine bond in L002 and the side chain of Q1455 from the LI loop of the p300 catalytic domain (Fig, 6). In general, L002 fits quite nicely into the acetyl-CoA-binding pocket (Fig. 6B and C), making key contacts with residues in helices a3, a4 and the LI loop. L002 could also be docked to the acetyl-Co.A -binding pockets of other acetyltratisferases with the docking scores of -47.3 (PCAF), -43 (p300), -34,5 (KAT5/Tip60), and -17 (MYST1) (the lower the scores, the higher the possible inhibitor-enzyme affinity). These docking scores correlate well with the inhibition data shown in Table 2. Discussion

The goal was to screen for compounds that inhibit p30Q and selectively kills TNBC cells. One lead compound, LG02, exhibited an IC₅₀ of 1.97 μΜ based on an in vitro HAT assay (Fig. 2), and preferentially suppressed cell proliferation of TNBC ceil lines compared to luminal BC cell line MCF7 and HMEC (Figs 3 and 4 and Table 1). Cell-based assays indicated that L002 could inhibit histone acetylation (Fig. 2).

A number of chemical inhibitors of p300 and CBP have been identified. These compounds include natural products curcumin (32), garcino! (33), and anacardic acid (30). These natural products exhibited moderate inhibitory potency and structure-activity relationship (SAR)-based approaches to improve selectivity and potency are quite challenging due to their complex chemical structures. A FITS of 69,000 compounds identified isothiazolones as inhibitors of PCAF and other acetyltransferases (34). These compounds appear to form covalent adducts with their targets and seem to induce irreversible inhibition and toxicity (34), thus limiting their potential applications. A series of analogs of the synthetic bisubstrate HAT inhibitor Lys-CoA have been described. These synthetic compounds are highly potent in inhibiting acetyltransferases (35), but they have relatively large molecular weights and are poorly cell-permeable. The most potent p300 inhibitor identified thus is the small molecule compound C646 (31). This compound inhibits p300 at submicromolar potency. Strong inhibition of the enzymatic activity of p300 by C646 has been demonstrated in diver's applications (36, 37). Nonetheless, C646 appears to be inactivated in the presence of serum (38). Furthermore, the requirement of the nitroaromatic moiety for C646 to inhibit p300 (31) might limit its in vivo application due to hepatotoxicity associated with nitroaromatic compounds (39). Thus, novel and potent chemical inhibitors of p300 could broaden the appeal of such compounds for further preclinical and clinical evaluation.

L002 is structurally distinct from any known HAT inhibitors. The two ring moieties (quinone imine and methyoxyphenyl group) are connected by the suifonyl group. The two rings are not in the same plane, and this three-dimensional arrangement appears to fit well in the acetyl-CoA pocket of the p300 catalytic domain (Fig. 6). Initial SAR assessment suggests that the absence of the oxygen atom in the methyoxyl group seems to enhance the inhibitory potency (Fig. 5). Further modifications of the compound could potentially lead to a more specific and potent p300 inhibitor. Future studies should not only focus on improving target selectivity and potency of L002 against p300, but its in vivo toxicity profile and anticancer efficacy should be vigorously evaluated. Additionally, although molecular docking results suggest that L002 might bind to the acetyl-CoA site, the biochemical mechanism of the inhibition of p300 by this compound should be elucidated.

Targeting p300 might have therapeutic implications in a range of different diseases including heart malfunction (41), diabetes mellitus (42) and HIV infection (43), probably due to the fact that p300 is a pleiotropic protein that is involved in diverse biological mechanisms ranging from cell survival, proliferation, metabolic pathways and viral infection. More than 400 protein-binding partners have been described for p300 and its paralog CBP, making them among the most connected interaction "hubs" in the cells (8). p300/CBP are found in mammals and Drosophila, and their placement in the interaction hubs indicates that they have diverse roles in a multicellular organism, some of which may be essential for cell and organism viability (8). Indeed, homozygous knockout of either p300 or CBP as well as their compound heterozygous knockout causes embryonic lethality in mice (8, 44, 45). These observations indicate that pharmacological inhibition of p300 and CBP might cause unintended toxicity. Thus, therapeutic strategies based on targeting p300/CBP might be more applicable against diseases that rely on excessive p300/CBP activity. The data demonstrated that TNBC cell lines are more susceptible to L002 than MCF7 or HMECs. Thus, a therapeutic window might exist for treating TNBC with L002. Although precisely how L002 selectively kills TNBC cells require further investigation, the importance of p300 in molecular pathways that sustain CSCs in TNBC provides a plausible explanation. In particular, because of the critical role of p300 in the JAK/STAT (46) and Wnt/p-catenin pathways (47-49) that underpin CSC self-renewal, survival and proliferation (20), novel small molecule inhibitors of p300 might be more effective therapies for TNBC and other solid tumors that are still refractory to currently available chemotherapies and targeted therapies. It is anticipated that L002 as a lead p300 inhibitor will be further developed as a useful chemical probe for studying the biological function of p300 and in preclinical animal model for treating TNBC. EXAMPLE 2

MATERIALS AND METHODS

Cell-based HTS assay

All cancer cell lines were obtained from ATCC, and HMECs were purchased from Invitrogen. HTS assays were adapted to 1,536-well format. In the final protocol, 500 cells were seeded per well in 3 μΐ, of medium and cultured overnight. Each compound from the Scripps drug discovery library (n=622,079 compounds) dissolved in DMSO was added (6.2 μΜ, final concentration with 0.6% DMSO). The cells were then incubated for 72 hr. Cell viability was assayed by adding 4 μΐ, of CellTiter-Glo reagent (Promega) per well. Luminescence from each well was quantified. The effect of treatment on cell survival was calculated for each compound using the equation: % Growth inhibition = [1- (RLU Test Compound - Median RLU High Control) / (Median RLU Low Control - Median RLU High Control )]* 100, where RLU Test Compound, RLU High Control, and RLU Low Control are the luminescence intensity of wells containing a test compound, 50 μΜ SAHA (suberoylanilide hydroxamic acids), and DMSO, respectively. The average percent inhibition of each compound, standard deviation and the hit cutoff were calculated as previously described (50). p300 acetyltransferase activity assay

In vitro high-throughput p300 acetyltransferase activity assays were done using a fluorescence assay as described above. Radioactivity-based filter-binding (HotSpot) HAT assays, in which [³H]-acetyl-CoA and histone H3 were used as substrates, were done by Reaction Biology Corporation. Cell viability, cell cycle and colony formation assays

Cell viability and cell cycle assays were done as described (26). In colony formation assay, cells were seeded in 6-well plates and exposed to various concentrations of L002 for 48 h. The treated cells were trypsinized and 3,500 cells per well were reseeded. Cells were cultured with medium containing L002 at the same concentrations as in the initial treatment. Medium with a proper concentration of L002 was changed very four days until colonies appeared in about two weeks. Colonies were stained with 1% methylene blue. [³H]- Thymidine incorporation assays were performed essentially as described previously (51). Briefly, HCT116 cells were treated for 24 h with the indicated concentrations of L002. The cells were then pulsed for 2 h with [³H] -thymidine and the incorporation quantitated and normalized to that of the vehicle (DMSO) treated cells.

Histone isolation and western blotting

A near confluent culture of various cell lines was exposed to DMSO or 30 μΜ of L002 (final concentration) 2 h before cell harvest. One hour before cell harvest, cells were mock-treated or exposed to trichostatin A (TSA, 0.2 μΜ final concentration). Histones were isolated from the treated cells using acid extraction method as described previously (27). The isolated histones were separated with conventional SDS-PAGE or acid-urea gel electrophoresis as described (27). Total histones were assessed by SDS-PAGE and gel- staining with colloidal blue (Invitrogen).

Histones acetylated at specific lysine residues were detected with a proper antibody (H4ac, Upstate 06-866; H3K9ac, Upstate 07-352). Other antibodies used in this study include those from Santa Cruz Biotechnology (anti-p53, DO-1, SC-126; anti-pSTAT3, B-7, SC-8059; anti-STAT3, C-20, SC-482), from Epitomics (anti-p53K382ac, 2485-1; JAK2, 2863-1; PCNA, 2714-1; anti-H2BK12ac, 755-1), from BD Biosciences (anti-Hsp60, H99020; anti- p21, 556431), from Abeam (anti-H3K18ac, abl l91), from Cell Signaling Technology (anti- pSTAT3, 9145), and from Upstate Biotechnology (anti-H3K4me3, 07-473).

In vivo efficacy of L002 against TNBC xenograft

MDA-MB-468 cells (5 x 10⁶) in 50 μΐ of PBS were mixed with 50 μΐ Matrigel, which was injected into the flank of a female NU/NU mouse (5-7 week old, Charles River). Palpable tumors developed in two to three weeks. Tumor dimensions were measured with a digital caliper. Tumor volume was calculated with the formula (W² x L) x 0.5, where W is the width and L the length of a tumor (W < L). When tumor volumes reached -100 mm³, mice were divided into three treatment cohorts (n=5): no treatment (group 1), DMSO (group 2) or L002 (group 3) at 0.5 mg in 100 μΐ DMSO per injection intraperitoneally (IP) twice weekly for three weeks. Tumor-bearing mice were monitored for two additional weeks after treatment termination. Data are shown as means ± SD. The two-tailed Student's t test was used to compare differences between treatment groups. The differences were considered statistically significant if P < 0.05. The animal protocol was approved by the University of Florida IACUC.

RESULTS

In vitro activity of L002 and its analogs

One compound (L002, ChemBridge ID 6625948) exhibited potent inhibition of the p300 catalytic domain in vitro with an IC₅₀ of 1.98 μΜ. Notably, at high concentrations, L002 completely suppressed the catalytic activity of p300, whereas anacardic acid (AA, a known HAT inhibitor (30)) exerted only -60% inhibition at 52 μΜ (Fig. 7A). We have identified seven additional compounds structurally related to L002 (Fig. 7B). All these analogs inhibited p300 with varying potency (Table 2). Independent biochemical experiments using radioisotope HAT activity assays confirmed the inhibitory effects of these compounds on p300 (Table 2). Among these compounds, L003, L004 and L007 were least potent, whereas other compounds showed less than a 2-fold difference in IC₅₀, compared to L002 (Table 2). Structural comparison of these analogs suggests that a functional (methyl, methoxyl or bromide) group opposite to the sulfonyi moiety in the phenyl ring is largely unimportant or detrimental to p300 inhibition.

We determined selectivity of these compounds among various human acetyltransferases. CBP, a HAT closely related to p300, was inhibited by some of these analogs, and in general, they were less potent to CBP than to p300 (Table 2). L002 also inhibited PCAF and GCN5, two proteins of GNAT (GCN5 -related N-acetyltransferase) family, while other compounds were much less effective (Table 2). In contrast, these compounds displayed no inhibition against the MYST family of HATs (KAT5 (Tip60), KAT7 (MYST2) and KAT6B (MYST4), Table 2).

To further assess target specificity of L002, we tested it against a panel of HDACs and histone methyltransferases (HMTs). L002 did not inhibit HDAC1 (class I), HDAC6 (class lib), and HDAC11 (class IV) (Fig. 13). Similarly, L002 did not display inhibitory effects against a panel of 8 diverse HMTs (DOT1, EZH1, G9a, PRMT1, SETD2, SET7-9, SMYD2, and SUV39H2; see Fig. 14). Of note, L002 was not flagged as a promiscuous inhibitor in HTS assays. It emerged as a hit only in this HTS assay among 25 HTS campaigns. Thus, L002 is a specific inhibitor of acetyltransferases.

L002 inhibits p300-mediated acetylation in cell-based assays

HTS hit compounds were tested for their effects on p300-mediated cellular mechanisms. MDA-MB-231 cells were exposed to a test compound at 30 μΜ for 2 h, and then treated with TSA one hour after the addition of a test compound to inhibit deacetylation. Data presented in Fig. 8 show that L002 inhibited acetylation of histone H4 with a much smaller effect on acetylation of histone H3 at lysine 9 (H3K9; Fig. 8A, lane 6). In contrast, hit#l (ChemBridge ID 6743374) had no effects on H4 or H3K9 acetylation (Fig. 8B, lane 4), despite the observation that both compounds exhibited potent in vitro inhibition of p300 with ICso of 1.98 μΜ for L002 and 0.78 μΜ for hit#l (data not shown). Additionally, L002 but not hit#l inhibited acetylation of H3K18 and H2BK12 in MDA-MB-231 cells (Fig. 12). Furthermore, we tested the effects of several hit compounds on histone acetylation in HCT116 cells. As shown in Fig. 12B, L002 inhibited acetylation of H4, H3K18 and H2BK12, whereas hit#l and two additional hits, #3 (Chembridge ID 5473210) and #4 (Chembridge ID 5861253), did not obviously inhibit histone acetylation at these sites. Since p300 has been shown to mediate the specific acetylation examined in these experiments including H2BK12ac, H3K18ac and H4ac (acetylation at K5/K8/K12/K16) (52), these cell- based assays provide a validation for inhibitory effects of L002 on p300. Interestingly, AA moderately reduced H2BK12ac, but seemed to have no obvious effects on H3K18ac and H4ac (Fig. 12B). These results suggest that L002 might be a more potent acetyltransferase inhibitor than AA, consistent with our in vitro HAT assay (Fig. 7).

In TNBC cell line MDA-MB-468, L002 markedly suppressed acetylation of histone H3 (H3ac) and H4 (H4ac) (Fig. 8A). Inhibition of histone acetylation at various lysine residues by L002 was also observed in other cancer call lines including MDA-MB-231 and HCT116 (Fig. 12). Together, these cell-based assays provide a validation for the inhibitory effects of L002 on histone acetylation mediated by p300 and other acetyltransferases. In addition to histones, p300 acetylates numerous other proteins. p53 is specifically acetylated at Lys382 by p300 (53). Acetylation at this site is elevated in response to DNA damage and other cellular stresses (54). To assess whether L002 could inhibit acetylation of p53 at Lys382 (K382ac), HCT116 cells expressing wt p53 were exposed to L002, the genotoxic drug etoposide (Etop) or a combination thereof. As shown in Fig. 8B, K382ac was readily detectable in cells exposed to 10 μΜ etoposide (lane 4). However, L002 markedly reduced the level of K382ac in a dose-dependent manner (compare lane 7, 10 and 13 with 4), suggesting again that the L002 specifically targets p300.

In addition to histones, p300 acetylates numerous other proteins. p53 is specifically acetylated at Lys382 by p300 (53). Acetylation at this site is elevated in response to DNA damage and other cellular stresses (54). To assess whether L002 could inhibit acetylation of p53 at Lys382 (K382ac), HCT116 cells were exposed to genotoxic drug etoposide. The cells were further exposed to L002, TSA or their combination. K382ac was readily detectable in cells exposed to 10 μΜ etoposide in the presence of TSA. However, L002 markedly reduced K382ac, suggesting again that the L002 specifically targets p300. Notably, L002 alone did not affect etoposide-induced p21 expression. However, L002 appeared to antagonize TSA- induced p21 expression, even when cells were exposed to etoposide, supporting the notion that L002 acts through inhibiting acetylation.

Genotoxic stress also induces p53 phosphorylation. Indeed, phosphorylation of p53 at Ser392 (p-S392), which is mediated by several kinases including PK , p38, and CK2/FACT (30), was markedly elevated in HCT116 cells exposed to etoposide (Fig. 8B). L002 did not exert any obvious inhibition of p53 Ser392 phosphorylation even at 30 μΜ when cells were exposed to2 μΜ of etoposide (compare lanes 6, 9 and 12 with 3 in the p53 p-S392 panel). In cells exposed to 10 μΜ of etoposide, slight reduction of p53 p-S392 levels was noted when cells were also treated with 10 or 30 μΜ of L002 (compare lanes 10 and 13 with 4). This is likely due to the reduced levels of total p53, as dose-dependent suppression of etoposide- induced p53 accumulation by L002 was clearly observed (see the p53 panel in Fig. 8B). These data suggest that L002 did not inhibit kinases that phosphorylate p53 S392; however, inhibition of p53 acetylation by L002 seems to compromise p53 protein stability, supporting the notion that acetylation antagonizes ubiquitin-mediated p53 degradation. Nonetheless, L002 did not affect etoposide-induced p21 expression despite attenuated p53 acetylation at Lys382 (Fig. 8B).

To further understand the cellular mechanism of action of L002, we examined the influence of L002 on STAT3 activation, which requires p300-mediated acetylation of STAT3 (17). The pancreatic cancer cell line MIA PaCa-2, in which STAT3 is constitutively active (55), was exposed to varying doses of L002 or L004. As shown in Fig. 8C, the levels of STAT3 phosphorylated at Y705 were reduced in a concentration-dependent manner by both L002 and L004, and the former seemed more potent. Collectively, these lines of evidence provide strong support for our hypothesis that inhibition of p300 is a specific cellular mechanism of action by L002.

Cytotoxicity of L002 to cancer cell lines

The differential cytotoxic effects of L002 to HMECs and MDA-MD-231 cells, as determined in the HTS assays, suggest that it selectively kills cancer cells. To further validate cytotoxicity of L002 to cancer cell lines, we tested sensitivity of a panel of cancer cell lines derived from diverse types of solid cancer to L002. As shown in Table 1 and Fig. 9, these cell lines exhibited varying sensitivity to L002. Notably, all four tested TNBC cell lines were highly susceptible to treatment with L002 with CC50 at low micromolar concentrations, whereas cell lines of luminal subtype BC were more resistant. We further examined the impact of L002 treatment on clonal growth of cancer cell lines. As shown in Fig. 9B, in the presence of 1 μΜ of L002, the clonal growth of MDA-MB-231 cells was completely suppressed, whereas colonies of MCF7 cells were unaffected, compared to the control (DMSO-treated cells). For MCF7 cells, a significant number of colonies survived at 5 μΜ of L002, although no colonies were detected at 10 μΜ (Fig. 9B). Inhibition of cell growth was reversible, as drug washout permitted recovery of treated cells (Fig. 9C). Nonetheless, MDA- MB-231 cells were killed within 24h of drug exposure (Fig. 9C).

Among these cell lines from solid tumors, SH-SY5Y (neuroblastoma) was most sensitive, whereas A549 (lung cancer) and MIA PaCa-2 (pancreatic cancer) were highly resistant to L002 (Table 3). L002 was further tested against the NCI-60 panel of human cancer cell lines. Strikingly, diverse leukemia and lymphoma cell lines were all extremely sensitive to L002 (Table 3). Among the solid tumor cell lines, most of the BC cell lines including MDA-MB-231, MCF7 and MDA-MB-435 were sensitive to L002. In contrast, cell lines of non- small cell lung and CNS cancer were largely insensitive to L002.

To explore the potential mechanism by which L002 kills cancer cells, we assessed effects of L002 treatment on cell cycle profiles of MDA-MB-231 cells. As shown in Fig. 10A, compared to treatment with DMSO, exposure of MDA-MB-231 cells to L002 increased cell numbers in the Gl phase of the cell cycle with a concomitant reduction of the percentage of cells in the S phase. The proportion of cells with sub-Gl DNA content, indicative of cells undergoing apoptosis, was higher in MDA-MB-231 cells exposed to L002 compared to those treated with DMSO. In HCTl 16 cells, L002 potently inhibited DNA replication, as measured in thymidine incorporation assays (Fig. 10B). Taken together, these results indicate that L002 can induce growth arrest and apoptosis in cancer cells to exert antiproliferative effects.

In vivo anticancer efficacy of L002

To test whether L002 could suppress tumor growth in vivo, L002 was administrated via IP to mice bearing tumor xenografts of TNBC cell line MDA-MB-468. Fig. 11A shows that L002 effectively suppressed tumor growth during systemic treatment and importantly tumors did not grow back after treatment termination. The twice-weekly dosing regimen was well tolerated, as the change of body weight was within 10% (Fig. 11B). Tumor sections from mice treated with DMSO or L002 were subjected to immunohistochemical staining with an antibody against acetylated histone H4 (H4ac). As shown in Fig. 11C, the levels of H4ac were markedly lower in tumors from mice treated with L002 in comparison to those from mice treated with DMSO. Altogether, these data represent the first in vivo proof-of-principle regarding the feasibility of acetyltransferase inhibition for treating solid malignancy.

Discussion

Here we reported the discovery of a new class of inhibitors of acetyltransferases.

These compounds share the same core chemical scaffold and potently inhibited p300 and related acetyltransferases in vitro and in cells. A number of chemical inhibitors of p300 and CBP have been identified. These compounds include natural products curcumin (32), garcinol (33), and anacardic acid (30). These natural products exhibited moderate inhibitory potency and structure-activity relationship (SAR)-based approaches to improve selectivity and potency are quite challenging due to their complex chemical structures. A HTS of 69,000 compounds identified isothiazolones as inhibitors of PCAF and other acetyltransferases (34). These compounds appear to form covalent adducts with their targets and seem to induce irreversible inhibition and toxicity (34), thus limiting their potential applications. A series of analogs of the synthetic bisubstrate HAT inhibitor Lys-CoA have been described. These synthetic compounds are highly potent in inhibiting acetyltransferases (35), but they have relatively large molecular weights and are poorly cell-permeable. The most potent p300 inhibitor identified thus far, C646, inhibits p300 at submicromolar potency (31). Inhibition of the enzymatic activity of p300 by C646 has been demonstrated in diverse applications (36, 37). Nonetheless, C646 appears to be inactivated in the presence of serum (38). Furthermore, the requirement of the nitroaromatic moiety for C646 to inhibit p300 (31) might limit its in vivo application due to hepatotoxicity associated with nitroaromatic compounds (39). Thus, novel and potent chemical inhibitors of p300 could broaden the appeal of such compounds for further preclinical and clinical evaluation.

L002 is structurally distinct from any known HAT inhibitors. The two ring moieties (quinone imine and methyoxyphenyl group) are connected by the suifonyi group. The two rings are not in the same plane, and this three-dimensional arrangement appears to fit well in the acetyl-CoA pocket of the p300 catalytic domain (Fig. 6). Initial SAR assessment suggests that the absence of the oxygen atom in the methyoxyl group enhances the inhibitory potency (Table 2). Furthermore, replacement of the methyoxy group with a bromine atom in L002 also markedly reduced the inhibitory potency against p300 (Table 2).

Interestingly, L002 and its analogs displayed differential inhibition to p300/CBP and other acetyltransferases. Most of the eight analogs showed potent inhibition to p300/CBP, whereas only L001 and L002 detectably inhibited PCAF and GCN5 (Table 2). In contrast, these compounds did not inhibit the MYST family of acetyltransferases (Table 2). Furthermore, L002 did not inhibit HDACs and a panel of diverse HMTs (Figs. 13 and 14). Although not extensively tested against kinases, L002 did not seem to impact p53 phosphorylation (Fig. 8B). The demonstrated selectivity of these compounds to different classes of acetyltransferases perhaps reflects their structural differences. Although these enzymes have divergent amino acid sequences, their catalytic domains share structural similarity in the central core associated with acetyl-CoA binding, but they also have pronounced differences (28).

Targeting p300 might have therapeutic implications in a range of different diseases including heart malfunction (41), diabetes mellitus (42) and HIV infection (43), probably due to the fact that p300 is a pleiotropic protein that is involved in diverse biological mechanisms ranging from cell survival, proliferation, metabolism and viral infection. More than 400 protein-binding partners have been described for p300/CBP, making them among the most connected interaction "hubs" in cells (8). p300/CBP are found in mammals and Drosophila, and their placement in the interaction hubs indicates that they have diverse roles in a multicellular organism, some of which may be essential for cell and organism viability (8). Indeed, homozygous knockout of either p300 or CBP as well as their compound heterozygous knockout causes embryonic lethality in mice (8, 44, 45). These observations indicate that pharmacological inhibition of p300/CBP might cause unintended toxicity. Thus, therapeutic strategies based on targeting p300/CBP might be more applicable against diseases that rely on excessive p300/CBP activity. Our data demonstrated that TNBC cell lines are more susceptible to L002 than luminal BC cell lines (Table 1). In vivo, L002 potently suppressed tumor growth of TNBC cell line MDA-MB-468 xenografts (Fig. 11). This represents the first demonstration of in vivo anticancer efficacy of acetyltransferase inhibitor using systemic drug administration. Although precisely why L002 is more potent to kill TNBC cells requires further investigation, the importance of p300 in pathways that sustain CSCs in TNBC provides a plausible explanation. In particular, because of the critical role of p300 in the JAK/STAT (46) and Wnt/p-catenin pathways (47-49) that underpin CSC self- renewal, survival and proliferation (20), chemical inhibitors of p300 might be more effective against TNBC and other cancers that are still refractory to currently available chemotherapies and targeted therapies .

Notably, among the NCI-60 panel of cancer cell lines, those derived from hematopoietic malignancies were highly sensitive to L002 (Table 3). This seems to mirror the more pronounced vulnerability of leukemias and lymphomas to agents targeting epigenetic pathways than solid cancers (56). Indeed, HDAC inhibitors SAHA (vorinostat) and romidepsin (ISTODAX) are already in clinical use for treating certain types of lymphoma. Significantly, agents targeting p300 acetyltransferase activity were effective in suppressing acute myelogenous leukemia (AML) in an ex vivo treatment approach using a mouse model (36). Since systemic administration of L002 efficiently suppressed tumor growth in vivo (Fig. 11), it is feasible to vigorously test the efficacy of L002 for treating hematopoietic malignancies using various animal models.

All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.

Note: * Cell growth inhibition did not surpass 50% at the highest concentration tested.

** Although this cell line was isolated from the pleural effusion of a patient with breast cancer, and has a gene expression profile consistent with a breast cancer origin, some studies demonstrated that it exhibits properties indicative of a melanoma origin.

(25%) If)^"5 (9%) (28%) (30%) (13%)

KAT5 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 ND ND

(4%) (2%) (-2%) (11%) (4%) (24%)

>1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 ND ND

MYST2/KAT7 (-46%) (1%) (-38%) (0%) (1%) (1%)

>1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 >1 x lO^-4 ND ND

MYST4/KAT6B (-21%) (-28%) (-18%) (-4%) (-34%) (-26%)

The compounds were tested in a 10-dose IC₅₀ mode with 3-fold serial dilution starting at 1 x 10^"4 M using the HotSpot HAT assays, in which [³H]-acetyl-CoA (at 3.08 x 10^"6 M) and histone H3 were used as substrates. When compound activity could not be fitted to an IC₅₀ curve, percent inhibition (100 % [DMSO control] - % activity at 1 x 10^"4 M of a test compound) is shown in parentheses. For L001 , L002, L004, L007 and L008, the IC₅₀ values derived from fluorescence assays are provided in parentheses. ND: not determined.

Note: * Cells were seeded in 96-well plate and 24h after seeding, cells were exposed to a single dose of 10 μΜ for 48h. Growth inhibition was determined based on the loss of total cellular proteins as measured by the binding of sulforhodamine B (SRB) dye to cellular proteins. REFERENCES

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Claims

CLAIMS I claim:

1. A method for treating cancer in a subject comprising administering an effective amount of a p300/CBP inhibitor selected from a compound of Formula I, Formula II or a compound selected from LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008:

Formula I

Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl. and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

LOOl

2. The method of claim 1, wherein the p300/CBP inhibitor is selective for cancer cells.

3. The method of claim 1, wherein the p300/CBP inhibitor is L001, L002, L003, L004, L005, L006a, L006b, L007 or L008, or an analog or derivative thereof.

4. The method of claim 1, wherein the p300/CBP inhibitor is L002, or an analog or derivative thereof.

5. The method of any one of claims 1-4, wherein the method further comprises administering radiation therapy and/or at least one additional anti-cancer agent.

6. The method of claim 5, wherein the anti-cancer agent is a chemotherapeutic agent selected from anthracyclines, platinum-based chemotherapy drugs, pyrimidine analogues, kinase inhibitors, alkylating agents, or a combination thereof.

7. The method of claim 6, wherein the chemotherapeutic agent is:

a) an anthracycline selected from doxorubicin, epirubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, carminomycin or detorubicin;

b) a platinum-based chemotherapy drug selected from carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate or satraplatin;

c) a pyrimidine analogue selected from 5-fluorouracil (5-FU), cytarabine or floxuridine;

d) an alkylating agent selected from nitrogen mustards such as cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine; nitrosourea compounds such as carmustine, lomustine, semustine and streptozotocin; busulfan; dacarbazine; procarbazine; altretamine; mitozolomide; or temozolomide; or

e) a tyrosine kinase inhibitor selected from the group consisting of sorafenib, sunitinib and imatinib.

8. The method of any one of claims 1-7, wherein the cancer is acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocydc leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immunobiastic lymphoma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, central nervous system (CNS) cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer, or breast cancer.

9. The method of claim 8, wherein the cancer is triple negative breast cancer.

10. The method of any one of claims 1-9, wherein the subject is human.

11. The method of any one of claims 1-10, wherein said compound has Formula I and:

i) R¹, R⁴, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are -H, R² and R⁵ are lower alkyl, R³ is an oxo (=0) group, m is 0-4 and n is 0-4;

ii) R¹, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iii) R¹, R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iv) R¹, R⁴, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4; or

v) R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R¹ is a lower alkyl group, m is 0-4 and n is 0-4.

12. The method of claim 11, wherein: n is 0 and m is 0; n is 0 and m is 1; n is 0 and m is 2; n is 0 and m is 3; n is 0 and m is 4; n is 1 and m is 0; n is 1 and m is 1 ; n is 1 and m is 2; n is 1 and m is 3; n is 1 and m is 4; n is 2 and m is 0; n is 2 and m is 1 ; n is 2 and m is 2; n is 2 and m is 3; n is 2 and m is 4; n is 3 and m is 0; n is 3 and m is 1 ; n is 3 and m is 2; n is 3 and m is 3; n is 3 and m is 4; n is 4 and m is 0; n is 4 and m is 1 ; n is 4 and m is 2; n is 4 and m is 3;or n is 4 and m is 4.

13. The method of claim 11, wherein said lower alkyl group is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, or cyclohexyl, all of which may be optionally substituted;

said lower alkoxy group is -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O- cyclopropyl, -O-butyl, -O-isobutyl, -0-tert-butyl, -O-cyclobutyl, -O-pentyl, -O- cyclopentyl, -O-hexyl, or -O-cyclohexyl, all of which may be optionally substituted; and n is 0 and m is 0 or 1.

14. The method of claim 13, wherein said lower alkyl groups and said lower alkoxy groups are unsubstituted.

15. The method of claim 14, wherein said lower alkyl groups are methyl, ethyl, propyl or isopropyl and said lower alkoxy groups are -O-methyl, -O-ethyl, -O-propyl or -O- isopropyl.

16. A method of inhibiting the growth, proliferation, or survival of a neoplastic cell comprising contacting the cell with an effective amount of a p300/CBP inhibitor selected from a compound of Formula I, Formula II or a compound selected from L001, L002, L003, L004, L005, L006a, L006b, L007 or L008:

Formula I

Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

17. The method of claim 16, wherein the p300/CBP inhibitor is selective for neoplastic cells.

18. The method of claim 16, wherein the p300/CBP inhibitor is L001, L002, L003, L004, L005, L006a, L006b, L007 or L008, or an analog or derivative thereof.

19. The method of claim 16, wherein the p300/CBP inhibitor is L002, or an analog or derivative thereof.

20. The method of any one of claims 16-19, wherein the method further comprises administering radiation therapy and/or at least one additional anti-cancer agent.

21. The method of claim 20, wherein the anti-cancer agent is a chemotherapeutic agent selected from anthracyclines, platinum-based chemotherapy drugs, pyrimidine analogues, kinase inhibitors, alkylating agents, or a combination thereof.

22. The method of claim 21 , wherein the chemotherapeutic agent is:

a) an anthracycline selected from doxorubicin, epirubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, carminomycin or detorubicin;

b) a platinum-based chemotherapy drug selected from carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate or satraplatin;

c) a pyrimidine analogue selected from 5-fluorouracil (5-FU), cytarabine or floxuridine; d) an alkylating agent selected from nitrogen mustards such as cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine; nitrosourea compounds such as carmustine, lomustine, semustine and streptozotocin; busulfan; dacarbazine; procarbazine; altretamine; mitozolomide; or temozolomide; or

e) a tyrosine kinase inhibitor selected from the group consisting of sorafenib, sunitinib and imatinib.

23. The method of any one of claims 16-22, wherein the cancer is acute lymphoblatic leukemia (ALL), acute myelogenous leukemia (AML), acute promyelocytie leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), acute monocytic leukemia (AMOL), hairy cell leukemia, large cell immuiioblastic lymphoma, plasmacytoma, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, brain cancer, lung cancer, central nervous system (CNS) cancer, melanoma, renal cancer, prostate cancer, colon cancer, ovarian cancer, or breast cancer.

24. The method of claim 23, wherein the cancer is triple negative breast cancer.

25. The method of any one of claims 16-24, wherein the subject is human.

26. The method of any one of claims 16-25, wherein said compound has Formula

I and:

i) R¹, R⁴, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are -H, R² and R⁵ are lower alkyl, R³ is an oxo (=0) group, m is 0-4 and n is 0-4;

ii) R¹, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iii) R¹, R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iv) R¹, R⁴, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4; or

v) R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R¹ is a lower alkyl group, m is 0-4 and n is 0-4.

27. The method of claim 26, wherein: n is 0 and m is 0; n is 0 and m is 1; n is 0 and m is 2; n is 0 and m is 3; n is 0 and m is 4; n is 1 and m is 0; n is 1 and m is 1 ; n is 1 and m is 2; n is 1 and m is 3; n is 1 and m is 4; n is 2 and m is 0; n is 2 and m is 1 ; n is 2 and m is 2; n is 2 and m is 3; n is 2 and m is 4; n is 3 and m is 0; n is 3 and m is 1 ; n is 3 and m is 2; n is 3 and m is 3; n is 3 and m is 4; n is 4 and m is 0; n is 4 and m is 1 ; n is 4 and m is 2; n is 4 and m is 3;or n is 4 and m is 4.

28. The method of claim 27, wherein said lower alkyl group is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, or cyclohexyl, all of which may be optionally substituted;

said lower alkoxy group is -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O- cyclopropyl, -O-butyl, -O-isobutyl, -0-tert-butyl, -O-cyclobutyl, -O-pentyl, -O- cyclopentyl, -O-hexyl, or -O-cyclohexyl, all of which may be optionally substituted; and n is 0 and m is 0 or 1.

29. The method of claim 28, wherein said lower alkyl groups and said lower alkoxy groups are unsubstituted.

30. The method of claim 29, wherein said lower alkyl groups are methyl, ethyl, propyl or isopropyl and said lower alkoxy groups are -O-methyl, -O-ethyl, -O-propyl or -O- isopropyl.

31. The method of claims 16-30, wherein said method is performed in vitro.

32. A pharmaceutical composition comprising a pharmaceutically acceptable carrier, a chemotherapeutic agent and a compound of Formula I, Formula II or a compound selected from L001, L002, L003, L004, L005, L006a, L006b, L007 or L008:

R⁹ R¹⁰ R¹ R²

Formula I Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl. In certain embodiments, R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), lower alkyl-, lower alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or lower perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,

or

33. The pharmaceutical composition of claim 32, wherein the p300/CBP inhibitor is LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008, or an analog or derivative thereof.

34. The pharmaceutical composition of claim 32, wherein the p300/CBP is L002, or an analog or derivative thereof.

35. The pharmaceutical composition of claim 32, wherein the anti-cancer agent is a chemotherapeutic agent selected from anthracyclines, platinum-based chemotherapy drugs, pyrimidine analogues, kinase inhibitors, alkylating agents, or a combination thereof.

36. The pharmaceutical composition of claim 32, wherein the chemotherapeutic agent is:

a) an anthracycline selected from doxorubicin, epirubicin, daunorubicin, aclarubicin, idarubicin, amrubicin, pirarubicin, valrubicin, zorubicin, carminomycin or detorubicin;

b) a platinum-based chemotherapy drug selected from carboplatin, cisplatin, nedaplatin, oxaliplatin, triplatin tetranitrate or satraplatin;

c) a pyrimidine analogue selected from 5-fluorouracil (5-fu), cytarabine or floxuridine;

d) an alkylating agent selected from nitrogen mustards such as cyclophosphamide, chlorambucil, uramustine, ifosfamide, melphalan and bendamustine; nitrosourea compounds such as carmustine, lomustine, semustine and streptozotocin; busulfan; dacarbazine; procarbazine; altretamine; mitozolomide; or temozolomide; or

e) a tyrosine kinase inhibitor selected from the group consisting of sorafenib, sunitinib and imatinib.

37. The pharmaceutical composition of claims 32-36, wherein said compound has Formula I and:

i) R¹, R⁴, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are -H, R² and R⁵ are lower alkyl, R³ is an oxo (=0) group, m is 0-4 and n is 0-4;

ii) R¹, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iii) R¹, R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iv) R¹, R⁴, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4; or

v) R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R¹ is a lower alkyl group, m is 0-4 and n is 0-4.

38. The pharmaceutical composition of claim 37, wherein: n is 0 and m is 0; n is 0 and m is 1 ; n is 0 and m is 2; n is 0 and m is 3; n is 0 and m is 4; n is 1 and m is 0; n is 1 and m is 1 ; n is 1 and m is 2; n is 1 and m is 3; n is 1 and m is 4; n is 2 and m is 0; n is 2 and m is 1 ; n is 2 and m is 2; n is 2 and m is 3; n is 2 and m is 4; n is 3 and m is 0; n is 3 and m is 1 ; n is 3 and m is 2; n is 3 and m is 3; n is 3 and m is 4; n is 4 and m is 0; n is 4 and m is 1 ; n is 4 and m is 2; n is 4 and m is 3;or n is 4 and m is 4.

39. The pharmaceutical composition of claim 37, wherein said lower alkyl group is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, or cyclohexyl, all of which may be optionally substituted;

said lower alkoxy group is -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O- cyclopropyl, -O-butyl, -O-isobutyl, -0-tert-butyl, -O-cyclobutyl, -O-pentyl, -O- cyclopentyl, -O-hexyl, or -O-cyclohexyl, all of which may be optionally substituted; and n is 0 and m is 0 or 1.

40. The pharmaceutical composition of claim 39, wherein said lower alkyl groups and said lower alkoxy groups are unsubstituted.

41. The pharmaceutical composition of claim 40, wherein said lower alkyl groups are methyl, ethyl, propyl or isopropyl and said lower alkoxy groups are -O-methyl, -O-ethyl, -O-propyl or -O-isopropyl.

42. A method of treating a disease or disorder comprising the administration of a therapeutically effective amount of a compound of Formula I, Formula II or a compound selected from LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008 to a subject having a disease or disorder responsive to inhibition of p300, PCAF, GCN5 and/or CBP:

R⁹ R 10 R¹ R²

Formula I Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

43. The method of claim 42, wherein the inhibitor is LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008, or an analog or derivative thereof.

44. The method of claim 42, wherein the inhibitor is L002, or an analog or derivative thereof.

45. The method of any one of claims 42-44, wherein the subject is human.

46. The method of any one of claims 42-45, wherein said compound has Formula

I and:

i) R¹, R⁴, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are -H, R² and R⁵ are lower alkyl, R³ is an oxo (=0) group, m is 0-4 and n is 0-4;

ii) R¹, R⁵, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4; iii) R¹, R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4;

iv) R¹, R⁴, R⁶, R⁷, R⁹ and R¹⁰ are -H, R² and R⁴ are lower alkyl, R³ is an oxo (=0) group, R⁸ is a lower alkoxy group or an alkoxy group, m is 0-4 and n is 0-4; or

v) R², R⁴, R⁵, R⁶ R⁷, R⁹ and R¹⁰ are -H, R³ is an oxo (=0) group, R¹ is a lower alkyl group, m is 0-4 and n is 0-4.

47. The method of claim 46, wherein: n is 0 and m is 0; n is 0 and m is 1; n is 0 and m is 2; n is 0 and m is 3; n is 0 and m is 4; n is 1 and m is 0; n is 1 and m is 1 ; n is 1 and m is 2; n is 1 and m is 3; n is 1 and m is 4; n is 2 and m is 0; n is 2 and m is 1 ; n is 2 and m is 2; n is 2 and m is 3; n is 2 and m is 4; n is 3 and m is 0; n is 3 and m is 1 ; n is 3 and m is 2; n is 3 and m is 3; n is 3 and m is 4; n is 4 and m is 0; n is 4 and m is 1 ; n is 4 and m is 2; n is 4 and m is 3;or n is 4 and m is 4.

48. The method of claim 46, wherein said lower alkyl group is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, pentyl, cyclopentyl, hexyl, or cyclohexyl, all of which may be optionally substituted;

said lower alkoxy group is -O-methyl, -O-ethyl, -O-propyl, -O-isopropyl, -O- cyclopropyl, -O-butyl, -O-isobutyl, -0-tert-butyl, -O-cyclobutyl, -O-pentyl, -O- cyclopentyl, -O-hexyl, or -O-cyclohexyl, all of which may be optionally substituted; and n is 0 and m is 0 or 1.

49. The method of claim 48, wherein said lower alkyl groups and said lower alkoxy groups are unsubstituted.

50. The method of claim 49, wherein said lower alkyl groups are methyl, ethyl, propyl or isopropyl and said lower alkoxy groups are -O-methyl, -O-ethyl, -O-propyl or -O- isopropyl.

51. The method of any one of claims 42-50, wherein said disease or disorder is associated with excessive activity of p300, PCAF, GCN5 and/or CBP.

52. The method of any one of claims 42-50, wherein said disease or disorder is HIV infection, infectious disease, heart disease, diabetes mellitus, inflammation or airway inflammation.

53. Use of a compound selected from Formula I, Formula II or a compound selected from LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008 for the treatment of cancer in a subject comprising administering an effective amount of a compound of:

Formula I

Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or a compound selected from LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008.

54. The use according to claim 53, further comprising the use of a chemotherapeutic agent or radiation for the treatment of said cancer.

55. Use of a compound selected from Formula I, Formula II or a compound selected from LOOl, L002, L003, L004, L005, L006a, L006b, L007 or L008 for the treatment of a disease or disorder responsive to inhibition of p300, PCAF, GCN5 and/or CBP in a subject comprising administering an effective amount of a compound of:

Formula I

Formula II

wherein R¹, R², R³ (if present), R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are each, independently, selected from hydrogen (H-), alkyl-, alkoxy-, carboxyl-, carboxy esters, amine, oxo, halo, or perhaloalkyl and n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or a compound selected from L001, L002, L003, L004, L005, L006a, L006b, L007 or L008.

56. The use according to claim 55, wherein said disease or disorder is HIV infection, infectious disease, heart disease, diabetes mellitus, inflammation or airway inflammation.