Additional file 1: Figure S1. of The Yin/Yan of CCL2: a minor role in neutrophil anti-tumor activity in vitro but a major role on the outgrowth of metastatic breast cancer lesions in the lung in vivo

Naïve BALB/c neutrophils can kill PyMT (FVB) tumor cells, but CCL2 does not increase killing. PyMT cells from FVB mice seeded with and without naïve BALB/c neutrophils (30 neutrophils: 1 tumor cell), in the absence and presence of CCL2. After 18-h at 37 °C, cells were lysed and luciferase was measured to determine tumor cell killing. Although from a different mouse strain, naïve BALB/c neutrophils were able to kill FVB PyMT tumor cells (p = 0.005). However, CCL2 did not enhance this effect (p = 0.347), Kruskal-Wallis test with Dunn’s test for multiple comparisons. Values are graphed as mean ± SD. Figure S2. Entrainment properties of less aggressive PyMT tumor cells on the metastatic outgrowth of more aggressive TGFβR2 knock out PyMT tumors. Female FVB mice (10 weeks old) were injected with either 15,000 PyMT breast cancer cells (MFP) or PBS (Non-tumor bearing) in the 4th mammary fat pad. Two weeks later either 1 × 106 TGFβR2 knockout PyMT (TbR2KO) breast cancer cells or PBS alone (in 200 μl) were delivered by tail vein injection to mice bearing PyMT tumors or into non-tumor bearing mice (t.v. TbR2KO). Three weeks later, mice were sacrificed and lungs were removed, fixed, H&E stained and the number of metastases counted. Analysis of variance with blocking (two experiments) was performed for an overall comparison (p < 0.001). Tukey’s honestly significant difference (HSD) for multiple comparisons among groups (adj. p = 0.009 for MFP-PBS vs. MFP + TbR2KO, adj. p < 0.001 for MFP-PBS vs. t.v. TbR2KO). NS = not significant, p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001. Values are graphed as mean ± SD. Figure S3. Intranasal delivery of CCL2 facilitates the recruitment of leukocytes into BAL fluid. 3A. BAL fluid isolated from mice receiving intranasal delivery of CCL2 showed an increase in CD8+ T cells as CCL2 delivery increased from 100 ng to 1000 ng. Data are shown as % CD45+ cells and as % total cells. 3B. BAL fluid from mice receiving intranasal delivery of CCL2 exhibited a trend toward increased numbers of neutrophils and NK cells with increasing concentrations. Data are shown as % live cells in BAL fluid. Figure S4. Blocking antibody to CCL2 reverses the neutrophil killing of 67NR cells in vitro. 67NR cells(T) expressing luciferase were seeded with and without neutrophils (E for effector cells) at a ratio of 30 neutrophils: 1 tumor cell in the presence of control IgG or blocking antibody to CCL2. After an 18-hs at 37 °C, cells were lysed and luciferase was measured to determine tumor cell killing. Anti-CCL2 (50 ng/ml BD Biosciences) reversed the 67NR tumor cell killing of BALB/c TEN, p < 0.01, Student’s t test, n = 5 per group. (PPTX 124 kb

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydro­isoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physico­chemical properties. We identified the 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5­(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physico­chemical properties compared to our best-in-class dihydro­isoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics

Structure-Based Discovery of Potent, Orally Bioavailable Benzoxazepinone-Based WD Repeat Domain 5 Inhibitors

The chromatin-associated protein WDR5 (WD repeat domain 5) is an essential cofactor for MYC and a conserved regulator of ribosome protein gene transcription. It is also a high-profile target for anti-cancer drug discovery, with proposed utility against both solid and hematological malignancies. We have previously discovered potent dihydro­isoquinolinone-based WDR5 WIN-site inhibitors with demonstrated efficacy and safety in animal models. In this study, we sought to optimize the bicyclic core to discover a novel series of WDR5 WIN-site inhibitors with improved potency and physico­chemical properties. We identified the 3,4-dihydrobenzo­[f]­[1,4]­oxazepin-5­(2H)-one core as an alternative scaffold for potent WDR5 inhibitors. Additionally, we used X-ray structural analysis to design partially saturated bicyclic P7 units. These benzoxazepinone-based inhibitors exhibited increased cellular potency and selectivity and favorable physico­chemical properties compared to our best-in-class dihydro­isoquinolinone-based counterparts. This study opens avenues to discover more advanced WDR5 WIN-site inhibitors and supports their development as novel anti-cancer therapeutics

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure–activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at submicromolar concentrations in vitro, increase levels of active RAS–GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase−extracellular regulated kinase (MAPK–ERK) pathway, resulting in a decrease in pERK1/2^T202/Y204 protein levels at higher compound concentrations

Discovery of Aminopiperidine Indoles That Activate the Guanine Nucleotide Exchange Factor SOS1 and Modulate RAS Signaling

Deregulated RAS activity, often the result of mutation, is implicated in approximately 30% of all human cancers. Despite this statistic, no clinically successful treatment for RAS-driven tumors has yet been developed. One approach for modulating RAS activity is to target and affect the activity of proteins that interact with RAS, such as the guanine nucleotide exchange factor (GEF) son of sevenless homologue 1 (SOS1). Here, we report on structure–activity relationships (SAR) in an indole series of compounds. Using structure-based design, we systematically explored substitution patterns on the indole nucleus, the pendant amino acid moiety, and the linker unit that connects these two fragments. Best-in-class compounds activate the nucleotide exchange process at submicromolar concentrations in vitro, increase levels of active RAS–GTP in HeLa cells, and elicit signaling changes in the mitogen-activated protein kinase−extracellular regulated kinase (MAPK–ERK) pathway, resulting in a decrease in pERK1/2^T202/Y204 protein levels at higher compound concentrations

Discovery of Potent 2‑Aryl-6,7-dihydro‑5<i>H</i>‑pyrrolo[1,2‑<i>a</i>]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5<i>H</i>-pyrrolo­[1,2-<i>a</i>]­imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer

Discovery of Potent 2‑Aryl-6,7-dihydro‑5<i>H</i>‑pyrrolo[1,2‑<i>a</i>]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5<i>H</i>-pyrrolo­[1,2-<i>a</i>]­imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer