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 WD repeat domain 5 inhibitors that demonstrate efficacy and safety in preclinical animal models.

WD repeat domain 5 (WDR5) is a core scaffolding component of many multiprotein complexes that perform a variety of critical chromatin-centric processes in the nucleus. WDR5 is a component of the mixed lineage leukemia MLL/SET complex and localizes MYC to chromatin at tumor-critical target genes. As a part of these complexes, WDR5 plays a role in sustaining oncogenesis in a variety of human cancers that are often associated with poor prognoses. Thus, WDR5 has been recognized as an attractive therapeutic target for treating both solid and hematological tumors. Previously, small-molecule inhibitors of the WDR5-interaction (WIN) site and WDR5 degraders have demonstrated robust in vitro cellular efficacy in cancer cell lines and established the therapeutic potential of WDR5. However, these agents have not demonstrated significant in vivo efficacy at pharmacologically relevant doses by oral administration in animal disease models. We have discovered WDR5 WIN-site inhibitors that feature bicyclic heteroaryl P7 units through structure-based design and address the limitations of our previous series of small-molecule inhibitors. Importantly, our lead compounds exhibit enhanced on-target potency, excellent oral pharmacokinetic (PK) profiles, and potent dose-dependent in vivo efficacy in a mouse MV4:11 subcutaneous xenograft model by oral dosing. Furthermore, these in vivo probes show excellent tolerability under a repeated high-dose regimen in rodents to demonstrate the safety of the WDR5 WIN-site inhibition mechanism. Collectively, our results provide strong support for WDR5 WIN-site inhibitors to be utilized as potential anticancer therapeutics.

Discovery of Potent Orally Bioavailable WD Repeat Domain 5 (WDR5) Inhibitors Using a Pharmacophore-Based Optimization.

WD repeat domain 5 (WDR5) is a nuclear scaffolding protein that forms many biologically important multiprotein complexes. The WIN site of WDR5 represents a promising pharmacological target in a variety of human cancers. Here, we describe the optimization of our initial WDR5 WIN-site inhibitor using a structure-guided pharmacophore-based convergent strategy to improve its druglike properties and pharmacokinetic profile. The core of the previous lead remained constant while a focused SAR effort on the three pharmacophore units was combined to generate a new in vivo lead series. Importantly, this new series of compounds has picomolar binding affinity, improved cellular antiproliferative activity and selectivity, and increased kinetic aqueous solubility. They also exhibit a desirable oral pharmacokinetic profile with manageable intravenous clearance and high oral bioavailability. Thus, these new leads are useful probes toward studying the effects of WDR5 inhibition.

Discovery and Structure-Based Optimization of Potent and Selective WD Repeat Domain 5 (WDR5) Inhibitors Containing a Dihydroisoquinolinone Bicyclic Core.

WD repeat domain 5 (WDR5) is a member of the WD40-repeat protein family that plays a critical role in multiple chromatin-centric processes. Overexpression of WDR5 correlates with a poor clinical outcome in many human cancers, and WDR5 itself has emerged as an attractive target for therapy. Most drug-discovery efforts center on the WIN site of WDR5 that is responsible for the recruitment of WDR5 to chromatin. Here, we describe discovery of a novel WDR5 WIN site antagonists containing a dihydroisoquinolinone bicyclic core using a structure-based design. These compounds exhibit picomolar binding affinity and selective concentration-dependent antiproliferative activities in sensitive MLL-fusion cell lines. Furthermore, these WDR5 WIN site binders inhibit proliferation in MYC-driven cancer cells and reduce MYC recruitment to chromatin at MYC/WDR5 co-bound genes. Thus, these molecules are useful probes to study the implication of WDR5 inhibition in cancers and serve as a potential starting point toward the discovery of anti-WDR5 therapeutics.

The efficacy and cardiac evaluation of aminomethyl tetrahydronaphthalene ketopiperazines: a novel class of potent MCH-R1 antagonists.

The design, synthesis, and biological studies of a novel class of MCH-R1 antagonists based on an aminotetrahydronaphthalene ketopiperazine scaffold is described. Compounds within this class promoted significant body weight reduction in mouse diet induced obesity studies. The potential for hERG blockage activity and QT interval studies in anesthetized dogs are discussed.

Aminomethyl tetrahydronaphthalene biphenyl carboxamide MCH-R1 antagonists--Increasing selectivity over hERG.

Aminomethyl tetrahydronaphthalene biphenyl carboxamide MCH-R1 antagonists with greater selectivity over hERG were identified. SAR studies addressing two distinct alternatives for structural modifications leading to improve hERG selectivity are described.

Aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists--Increasing selectivity over hERG.

A direct correlation between hERG binding and QTc prolongation was established for a series of aminomethyl tetrahydronaphthalene ketopiperazine MCH-R1 antagonists. Compounds within this class with greater selectivity over hERG were developed. Compound 4h proved to have the best profile, with MCH-R1 Ki = 16 nm and hERG IC50 = 25 microM.

Novel pyrazolopiperazinone- and pyrrolopiperazinone-based MCH-R1 antagonists.

The synthesis and biological testing of novel classes of potent melanin-concentrating hormone (MCH-R1) antagonists based on pyrazolopiperazinone and pyrrolopiperazinone scaffolds are described.

Identification of substituted 4-aminopiperidines and 3-aminopyrrolidines as potent MCH-R1 antagonists for the treatment of obesity.

A substituted 4-aminopiperidine was identified as showing activity in an MCH assay from an HTS effort. Subsequent structural modification of the scaffold led to the identification of a number of active MCH antagonists. 3,5-Dimethoxy-N-(1-(naphthalen-2-ylmethyl)piperidin-4-yl)benzamide (5c) was among those with the highest binding affinity to the MCH receptor (K(i)=27nM), when variations were made at benzoyl and naphthylmethyl substitution sites from the initial HTS hit. Further optimization via piperidine ring contraction resulted in enhanced MCH activity in a 3-aminopyrrolidine series, where (R)-3,5-dimethoxy-N-(1-(naphthalen-2-ylmethyl)-pyrrolidin-3-yl)benzamide (10i) was found to be an excellent MCH antagonist (K(i)=7nM).

Design and synthesis of substituted quinolines as novel and selective melanin concentrating hormone antagonists as anti-obesity agents.

A novel series of substituted quinoline analogs were designed and synthesized as potent and selective melanin concentrating hormone (MCH) antagonists. These analogs show potent (nM) activity (12a-k) with a moderate selectivity. Conversely, the conformationally constrained thienopyrimidinone analogs (18a-g) showed improved activity in MCH-1R and selectivity over 5HT2C.

Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation.

OBJECTIVE: To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. SAMPLE POPULATION: Blood samples from 6 Thoroughbreds. PROCEDURE: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. RESULTS: In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours. CONCLUSIONS: Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.

Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody.

OBJECTIVE: To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. SAMPLE POPULATION: Platelets obtained from 6 Thoroughbreds. PROCEDURE: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion. RESULTS: Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817-activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of equine platelets can be detected by use of fluorescent-labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders.

Normal and perturbed endothelial cells from canine femoral arteries and femoral veins exhibit heterogeneity in hemostatic properties and growth characteristics.

BACKGROUND: We sought to examine the heterogeneity of endothelial cells from the same anatomic site but different vascular systems and described von Willebrand factor (vWF) release and morphological change in response to injury-associated factor in femoral vessels from canine in vitro. METHODS: Levels of hemostatic factors (vWF, plasminogen activator inhibitor type 1(PAI-1), antithrombin III (ATIII), in tissue sections and cultured endothelial cells of canine femoral arteries and canine femoral veins were compared by the immunohistochemistry technique. In addition to comparing cell growth density and cell protein contents, cultured femoral arterial endothelial cells (FAECs) and cultured femoral venous endothelial cells (FVECs) were incubated with a series concentration of basic fibroblast factor (bFGF) (1, 10, 100 ng/ml) for up to 48 hours to test the amount of vWF secretion and morphological change. RESULTS: Both in tissue sections and cultured cells, the levels of vWF are higher in FVECs than in FAECs. We were unable to differentiate the level of PAI-1 and ATIII difference between FAECs and FVECs. bFGF (10 ng/ml) significantly increased vWF secretion from cultured FAECs but not from FVECs. The size of cultured FAECs is smaller than of FVECs; however, FAECs have higher amounts of protein contents than FVECs. CONCLUSIONS: These comparative studies provide evidence indicating that the characteristics of FVECs differ from those of FAECs. These differences may be indicated heterogeneity with either inherited or acquired thrombotic disease.

Anti-A isoagglutinins in two blood type B cats are IgG and IgM.

Sera from two blood type B cats had strong isoagglutinating and isohemolyzing titers against blood type A erythrocytes. In order to determine the class of the immunoglobulins, sera from the cats were pooled, ammonium sulfate precipitated, and gel filtered using sepharose 6B to separate the immunoglobulins by molecular size. The immunoglobulin concentrate separated into two fractions. The initial part of the first fraction was shown in an ELISA to contain IgM and to be devoid of IgG by immunoelectrophoresis and to have agglutinating activity (a titer of 1:4 with a protein concentration of 0.8 mg/ml). Treating this fraction with the reducing agent dithiothreitol (DTT) eliminated agglutinating activity. The latter portion of the second column fraction was shown to contain IgG by immunoelectrophoresis, and to be devoid of IgM by ELISA. Agglutinating activity was also present in the second fraction (a titer of 1:2 with a protein concentration of 1.9 mg/ml); hemagglutinating activity was not decreased by DTT treatment. These studies show that the predominant anti-A isoagglutinating activity in pooled sera from two blood type B cats is IgM and that some isoagglutinin activity can be associated with IgG.