Darryl Mcconnell

Potent and long-acting dimeric inhibitors of influenza virus neuraminidase are effective at a once-weekly dosing regimen.

ABSTRACT Dimeric derivatives (compounds 7 to 9) of the influenza virus neuraminidase inhibitor zanamivir (compound 2), which have linking groups of 14 to 18 atoms in length, are approximately 100-fold more potent inhibitors of influenza virus replication in vitro and in vivo than zanamivir. The observed optimum linker length of 18 to 22 Å, together with observations that the dimers cause aggregation of isolated neuraminidase tetramers and whole virus, indicate that the dimers benefit from multivalent binding via intertetramer and intervirion linkages. The outstanding long-lasting protective activities shown by compounds 8 and 9 in mouse influenza infectivity experiments and the extremely long residence times observed in the lungs of rats suggest that a single low dose of a dimer would provide effective treatment and prophylaxis for influenza virus infections.

Structure-Based Design of an in Vivo Active Selective BRD9 Inhibitor

Components of the chromatin remodelling switch/sucrose nonfermentable (SWI/SNF) complex are recurrently mutated in tumors, suggesting that altering the activity of the complex plays a role in oncogenesis. However, the role that the individual subunits play in this process is not clear. We set out to develop an inhibitor compound targeting the bromodomain of BRD9 in order to evaluate its function within the SWI/SNF complex. Here, we present the discovery and development of a potent and selective BRD9 bromodomain inhibitor series based on a new pyridinone-like scaffold. Crystallographic information on the inhibitors bound to BRD9 guided their development with respect to potency for BRD9 and selectivity against BRD4. These compounds modulate BRD9 bromodomain cellular function and display antitumor activity in an AML xenograft model. Two chemical probes, BI-7273 (1) and BI-9564 (2), were identified that should prove to be useful in further exploring BRD9 bromodomain biology in both in vitro and in vivo settings.

Microwave-assisted parallel synthesis of fused heterocycles in a novel parallel multimode reactor.

New rotor types using disposable glass vials for small-scale parallel synthesis in multimode microwave reactors are introduced. One rotor comprises 16 groups of four vials, whereas the second uses four silicon carbide plates with a 6 x 4 matrix to process the vials. Both rotors achieve utmost temperature homogeneity upon microwave irradiation and can be used for microwave-mediated reactions at temperatures of up to 200 degrees C and pressures of 20 bar. The generation of three different heterocycle libraries furnishing thiophenes, oxindoles, and benzimidazoles using the new rotor types is described.

Sensitivity and engineered resistance of myeloid leukemia cells to BRD9 inhibition

Here we show that acute myeloid leukemia (AML) cells require the BRD9 subunit of the SWI/SNF chromatin remodeling complex to sustain MYC transcription, rapid cell proliferation, and a block in differentiation. Based on these observations, we derived small-molecule inhibitors of the BRD9 bromodomain, which selectively suppressed the proliferation of mouse and human AML cell lines. To establish these effects as on-target, we engineered a bromodomain-swap allele of BRD9, which retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells conferred resistance to the anti-proliferative effects of our compound series, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. Our study provides the first evidence for a role of BRD9 in cancer and reveals a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells.

Direct NMR Probing of Hydration Shells of Protein Ligand Interfaces and Its Application to Drug Design

Fragment-based drug design exploits initial screening of low molecular weight compounds and their concomitant affinity improvement. The multitude of possible chemical modifications highlights the necessity to obtain structural information about the binding mode of a fragment. Herein we describe a novel NMR methodology (LOGSY titration) that allows the determination of binding modes of low affinity binders in the protein-ligand interface and reveals suitable ligand positions for the addition of functional groups that either address or substitute protein-bound water, information of utmost importance for drug design. The particular benefit of the methodology and in contrast to conventional ligand-based methods is the independence of the molecular weight of the protein under study. The validity of the novel approach is demonstrated on two ligands interacting with bromodomain 1 of bromodomain containing protein 4, a prominent cancer target in pharmaceutical industry.

Abstract LB-206: A bromodomain-swap allele demonstrates that on-target chemical inhibition of BRD9 limits the proliferation of acute myeloid leukemia cells

Recent studies have revealed vital roles of SWI/SNF complexes in leukemia and a variety of other cancers, making this chromatin remodeler a candidate drug target in human malignancy. Chemical modulation of SWI/SNF activity, however, remains to be achieved. Given the success of pharmacological bromodomain inhibition, we evaluated the role of bromodomain-carrying SWI/SNF subunits and identified Bromodomain-containing protein 9 (BRD9) as critical for the growth of Acute Myeloid Leukemia (AML). In AML cells, BRD9 binds the enhancer of the MYC proto-oncogene and sustains MYC transcription, rapid cell proliferation, as well as a block in differentiation. Based on these observations, we derived a small-molecule inhibitor of the BRD9 bromodomain, which partially displaces BRD9 from MYC enhancer elements and selectively suppresses the proliferation of mouse and human AML cell lines. Given the known role of other bromodomains, namely those of BRD4, in leukemia growth, ruling out potential off-target activity of our BRD9 inhibitor was critical. Traditionally, bromodomain inhibitor selectivity is tested using in vitro binding assays that examine a subset of other bromodomains. To sample the entire space of potential off-target proteins, we sought an in-cell selectivity assay. To this end, we engineered a bromodomain-swap allele of BRD9, which retains functionality despite a radically altered bromodomain pocket. Expression of this allele in AML cells confers resistance to the anti-proliferative effects of our BRD9 inhibitor, thus establishing BRD9 as the relevant cellular target. Furthermore, we used an analogous domain-swap strategy to generate an inhibitor-resistant allele of EZH2. Our study provides the first evidence for a role of BRD9 in cancer and further highlights a simple genetic strategy for constructing resistance alleles to demonstrate on-target activity of chemical probes in cells. Citation Format: Anja F. Hohmann, Laetitia J. Martin, Jessica Minder, Jae-Seok Roe, Junwei Shi, Steffen Steurer, Gerd Bader, Darryl McConnell, Mark Pearson, Thomas Gerstberger, Teresa Gottschamel, Diane Thompson, Yutaka Suzuki, Manfred Koegl, Christopher Vakoc. A bromodomain-swap allele demonstrates that on-target chemical inhibition of BRD9 limits the proliferation of acute myeloid leukemia cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-206.

Abstract 2330: BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy

Background: Inhibitors of apoptosis proteins (IAPs) regulate cellular apoptosis by interfering with the proteolytic activities of caspases. IAP inhibitors (SMAC mimetics) have been developed to restore the defective apoptosis that characterizes many tumour cells. Emerging evidence demonstrates that IAPs are critical components of immune-modulatory pathways that control innate and adaptive immunity. Accordingly, SMAC mimetics hold the promise of both inducing tumour cell killing and stimulating the immune system to recognize and eliminate dying tumour cells. Here we show that BI5 primes immune components and synergises with PD-1 checkpoint inhibitors to promote eradication of syngeneic tumors. Methods: Here we report the efficacy and modulation of the immune response by a potent and selective SMAC mimetic, BI5. We characterised the effect of BI5 on tumor growth inhibition as a single agent and in combination with an anti-PD-1 antibody in syngeneic mouse tumor models. A detailed 17-colour multi-color flow cytometry analysis was used to investigate the mechanisms by which the SMAC mimetic interacts with anti-PD-1 therapy in vivo. Results: Treatment of the syngeneic mouse tumor models MBT-2 and EMT-6 with the SMAC mimetic in combination with an anti-PD-1 antibody results in remarkable tumor regressions in vivo. Importantly, the combined effect of the SMAC mimetic and anti-PD-1 on tumor growth was dependent on the adaptive immune system in vivo. Mechanistic studies show that degradation of IAP triggers tumor cell death, which leads to a potent activation of dendritic cells in the draining lymph nodes and a subsequent influx of T and NK cells into the tumor microenvironment. Interestingly, in the presence of the SMAC mimetic alone, an induction of PD-1 expression on tumor-infiltrating CD8+ T cells was observed, which in turn resulted in the exhaustion of these cells and tumor outgrowth. In the presence of the anti-PD-1 antibody, T cells are reactivated leading to potent and long term tumor eradication. Conclusion: We show that our SMAC mimetic leads to a potent induction of immunogenic cell death and sets up a “virtuous cycle” by potentiating dendritic cell and T cell mediated immune responses that further promote induction of cell death. These effects are potentiated by checkpoint inhibitors, leading to long term tumor control. Tumours with minimal T-cell infiltration are poorly responsive to PD-1 monotherapy. These studies indicate that SMAC mimetics, such as BI5, represent promising and tolerated combination partners for checkpoint inhibitors in patients that lack a strong immune inflammatory signature. Citation Format: Markus Reschke, Maria Antonietta Impagnatiello, Ulrich Reiser, Dirk Scharn, Walter Spevak, Alexander Savchenko, Andreas Wernitznig, Martina Sykora, Rebecca Langlois, Elisabeth Zier, Daniel Zach, Sabine Kallenda, Pilar Garin-Chesa, Jens Quant, Mark Pearson, Darryl McConnell, Norbert Kraut, Juergen Moll. BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2017-2330

Abstract 4630: Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance

Background: Cyclin-dependent kinase 8 (CDK8) is part of the mediator complex that can either positively or negatively influence transcription. CDK8 is known to phosphorylate signal transducer and activator of transcription 1 (STAT1) at the position Ser727. STAT1 activity is regulated by JAK-mediated phosphorylation of tyrosine701 which leads to dimerization, nuclear translocation and IFN-γ induced phosphorylation mediated by CDK8. Introduction of an alanine mutation at the phosphorylation site STAT1-S727 results in enhanced NK cell cytotoxicity accompanied by increased levels of perforin and granzyme B (Putz et al. 2013). Method: Here we present the discovery and development of potent and selective CDK8 inhibitors guided by crystallography. The inhibitory effect of optimized compounds BI 9811 and BI 1347 on STAT1 phosphorylation and perforin release was investigated in the human NK cell line NK-92MI. Direct effects on cancer cells were furthermore analyzed in a broad panel of cell lines. The compound BI 1347 was profiled in vivo in the orthotopic B16-F10 melanoma mouse model. Results: Highly potent and selective CDK8 inhibitors were identified with an IC50 of below 10 nM in a biochemical kinase assay, which translated in a potent down regulation of the STAT1- Ser727 signal and in increased perforin and granzyme B secretion. BI 9811 and BI 1347 were highly selective for CDK8, as tested in a broad kinase panel and showed no cytotoxic activity on NK cells and most cancer cell lines, which distinguishes this compound class from published CDK8 inhibitors. A representative molecule out of this compound class demonstrated in vivo biomarker modulation and survival increase in the murine B16-F10 melanoma mouse model. Conclusion: We developed potent CDK8 inhibitors that show activation of NK cells that translates into biomarker modulation (pSTAT1Ser727) and in vivo efficacy. Citation Format: Marco H. Hofmann, Harald Engelhardt, Sebastian Carotta, Heribert Arnhof, Dirk Scharn, Marc Kerenyi, Moritz Mayer, Gerhard Gmaschitz, Georg Egger, Christian Engelhardt, Michael Sanderson, Maria A. Impagnatiello, Renate Schnitzer, Mark Pearson, Darryl McConnell, Norbert Kraut, Jurgen Moll. Development of selective and potent CDK8 inhibitors that increase NK cell activity, which translates in tumor surveillance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4630. doi:10.1158/1538-7445.AM2017-4630