Michael T. Rooney

Pharmacodynamic Profiling of a Siderophore-Conjugated Monocarbam in Pseudomonas aeruginosa: Assessing the Risk for Resistance and Attenuated Efficacy

ABSTRACT The objective of this study was to investigate the risk of attenuated efficacy due to adaptive resistance for the siderophore-conjugated monocarbam SMC-3176 in Pseudomonas aeruginosa by using a pharmacokinetic/pharmacodynamic (PK/PD) approach. MICs were determined in cation-adjusted Mueller-Hinton broth (MHB) and in Chelex-treated, dialyzed MHB (CDMHB). Spontaneous resistance was assessed at 2× to 16× the MIC and the resulting mutants sequenced. Efficacy was evaluated in a neutropenic mouse thigh model at 3.13 to 400 mg/kg of body weight every 3 h for 24 h and analyzed for association with free time above the MIC (fT>MIC). To closer emulate the conditions of the in vivo model, we developed a novel assay testing activity mouse whole blood (WB). All mutations were found in genes related to iron uptake: piuA, piuC, pirR, fecI, and pvdS. Against four P. aeruginosa isolates, SMC-3176 displayed predictable efficacy corresponding to the fT>MIC using the MIC in CDMHB (R2 = 0.968 to 0.985), with stasis to 2-log kill achieved at 59.4 to 81.1%. Efficacy did not translate for P. aeruginosa isolate JJ 4-36, as the in vivo responses were inconsistent with fT>MIC exposures and implied a threshold concentration that was greater than the MIC. The results of the mouse WB assay indicated that efficacy was not predictable using the MIC for JJ 4-36 and four additional isolates, against which in vivo failures of another siderophore-conjugated β-lactam were previously reported. SMC-3176 carries a risk of attenuated efficacy in P. aeruginosa due to rapid adaptive resistance preventing entry via the siderophore-mediated iron uptake systems. Substantial in vivo testing is warranted for compounds using the siderophore approach to thoroughly screen for this in vitro-in vivo disconnect in P. aeruginosa.

Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP Antagonists Resulting in a Clinical Candidate for the Treatment of Cancer (AZD5582)

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

Discovery of Efficacious Pseudomonas aeruginosa-Targeted Siderophore-Conjugated Monocarbams by Application of a Semi-mechanistic Pharmacokinetic/Pharmacodynamic Model

To identify new agents for the treatment of multi-drug-resistant Pseudomonas aeruginosa, we focused on siderophore-conjugated monocarbams. This class of monocyclic β-lactams are stable to metallo-β-lactamases and have excellent P. aeruginosa activities due to their ability to exploit the iron uptake machinery of Gram-negative bacteria. Our medicinal chemistry plan focused on identifying a molecule with optimal potency and physical properties and activity for in vivo efficacy. Modifications to the monocarbam linker, siderophore, and oxime portion of the molecules were examined. Through these efforts, a series of pyrrolidinone-based monocarbams with good P. aeruginosa cellular activity (P. aeruginosa MIC90 = 2 μg/mL), free fraction levels (>20% free), and hydrolytic stability (t1/2 ≥ 100 h) were identified. To differentiate the lead compounds and enable prioritization for in vivo studies, we applied a semi-mechanistic pharmacokinetic/pharmacodynamic model to enable prediction of in vivo efficacy from in vitro data.

Discovery of bacterial NAD⁺-dependent DNA ligase inhibitors: improvements in clearance of adenosine series.

Optimization of clearance of adenosine inhibitors of bacterial NAD(+)-dependent DNA ligase is discussed. To reduce Cytochrome P-450-mediated metabolic clearance, many strategies were explored; however, most modifications resulted in compounds with reduced antibacterial activity and/or unchanged total clearance. The alkyl side chains of the 2-cycloalkoxyadenosines were fluorinated, and compounds with moderate antibacterial activity and favorable pharmacokinetic properties in rat and dog were identified.

SAR and Structural Analysis of Siderophore-Conjugated Monocarbam Inhibitors of Pseudomonas aeruginosa PBP3

A main challenge in the development of new agents for the treatment of Pseudomonas aeruginosa infections is the identification of chemotypes that efficiently penetrate the cell envelope and are not susceptible to established resistance mechanisms. Siderophore-conjugated monocarbams are attractive because of their ability to hijack the bacterias iron uptake machinery for transport into the periplasm and their inherent stability to metallo-β-lactamases. Through development of the SAR we identified a number of modifications to the scaffold that afforded active anti-P. aeruginosa agents with good physicochemical properties. Through crystallographic efforts we gained a better understanding into how these compounds bind to the target penicillin binding protein PBP3 and factors to consider for future design.

Pyridodiazepine Amines Are Selective Therapeutic Agents for Helicobacter pylori by Suppressing Growth through Inhibition of Glutamate Racemase but Are Predicted To Require Continuous Elevated Levels in Plasma To Achieve Clinical Efficacy

ABSTRACT A pyridodiazepine amine inhibitor of Helicobacter pylori glutamate racemase (MurI) was characterized. The compound was selectively active against H. pylori, and growth suppression was shown to be mediated through the inhibition of MurI by several methods. In killing kinetics experiments, the compound showed concentration-independent activity, with about a 2-log loss of viability in 24 h. A demonstration of efficacy in a mouse infection model was attempted but not achieved, and this was attributed to the failure to attain extended exposure levels above the MIC for >95% of the time. This index and magnitude were derived from pharmacokinetic-pharmacodynamic (PK-PD) studies with amoxicillin, another inhibitor of peptidoglycan biosynthesis that showed slow killing kinetics similar to those of the pyridodiazepine amines. These studies indicate that MurI and other enzymes involved in peptidoglycan biosynthesis may be less desirable targets for monotherapy directed against H. pylori if once-a-day dosing is required.

Identification and Optimization of Benzimidazole Sulfonamides as Orally Bioavailable Sphingosine 1-Phosphate Receptor 1 Antagonists with in Vivo Activity.

We report here a novel series of benzimidazole sulfonamides that act as antagonists of the S1P1 receptor, identified by exploiting an understanding of the pharmacophore of a high throughput screening (HTS)-derived series of compounds described previously. Lead compound 2 potently inhibits S1P-induced receptor internalization in a cell-based assay (EC50 = 0.05 μM), but has poor physical properties and metabolic stability. Evolution of this compound through structure-activity relationship development and property optimization led to in vivo probes such as 4. However, this compound was unexpectedly found to be a potent CYP3A inducer in human hepatocytes, and thus further chemistry efforts were directed at addressing this liability. By employing a pregnane X receptor (PXR) reporter gene assay to prioritize compounds for further testing in human hepatocytes, we identified lipophilicity as a key molecular property influencing the likelihood of P450 induction. Ultimately, we have identified compounds such as 46 and 47, which demonstrate the desired S1P1 antagonist activity while having greatly reduced risk of CYP3A induction in humans. These compounds have excellent oral bioavailability in preclinical species and exhibit pharmacodynamic effects of S1P1 antagonism in several in vivo models following oral dosing. Relatively modest antitumor activity was observed in multiple xenograft models, however, suggesting that selective S1P1 antagonists would have limited utility as anticancer therapeutics as single agents.

Discovery of heterocyclic sulfonamides as sphingosine 1-phosphate receptor 1 (S1P1) antagonists

We have discovered a novel class of heterocyclic sulfonamides that act as antagonists of the S1P1 receptor. While members of this series identified from a high-throughput screen showed promising levels of potency in a cell-based assay measuring the inhibition of receptor internalization, most compounds were excessively lipophilic and contained an oxidation-prone thioether moiety. As a result, such compounds suffered from poor physical properties and metabolic stability, limiting their utility as in vivo probes. By removing the thioether group and systematically developing an understanding of structure-activity relationships and the effects of lipophilicity on potency within this series, we have been able to identify potent compounds with vastly improved physical properties. A representative enantiopure triazole sulfonamide (33) has measurable bioavailability following a low (3mg/kg) oral dose in rat, highlighting an achievement of the early hit-to-lead efforts for this series.

Abstract B100: Structure-based design of AZ7732 a novel in vivo active beta-alanine-derived pan-IAP inhibitor.

Both monomeric and dimeric SMAC (Second Mitochondria-derived Activator of Caspaces) mimetics acting as IAP (Inhibitor of apoptosis proteins) anatgonists have been reported in the clinic as well as extensively in the literature (1). The first four amino acids in the N-terminal of SMAC (AVPI) are critical for binding to IAP proteins. Reported medicinal chemistry exploration of the AVPI template has primarily consisted of variation to the VPI position in the amino-terminal of the SMAC peptide. Structural illucidation of XIAP bound to IAP inhibitors has revealed a critical role for the alanine with dense hydrogen bonding, electrostatic and hydrophibic complementarity with the protein. To our knowledge, exploration of alanine modifications has been limited and generally led to significant reduction in potency. Using the molecular modeling software SuperStar(2), we investigated the publically available co-crystal structures of Smac-mimetics with cIAP1 and hypothesized that homologating the basic amine might be tolerated. Applying this strategy, we report on the successful transfer of a beta-alanine warhead to a number of monomeric scaffolds. The resulting novel monomers maintained cIAP1/2 potency albeit with a reduction in xIAP potency. We report here the first co-crystal structure of xIAP baculoviral IAP repeat 3 domain (BIR3) with a beta-alanine derived monomer. Examination of the binding site contacts in the co-crystal structure provided further insight into the optimization of the warhead. Herein we describe the synthesis, SAR and SPR of this novel warhead and the discovery of beta-alanine derived pan-IAP inhibitors. We show that the SAR can be transferred to dimers and is invariant to the position of dimerization. We report our efforts to optimize the series and mitigate Cyp3A4 inhibition. This work led to the discovery of AZ7732, a novel dimeric SMAC-mimetic; a pan inhibitor of IAPs (cIAP BIR3 IC50 = 12 nM, XIAP BIR3 IC50 = 13 nM, and XIAP BIR2 IC50 = 30 nM); potent in cells as a single agent (MDA-MB231 cIAP degradation IC50 = 0.2 nM, GI50 = 0.4 nM) and is synergistic in vitro in combination with gemcitabine. AZ7732 has favorable in vivo PK with physical properties suitable for IV dosing. AZ7732 is active in vivo as a single agent. Once weekly dosing in MDA-MB231 led to dose-dependent tumor growth inhibition with stasis achieved at 2.5 mpk, ¼ MTD. In conclusion, Structure-based design and medicinal chemistry efforts have successfully identified novel monomeric and dimeric SMAC mimetics leading to the discovery of a novel in vivo active dimeric pan-IAP inhibitor. (1) Fulda et al, Nat. Rev. Drug Disc., 11, 109-123, 2012. (2) M. L. Verdonk, et al, J. Mol. Biol., 289, 1093-1108, 1999 Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B100. Citation Format: Jamal C. Saeh, Brian Aquila, Daniel Russell, Edward Hennessy, Alex Hird, Melissa Vasbinder, Andrew Ferguson, Bin Yang, Maureen Hattersley, Naomi Laing, Terry MacIntyre, Troy Patterson, Galina Repik, Michael Rooney, Haiyun Wang, Dave Witson, Li Sha, Donald Cook, Paula Lewis, John Lee, Danyang Li, Victor Kamhi, Vibha Oza, Charles Omer. Structure-based design of AZ7732 a novel in vivo active beta-alanine-derived pan-IAP inhibitor. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B100.

Abstract 2353: Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208.

The PIM serine/threonine kinase family, composed of three highly homologous members, PIM-1, PIM-2 and PIM-3, are upregulated in leukemias and lymphomas, including AML, NHL and CLL, highlighting the potential of these kinases as therapeutic targets in these indications. Over-expression of PIM-1 or PIM-3 has also been observed several solid tumors, in particular prostate, pancreatic, gastric, bladder and hepatocellular cancers. PIM kinases are downstream effectors of many cytokine and growth factor signaling pathways and are direct transcriptional targets of STAT transcription factors activated by these pathways, thereby mediating cell proliferation and survival. We have identified novel, potent and highly selective inhibitors of the PIM family kinases. The synthesis, X-ray crystallographic binding mode, and SAR of this benzylidene-1,3-thiazolidine-2,4-dione series are described. Examples from this series exhibit single digit nanomolar potency against all three PIMs, and have been shown to be selective across a panel of more than 440 kinases, with inhibition found for only approximately 3% of the panel, and at least 10-fold selectivity over kinases outside the PIM family. The compounds additionally are stable in rat microsomes, have high aqueous solubilities, and are not potent against the hERG ion channel. The series has antiproliferative activity in a panel of AML cells, has excellent pre-clinical pharmacokinetic properties, and lead compounds have shown strong tumor growth inhibition in vivo in mouse AML xenograft models. From this series, AZD1208 has recently entered Phase I clinical trials for AML and solid tumors. Citation Format: Michelle L. Lamb, Les A. Dakin, Michael H. Block, Huawei Chen, Erin Code, James E. Dowling, Xiaomei Feng, Andrew D. Ferguson, Isabelle Green, Alexander W. Hird, Tina Howard, Dennis Huszar, Erika K. Keeton, Paul D. Lyne, Hannah Pollard, Michael Rooney, Jon Read, Allan J. Wu, Tao Zhang, Xiaolan Zheng. Novel inhibitors of PIM-1, PIM-2, and PIM-3 protein kinases: medicinal chemistry leading to AZD1208. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2353. doi:10.1158/1538-7445.AM2013-2353