Anjaneyulu Sheri

Penicillin Sulfone Inhibitors of Class D β-Lactamases

ABSTRACT OXA β-lactamases are largely responsible for β-lactam resistance in Acinetobacter spp. and Pseudomonas aeruginosa, two of the most difficult-to-treat nosocomial pathogens. In general, the β-lactamase inhibitors used in clinical practice (clavulanic acid, sulbactam, and tazobactam) demonstrate poor activity against class D β-lactamases. To overcome this challenge, we explored the abilities of β-lactamase inhibitors of the C-2- and C-3-substituted penicillin and cephalosporin sulfone families against OXA-1, extended-spectrum (OXA-10, OXA-14, and OXA-17), and carbapenemase-type (OXA-24/40) class D β-lactamases. Three C-2-substituted penicillin sulfone compounds (JDB/LN-1-255, JDB/LN-III-26, and JDB/ASR-II-292) showed low Ki values for the OXA-1 β-lactamase (0.70 ± 0.14 → 1.60 ± 0.30 μM) and demonstrated significant Ki improvements compared to the C-3-substituted cephalosporin sulfone (JDB/DVR-II-214), tazobactam, and clavulanic acid. The C-2-substituted penicillin sulfones JDB/ASR-II-292 and JDB/LN-1-255 also demonstrated low Kis for the OXA-10, -14, -17, and -24/40 β-lactamases (0.20 ± 0.04 → 17 ± 4 μM). Furthermore, JDB/LN-1-255 displayed stoichiometric inactivation of OXA-1 (the turnover number, i.e., the partitioning of the initial enzyme inhibitor complex between hydrolysis and enzyme inactivation [tn] = 0) and tns ranging from 5 to 8 for the other OXA enzymes. Using mass spectroscopy to study the intermediates in the inactivation pathway, we determined that JDB/LN-1-255 inhibited OXA β-lactamases by forming covalent adducts that do not fragment. On the basis of the substrate and inhibitor kinetics of OXA-1, we constructed a model showing that the C-3 carboxylate of JDB/LN-1-255 interacts with Ser115 and Thr213, the R-2 group at C-2 fits between the space created by the long B9 and B10 β strands, and stabilizing hydrophobic interactions are formed between the pyridyl ring of JDB/LN-1-255 and Val116 and Leu161. By exploiting conserved structural and mechanistic features, JDB/LN-1-255 is a promising lead compound in the quest for effective inhibitors of OXA-type β-lactamases.

Antiviral Efficacy and Host Innate Immunity Associated with SB 9200 Treatment in the Woodchuck Model of Chronic Hepatitis B.

SB 9200, an oral prodrug of the dinucleotide SB 9000, is being developed for the treatment of chronic hepatitis B virus (HBV) infection and represents a novel class of antivirals. SB 9200 is thought to activate the viral sensor proteins, retinoic acid-inducible gene 1 (RIG-I) and nucleotide-binding oligomerization domain-containing protein 2 (NOD2) resulting in interferon (IFN) mediated antiviral immune responses in virus-infected cells. Additionally, the binding of SB 9200 to these sensor proteins could also sterically block the ability of the viral polymerase to access pre-genomic RNA for nucleic acid synthesis. The immune stimulating and direct antiviral properties of SB 9200 were evaluated in woodchucks chronically infected with woodchuck hepatitis virus (WHV) by daily, oral dosing at 15 and 30 mg/kg for 12 weeks. Prolonged treatment resulted in 2.2 and 3.7 log10 reductions in serum WHV DNA and in 0.5 and 1.6 log10 declines in serum WHV surface antigen from pretreatment level with the lower or higher dose of SB 9200, respectively. SB 9200 treatment also resulted in lower hepatic levels of WHV nucleic acids and antigen and reduced liver inflammation. Following treatment cessation, recrudescence of viral replication was observed but with dose-dependent delays in viral relapse. The antiviral effects were associated with dose-dependent and long-lasting induction of IFN-α, IFN-β and IFN-stimulated genes in blood and liver, which correlated with the prolonged activation of the RIG-I/NOD2 pathway and hepatic presence of elevated RIG-I protein levels. These results suggest that in addition to a direct antiviral activity, SB 9200 induces antiviral immunity during chronic hepadnaviral infection via activation of the viral sensor pathway.

Efficient inhibition of class A and class D β-lactamases by Michaelis complexes

A 6-alkylidiene penam sulfone, SA-1-204, is an efficient inhibitor of both SHV-1 and OXA-1 β-lactamases with KI = 42 ± 4nm and 1.0 ± 0.1 μm, respectively. To gain insight into the reaction chemistry of SA-1-204, the reactions between this inhibitor and SHV-1 and OXA-1 were studied by Raman spectroscopy in single crystals and in solution. Raman signatures characteristic of the unreacted β-lactam ring show that in both phases the inhibitor binds as a noncovalent Michaelis-like complex. This complex is present as the major population for periods of up to an hour. On longer time scales, the Raman data show that β-lactam ring opening eventually leads to a complex mixture of reaction products. However, the data clearly demonstrate that the key species for inhibition on the time scale of bacterial half-lives is the noncovalent complex preceding acylation.

Why Clinically Used Tazobactam and Sulbactam Are Poor Inhibitors of OXA-10 β-Lactamase: Raman Crystallographic Evidence†

The clinically used inhibitors tazobactam and sulbactam are effective in the inhibition of activity of class A beta-lactamases, but not for class D beta-lactamases. The two inhibitors exhibit a complex multistep profile for their chemistry of inhibition with class A beta-lactamases. To compare the inhibition profiles for class A and D enzymes, the reactions were investigated within OXA-10 beta-lactamase (a class D enzyme) crystals using a Raman microscope. The favored reaction pathway appears to be distinctly different from that for class A beta-lactamases. In contrast to the case of class A enzymes that favor the formation of a key enamine species, the OXA-10 enzyme forms an alpha,beta-unsaturated acrylate (acid or ester). Quantum mechanical calculations support the likely product as the adduct of Ser115 to the acrylate. Few enamine-like species are formed by sulbactam or tazobactam with this enzyme. Taken together, our results show that the facile conversion of the initial imine, formed upon acylation of the active site Ser67, to the cis- and/or trans-enamine is disfavored. Instead, there is a significant population of the imine that could either experience cross-linking to a second nucleophile (e.g., Ser115) or give rise to the alpha,beta-unsaturated product and permanent inhibition. Alternatively, the imine can undergo hydrolysis to regenerate the catalytically active OXA-10 enzyme. This last process is the dominant one for class D beta-lactamases since the enzyme is not effectively inhibited. In contrast to sulbactam and tazobactam, the reactions between oxacillin or 6alpha-hydroxyisopropylpenicillinate (both substrates) and OXA-10 beta-lactamase appear much less complex. These compounds lead to a single acyl-enzyme species, the presence of which was confirmed by Raman and MALDI-TOF experiments.

The Importance of the trans‐Enamine Intermediate as a β‐Lactamase Inhibition Strategy Probed in Inhibitor‐Resistant SHV β‐Lactamase Variants

The ability of bacteria to express inhibitor‐resistant (IR) β‐lactamases is stimulating the development of novel inhibitors of these enzymes. The 2′β‐glutaroxypenicillinate sulfone, SA2‐13, was previously designed to enhance the stabilization of the deacylation‐refractory, trans‐enamine inhibitory intermediate. To test whether this mode of inhibition can overcome different IR mutations, we determined the binding mode of SA2‐13 through X‐ray crystallography, obtaining co‐crystals of the inhibitor–protein complex by soaking crystals of the IR sulfhydryl variable (SHV) β‐lactamase variants S130G and M69V with the inhibitor. The 1.45 Å crystal structure of the S130G SHV:SA2‐13 complex reveals that SA2‐13 is still able to form the stable trans‐enamine intermediate similar to the wild‐type complex structure, yet with its carboxyl linker shifted deeper into the active site in the space vacated by the S130G mutation. In contrast, data from crystals of the M69V SHV:SA2‐13 complex at 1.3 Å did not reveal clear inhibitor density indicating that this IR variant disfavors the trans‐enamine conformation, likely due to a subtle shift in A237.

Structures of SHV-1 β-lactamase with penem and penam sulfone inhibitors that form cyclic intermediates stabilized by carbonyl conjugation.

Bacterial β-lactamase enzymes are in large part responsible for the decreased ability of β-lactam antibiotics to combat infections. The inability to overcome β-lactamase mediated resistance spurred the development of inhibitors with penems and penam sulfones being amongst the most potent and broad spectrum mechanism-based inactivators. These inhibitors form covalent, “suicide-type” inhibitory intermediates that are attached to the catalytic S70 residue. To further probe the details of the mechanism of β-lactamase inhibition by these novel compounds, we determined the crystal structures of SHV-1 bound with penem 1, and penam sulfones SA1-204 and SA3-53. Comparison with each other and with previously determined crystal structures of members of these classes of inhibitors suggests that the final conformation of the covalent adduct can vary greatly amongst the complex structures. In contrast, a common theme of carbonyl conjugation as a mechanism to avoid deacylation emerges despite that the penem and penam sulfone inhibitors form different types of intermediates. The detailed insights gained from this study could be used to further improve new mechanism-based inhibitors of these common class A serine β-lactamases.

SB 9200, a novel agonist of innate immunity, shows potent antiviral activity against resistant HCV variants

SB 9200 is a novel, first‐in‐class oral modulator of innate immunity that is believed to act via the activation of the RIG‐I and NOD2 pathways. SB 9200 has broad‐spectrum antiviral activity against RNA viruses including hepatitis C virus (HCV), norovirus, respiratory syncytial virus, and influenza and has demonstrated activity against hepatitis B virus (HBV) in vitro and in vivo. In phase I clinical trials in chronically infected HCV patients, SB 9200 has been shown to reduce HCV RNA by up to 1.9 log10. Here, we demonstrate the antiviral activity of SB 9200 against a HCV replicon system and patient derived virus. Using the HCV capture‐fusion assay, we show that SB 9200 is active against diverse HCV genotypes and is also effective against HCV derived from patients who relapse following direct‐acting antiviral treatment, including viruses containing known NS5A resistance‐associated sequences. These data confirm the broad antiviral activity of SB 9200 and indicate that it may have clinical utility in HCV patients who have failed to respond to current antiviral regimens.

Abstract B40: Nucleotide analogs as novel STING agonists for immuno-oncology

Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. Recent evidence suggests that the activation of Stimulator of Interferon Genes (STING) pathway in tumor cells and/or antigen presenting cells (APCs) within the tumor microenvironment (TME) can induce type I Interferon production leading to apoptosis of tumor cells, as well as, induction of adaptive immune response (through priming of CD8+ T cells to tumor-associated antigens) thereby providing a powerful anti-cancer strategy. Therefore, therapeutic agents that activate STING signaling pathway in tumor cells and APCs in the TME are urgently needed. Herein, we describe the discovery of highly potent and selective first-in-class STING agonists for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of different cyclic dinucleotides bound to STING, a focused library of nucleotide compounds was prepared using standard phosphoramidite chemistry. The compounds were screened for induction of Interferon regulatory factor (IRF), Interferon-stimulated gene 54 (ISG54), and NF-KB using reporter assays. We used HEK293 cell line stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for initial hit discovery and the actives were further characterized in PBMCs and THP1 cells. The IRF and NF-kB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50 of the compounds were ascertained using Xlfit. Lead STING agonists were further evaluated for: (a) Binding affinity: Binding assays were conducted by Differential Scanning Fluorimetry (DSF) and Tm was calculated using Thermal Shift software, (b) Induction of pathogen recognition receptors (PRRs), ISGs and Programmed Death Ligands 1 & 2 (PDL1, PDL2) genes: THP1 cells and PBMCs were treated with various concentrations of lead compounds or 2,939-cGAMP or DMSO and the gene expression of different PRRs, ISGs, PDL1, and PDL2 was determined by quantitative RT-PCR using ΔΔct method, (c) Apoptosis-inducing activity: PBMCs and THP1 cells were treated with various concentrations of lead compounds, 29,39-cGAMP, or DMSO control and the apoptotic activity was evaluated using Caspase-Glo® 3/7 Assay (Promega), and (d) In vitro anti-tumor activity: STING-dependent anti-tumor activity of lead compounds in various tumor cell lines was assessed by either high-content imaging or through Cell titer Glo® Cytotoxicity Assay (Promega). Cell survival was calculated based upon % reduction of live cells compared to DMSO control. CC50 of the compounds were generated by curve fit in Xlfit. Results: Through in vitro assays in conjunction with Structure Activity Relationship (SAR) studies, we have identified several highly potent and selective first-in-class STING agonists. A promising lead nucleotide compound SB 11285 caused STING-dependent induction of: (a) IRF with an EC50 of 2 nM that is 1000-fold more potent than the natural STING agonist 29,39-cGAMP, (b) NF-kB with an EC50 of 200 nM that is >200-fold more potent than 29,39-cGAMP, (c) selective apoptosis of human monocyte leukemic cell lines (CC50, 500 nM) as compared to normal PBMCs through induction of IFN, and NF-kB signaling, and (d) expression of various PRRs and ISGs including RIG-I, MDA-5, LGP2, ISG54 and OAS-1, as well as, PDL1 and PDL2. Finally, SB 11285 showed potent in vitro anti-tumor activity in multiple tumor cell lines. Conclusion: We have discovered highly potent first-in-class STING agonists that show excellent selectivity in induction of IFN, NF-KB, ISGs, and PRRs, and apoptosis of tumor-derived cell lines. The lead STING agonist SB 11285 has potent immune-modulating, as well as, anti-tumor activities and is being advanced for additional preclinical studies for application in immuno-oncology. Citation Format: Sreerupa Challa, Shenghua Zhou, Anjaneyulu Sheri, Seetharamaiyer Padmanabhan, Samantha Delaney, Geeta Meher, Dillon Cleary, Vishal Nair, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Nucleotide analogs as novel STING agonists for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B40.

Abstract B39: Novel dinucleotides that activate STING signaling for immuno-oncology

Background: Immunotherapy has recently emerged as a transformative approach for the treatment of cancer; nevertheless, many patients remain unresponsive to treatment. It is being recognized that induction of type I interferons (IFN) and interferon-stimulated genes (ISGs) in tumor cells and within the tumor microenvironment (TME) is essential for modulating the host-immune response and inducing apoptosis of tumor cells. Furthermore, the antigen-presenting cells within TME can cause induction of adaptive immune response, through priming of CD8+ T cells and tumor killing. Importantly, the DNA released from damaged cells and cancer cells can be sensed by cyclic GMP-AMP synthase (cGAS) leading to the synthesis of cyclic-GMP-AMP (29,39-cGAMP), a second messenger that activates Stimulator of Interferon Genes (STING) pathway resulting in the production of type I IFN and ISGs. The cumulative effects of activation of innate and adaptive immune response can result in potent anti-cancer effects. Therefore, therapeutic agents that activate the cGAS-STING signaling pathway in tumor cells and TME are urgently needed. Herein, we describe the discovery of novel potent, first-in-class small molecules for application in immuno-oncology. Methods: Using structure-guided drug design, in conjunction with published crystal structures of cyclic dinucleotides bound to STING, a focused library of dinucleotide compounds was synthesized using phosphoramidite chemistry and evaluated for: (a) Induction of IFN signaling: The compounds were screened for the induction of Interferon regulatory factor (IRF), ISG54, and NF-κB using reporter assays. We used HEK293 cell line (SZ14) stably expressing ISG54 (ISRE)-promoter-driven firefly luciferase reporter gene for screening and the active compounds were further characterized in THP1 cells and human primary PBMCs. The IRF, ISG54, and NF-κB induction was calculated from % fold-change in luminescence compared to DMSO-treated cells and EC50s of the compounds were ascertained to identify active compounds, (b) Expression of IFN-β and IRF7 in THP1 cells: THP1 cells were treated with active compounds or controls for 22hrs. RNA was extracted and the expression of IFN-β, IRF7, was ascertained using semi-quantitative RT-PCR, (c) Induction of pathogen recognition receptors (PRRs) including RIG-I, MDA5, LGP2, and OAS-1 and ISG54: THP1 cells and PBMCs were treated with active compounds, 29,39-cGAMP (control), or DMSO and the gene expression of different PRRs, ISGs, was determined by quantitative RT-PCR using ΔΔct method, (d) Induction of cGAS-STING signaling using reporter assays: HEK293 cells stably expressing ISG54 were transfected with plasmids encoding human cGAS (wild-type, or K384A, K400A, or K411A mutants) and treated with active compounds, poly (dA:dT) (positive control), or DMSO for 21 hrs. ISG54 induction was calculated as fold-change in luminescence compared to DMSO-treated controls. (e) Cytotoxicity assays: THP1 cells were treated with active compounds or DMSO control with Lipofectamine and cytotoxicity assessed using the CellTiter-Glo® Luminescent assays. Cytotoxicity was calculated from %-fold change in luminescence compared to DMSO-treated sample. Results: Through in vitro assays in conjunction with Structure Activity Relationship studies, we have identified potent compounds that activate cGAS-STING signaling pathway for induction of IRF, IFN, and NF-κB. These compounds also cause induction of expression of PRRs, including RIG-I, MDA5, LGP2, as well as, ISG54 and OAS-1. Conclusion: We have discovered potent, first-in-class agents that cause induction of IFN, NF-κB, ISGs, and PRRs. Further optimization and preclinical evaluation of the compounds for application in immuno-oncology is underway. Citation Format: Shenghua Zhou, Sreerupa Challa, Seetharamaiyer Padmanabhan, Anjaneyulu Sheri, Samantha Delaney, Geeta Meher, Dillon Cleary, Rayomand Gimi, Santosh Khedkar, Radhakrishnan Iyer. Novel dinucleotides that activate STING signaling for immuno-oncology. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B39.