Selection Of Lead Compounds Research Articles

P.42Discovery of novel small molecule treatment options for FSHD

There is wide support for the pathogenesis model in which gain-of-function of the DUX4 gene in skeletal muscle cells underlies FSHD progression. DUX4 is a transcription factor whose expression is normally restricted to early embryonic development. Expression of DUX4 in muscle tissue of FSHD patients initiates a transcription cascade ultimately resulting in overt pathology. Drug discovery efforts in FSHD have been hampered by the very low and stochastic expression of DUX4 in a small percentage of myonuclei. Because the regulatory pathways involved in the activation of DUX4 are largely unknown, we implemented a phenotypic approach to identify novel small molecules and drug targets to repress DUX4. In order to build a predictive and translatable assay, we used primary patient-derived myocytes to develop and validate a high-content screening assay allowing automated sensitive detection and quantification of endogenous, non-stimulated DUX4 protein and myotube fusion efficiency. Our assay confirmed that the expression of DUX4 is dynamically regulated during myogenic differentiation and showed that effects on fusion are associated with potentially false-positive signals on the DUX4 readout. The excellent assay performance enabled high-throughput screening, resulting in the discovery and optimization of multiple hit series with confirmed activity in secondary assays. RNA-seq studies with selected compounds revealed normalization of the FSHD transcription signature. We applied a number of chemoproteomic approaches to deconvolute the mode of action of the phenotypic hit series, resulting in the discovery of novel targets to repress DUX4. In order to support the pharmacokinetic/pharmacodynamic strategy, we developed a xenograft animal model in which we could demonstrate in vivo repression of DUX4 after oral dosing of a selected lead compound from one of the series. Together, these results form the basis for building a diverse FSHD-focused product pipeline.

Abstract 1: A small molecule inhibits Nrf2 expression and activity in cancer cells

Abstract Activation of the Nrf2-Keap1-ARE pathway is a master defense mechanism protecting against oxidative and electrophilic stress. Nrf2 was traditionally considered cancer-preventing, as Nrf2 deficiency enhances susceptibility to carcinogens in a variety of mouse models. However, increasing evidence suggests a tumor-promoting role for Nrf2 in established tumors: gain of function mutations in NFE2L2, which activates the Nrf2 pathway, or loss of function mutations in its negative regulator, Keap1, have been found most frequently in lung cancer. Constitutive activation of the Nrf2 pathway is associated with chemoresistance and poor survival. Nrf2 inhibitors, therefore, become potential novel drugs for cancer treatment and needed tool compounds to study the Nrf2 pathway in cancer. To identify Nrf2 inhibitors, we conducted a high throughput screen with a diverse set of small molecules (~6500 compounds). 890 compounds were identified as hits, and a funnel strategy was applied to filter through the hits and select lead compounds. Although other known Nrf2 inhibitors were identified, a potentially novel compound, MSU225, was chosen as a lead for further evaluation. MSU225 downregulated tBHQ (tert-butyl hydroquinone)-induced Nrf2 transcriptional activity in a luciferase assay without inducing cell death. When A549 human lung cancer cells, in which Keap1 is mutated inducing constitutive activation of Nrf2, were treated with 5 µM MSU225, the mRNA expression of HMOX1, NQO1, GST1A1, GCLC, GCLM, and UGT1A6, all downstream targets of Nrf2, were decreased. Moreover, MSU225 significantly (p<0.05) decreased the protein level of Nrf2 in A549 cells. The effect of MSU225 on Nrf2 protein levels was blocked by the proteasome inhibitor MG132, suggesting MSU225 enhances Nrf2 degradation through the proteasome system. In addition, MSU225 inhibited the growth of human lung cancer cells (A549, H460, A427) in soft agar. Cells addicted to Nrf2 were more susceptible to MSU225 for suppression of cell proliferation than cells not addicted to Nrf2. MSU225 also sensitized A549 cells to carboplatin treatment. Our results suggest that MSU225 is a novel Nrf2 inhibitor, and additional in vivo studies will be done to determine if it can be used to treat experimental lung cancer. Citation Format: Di Zhang, Corbin Livingston, Thomas Dexheimer, Edmund Ellsworth, Aaron Odom, Karen Liby. A small molecule inhibits Nrf2 expression and activity in cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1.

Molecular dynamics simulations of the interaction of Mouse and Torpedo acetylcholinesterase with covalent inhibitors explain their differential reactivity: Implications for drug design

Although the three-dimensional structures of mouse and Torpedo californica acetylcholinesterase are very similar, their responses to the covalent sulfonylating agents benzenesulfonyl fluoride and phenylmethylsulfonyl fluoride are qualitatively different. Both agents inhibit the mouse enzyme effectively by covalent modification of its active-site serine. In contrast, whereas the Torpedo enzyme is effectively inhibited by benzenesulfonyl fluoride, it is almost completely resistant to phenylmethylsulfonyl fluoride. A bottleneck midway down the active-site gorge in both enzymes restricts access of ligands to the active site at the bottom of the gorge. Molecular dynamics simulations revealed that the mouse enzyme is substantially more flexible than the Torpedo enzyme, suggesting that enhanced ‘breathing motions’ of the mouse enzyme relative to the Torpedo enzyme may explain why phenylmethylsulfonyl fluoride can reach the active site in mouse acetylcholinesterase, but not in the Torpedo enzyme. Accordingly, we performed docking of the two sulfonylating agents to the two enzymes, followed by molecular dynamics simulations. Whereas benzenesulfonyl fluoride closely approaches the active-site serine in both mouse and Torpedo acetylcholinesterase in such simulations, phenylmethylsulfonyl fluoride is able to approach the active-site serine of mouse acetylcholinesterase, but remains trapped above the bottleneck in the Torpedo enzyme. Our studies demonstrate that reliance on docking tools in drug design can produce misleading information. Docking studies should, therefore, also be complemented by molecular dynamics simulations in selection of lead compounds. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:CHEMBIOINT:2.

Application of an in Vitro Blood-Brain Barrier Model in the Selection of Experimental Drug Candidates for the Treatment of Huntington's Disease.

Huntington's disease (HD) is a neurodegenerative disease caused by polyglutamine expansion in the huntingtin protein. For drug candidates targeting HD, the ability to cross the blood-brain barrier (BBB) and reach the site of action in the central nervous system (CNS) is crucial for achieving pharmacological activity. To assess the permeability of selected compounds across the BBB, we utilized a two-dimensional model composed of primary porcine brain endothelial cells and rat astrocytes. Our objective was to use this in vitro model to rank and prioritize compounds for in vivo pharmacokinetic and brain penetration studies. The model was first characterized using a set of validation markers chosen based on their functional importance at the BBB. It was shown to fulfill the major BBB characteristics, including functional tight junctions, high transendothelial electrical resistance, expression, and activity of influx and efflux transporters. The in vitro permeability of 54 structurally diverse known compounds was determined and shown to have a good correlation with the in situ brain perfusion data in rodents. We used this model to investigate the BBB permeability of a series of new HD compounds from different chemical classes, and we found a good correlation with in vivo brain permeation, demonstrating the usefulness of the in vitro model for optimizing CNS drug properties and for guiding the selection of lead compounds in a drug discovery setting.

Identification of new DNA gyrase inhibitors based on bioactive compounds from streptomyces: structure-based virtual screening and molecular dynamics simulations approaches

DNA gyrase enzyme has vital role in bacterial survival and can be considered as a potential drug target. Owing to the appearance of resistance to gyrase-targeted drugs, especially fluoroquinolone, screening new compounds which bind more efficiently to the mutant binding pocket is essential. Hence, in this work, using Smina Autodock and through structure-based virtual screening of StreptomeDB, several natural products were discovered based on the SimocyclinoneD8 (SD8) binding pocket of GyrA subunit of DNA gyrase. After evaluation of binding affinity, binding modes, critical interactions and physicochemical and pharmaceutical properties, three lead compounds were selected for further analysis. Afterward 60 ns molecular dynamics simulations were performed and binding free energies were calculated by the molecular mechanics/Poisson–Boltzmann surface area method. Also, interaction of the selected lead compounds with the mutated GyrA protein was evaluated. Results indicated that all of the selected compounds could bind to the both wild-type and mutated GyrA with the binding affinities remarkably higher than SimocyclinoneD8. Interestingly, we noticed that the selected compounds comprised angucycline moiety in their structure which could sufficiently interact with GyrA and block the DNA binding pocket of DNA gyrase, in silico. In conclusion, three DNA gyrase inhibitors were identified successfully which were highly capable of impeding DNA gyrase and can be considered as potential drug candidates for treatment of fluoroquinolone-resistant strains.Communicated by Ramaswamy H. Sarma

Rufomycin Targets ClpC1 Proteolysis in Mycobacterium tuberculosis and M. abscessus.

ClpC1 is an emerging new target for the treatment of Mycobacterium tuberculosis infections, and several cyclic peptides (ecumicin, cyclomarin A, and lassomycin) are known to act on this target. This study identified another group of peptides, the rufomycins (RUFs), as bactericidal to M. tuberculosis through the inhibition of ClpC1 and subsequent modulation of protein degradation of intracellular proteins. Rufomycin I (RUFI) was found to be a potent and selective lead compound for both M. tuberculosis (MIC, 0.02 μM) and Mycobacterium abscessus (MIC, 0.4 μM). Spontaneously generated mutants resistant to RUFI involved seven unique single nucleotide polymorphism (SNP) mutations at three distinct codons within the N-terminal domain of clpC1 (V13, H77, and F80). RUFI also significantly decreased the proteolytic capabilities of the ClpC1/P1/P2 complex to degrade casein, while having no significant effect on the ATPase activity of ClpC1. This represents a marked difference from ecumicin, which inhibits ClpC1 proteolysis but stimulates the ATPase activity, thereby providing evidence that although these peptides share ClpC1 as a macromolecular target, their downstream effects are distinct, likely due to differences in binding.

Therapeutic value of steroidal alkaloids in cancer: Current trends and future perspectives.

Discovery and development of new potentially selective anticancer agents are necessary to prevent a global cancer health crisis. Currently, alternative medicinal agents derived from plants have been extensively investigated to develop anticancer drugs with fewer adverse effects. Among them, steroidal alkaloids are conventional secondary metabolites that comprise an important class of natural products found in plants, marine organisms and invertebrates, and constitute a judicious choice as potential anti‐cancer leads. Traditional medicine and modern science have shown that representatives from this compound group possess potential antimicrobial, analgesic, anticancer and anti‐inflammatory effects. Therefore, systematic and recapitulated information about the bioactivity of these compounds, with special emphasis on the molecular or cellular mechanisms, is of high interest. In this review, we methodically discuss the in vitro and in vivo potential of the anticancer activity of natural steroidal alkaloids and their synthetic and semi‐synthetic derivatives. This review focuses on cumulative and comprehensive molecular mechanisms, which will help researchers understand the molecular pathways involving steroid alkaloids to generate a selective and safe new lead compound with improved therapeutic applications for cancer prevention and therapy. In vitro and in vivo studies provide evidence about the promising therapeutic potential of steroidal alkaloids in various cancer cell lines, but advanced pharmacokinetic and clinical experiments are required to develop more selective and safe drugs for cancer treatment.

Current status and future perspective of computational toxicology in drug safety assessment under ontological intellection.

For the safety assessment of pharmaceuticals, initial data management requires accurate toxicological data acquisition, which is based on regulatory safety studies according to guidelines, and computational systems have been developed under the application of Good Laboratory Practice (GLP). In addition to these regulatory toxicology studies, investigative toxicological study data for the selection of lead compound and candidate compound for clinical trials are directed to estimation by computational systems such as Quantitative Structure-Activity Relationship (QSAR) and related expert systems. Furthermore, in the "Go" or "No-Go" decision of drug development, supportive utilization of a scientifically interpretable computational toxicology system is required for human safety evaluation. A pharmaceutical safety evaluator as a related toxicologist who is facing practical decision needs not only a data-driven Artificial Intelligence (AI) system that calls for the final consequence but also an explainable AI that can provide comprehensive information necessary for evaluation and can help with decision making. Through the explication and suggestion of information on the mechanism of toxic effects to safety assessment scientists, a subsidiary partnership system for risk assessment is ultimately to be a powerful tool that can indicate project-vector with data weight for the corresponding counterparts. To bridge the gaps between big data and knowledge, multi-dimensional thinking based on philosophical ontology theory is necessary for handling heterogeneous data for integration. In this review, we will explain the current state and future perspective of computational toxicology related to drug safety assessment from the viewpoint of ontology thinking.

Antibacterial activity of di-butyl phthalate isolated from Begonia malabarica

Natural products from medicinal plants either as pure compounds or as standardized extracts provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. Begonia malabarica Lam. is one of the medicinally important herbs belonging to the family of Begoniaceae. It is reported that the leaves are used to treat respiratory infections, diarrhea, blood cancer and skin diseases. The whole plant possesses a variety of secondary metabolites. Isolation of such compounds will add more novel bioactive structures that are of interest to screen and select potential lead compounds to discover and develop drugs in the modern pharmaceutical sector. Against this backdrop, Begonia malabarica was extracted with various solvents and bioactive compounds were isolated using chromatographic techniques. One of the bioactive compounds isolated from it was colorless or pale yellow oily compound that is soluble in chloroform. The structure of the compound was elucidated as di-butyl phthalate with the help of spectral data such as FT-IR, MS, 1 H-NMR and 13C-NMR. This is the first report to the family of Begoniaceae. The compound is reported to have antibacterial and anticancer properties.

In silico ADME in drug design – enhancing the impact

<p>Each year the pharmaceutical industry makes thousands of compounds, many of which do not meet the desired efficacy or pharmacokinetic properties, describing the absorption, distribution, metabolism and excretion (ADME) behavior. Parameters such as lipophilicity, solubility and metabolic stability can be measured in high throughput in vitro assays. However, a compound needs to be synthesized in order to be tested. In silico models for these endpoints exist, although with varying quality. Such models can be used before synthesis and, together with a potency estimation, influence the decision to make a compound. In practice, it appears that often only one or two predicted properties are considered prior to synthesis, usually including a prediction of lipophilicity. While it is important to use all information when deciding which compound to make, it is somewhat challenging to combine multiple predictions unambiguously. This work investigates the possibility of combining in silico ADME predictions to define the minimum required potency for a specified human dose with sufficient confidence. Using a set of drug discovery compounds,in silico predictions were utilized to compare the relative ranking based on minimum potency calculation with the outcomes from the selection of lead compounds. The approach was also tested on a set of marketed drugs and the influence of the input parameters investigated.</p>

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes.

Mycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X‐ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1–TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

Determinants of the Inhibition of DprE1 and CYP2C9 by Antitubercular Thiophenes

AbstractMycobacterium tuberculosis (Mtb) DprE1, an essential isomerase for the biosynthesis of the mycobacterial cell wall, is a validated target for tuberculosis (TB) drug development. Here we report the X‐ray crystal structures of DprE1 and the DprE1 resistant mutant (Y314C) in complexes with TCA1 derivatives to elucidate the molecular basis of their inhibitory activities and an unconventional resistance mechanism, which enabled us to optimize the potency of the analogs. The selected lead compound showed excellent in vitro and in vivo activities, and low risk of toxicity profile except for the inhibition of CYP2C9. A crystal structure of CYP2C9 in complex with a TCA1 analog revealed the similar interaction patterns to the DprE1–TCA1 complex. Guided by the structures, an optimized molecule was generated with differential inhibitory activities against DprE1 and CYP2C9, which provides insights for development of a clinical candidate to treat TB.

Abstract 3022: Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors

Abstract Mutations in several components of the canonical Wnt pathway function as drivers in a range of common solid tumor cancers, including about 90% of sporadic colorectal cancers (CRCs), the third most common cancer in the US. In particular, activation of β-catenin (β-cat), a key Wnt signaling component, can initiate tumorigenesis in the intestine and promote cancer stem cell metastasis6,7. As such, targeting the Wnt pathway is an attractive approach to the development of cancer therapeutics. However, Wnt inhibitors tested to date, including those that disrupt β-cat activity, have either had limited efficacy or unacceptable side effect profiles1. Here, we developed a potent and robust, selective lead compound targeting oncogenic Wnt activity by disrupting the highly specific interaction of β-cat with its co-activators BCL9/B9L, which has been shown to abrogate transactivation. By using hydrocarbon stapled peptide technology and an acutely sensitive high throughput biochemical screening assay, we have identified several promising peptides that possess a strong pharmacokinetic profile. These peptides (i) exhibit robust activity in mouse models; (ii) inhibit cancer cell proliferation, metastasis, and stem cell progression; and (iii) modulate the immune microenvironment, demonstrating the feasibility of such peptide development and selection. Synergy is demonstrated between the stapled peptides and anti-PD1 antibodies, which has significant implications for combination therapy with commercial immune checkpoint blockers that have become standard of care for second-line melanoma treatment. These peptides represent a promising new potential therapeutic class for patients with CRC, for which traditional chemotherapy and targeted inhibitors have had limited therapeutic success. Citation Format: David Zhu, Joy Jin. Novel hydrocarbon stapled peptide inhibitors suppresses β-catenin dependent cancer growth and immunomodulates colorectal tumors [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 3022. doi:10.1158/1538-7445.AM2017-3022

Evaluating the Contribution of Transition-State Destabilization to Changes in the Residence Time of Triazole-Based InhA Inhibitors.

A critical goal of lead compound selection and optimization is to maximize target engagement while minimizing off-target binding. Since target engagement is a function of both the thermodynamics and kinetics of drug-target interactions, it follows that the structures of both the ground states and transition states on the binding reaction coordinate are needed to rationally modulate the lifetime of the drug-target complex. Previously, we predicted the structure of the rate-limiting transition state that controlled the time-dependent inhibition of the enoyl-ACP reductase InhA. This led to the discovery of a triazole-containing diphenyl ether with an increased residence time on InhA due to transition-state destabilization rather than ground-state stabilization. In the present work, we evaluate the inhibition of InhA by 14 triazole-based diphenyl ethers and use a combination of enzyme kinetics and X-ray crystallography to generate a structure-kinetic relationship for time-dependent binding. We show that the triazole motif slows the rate of formation for the final drug-target complex by up to 3 orders of magnitude. In addition, we identify a novel inhibitor with a residence time on InhA of 220 min, which is 3.5-fold longer than that of the INH-NAD adduct formed by the tuberculosis drug, isoniazid. This study provides a clear example in which the lifetime of the drug-target complex is controlled by interactions in the transition state for inhibitor binding rather than the ground state of the enzyme-inhibitor complex, and demonstrates the important role that on-rates can play in drug-target residence time.

Enhanced Simulations and Drug Discovery of a Muscarinic G-Protein-Coupled Receptor

G-protein-coupled receptors (GPCRs) represent primary targets of about one third of currently marketed drugs. However, GPCR drug discovery has suffered from major challenges, including the highly flexible nature of the receptors and the toxicity of agonist or antagonist drugs that are designed to bind the conserved “orthosteric” site. Here, we have performed all-atom enhanced simulations using the robust Gaussian accelerated molecular dynamics (GaMD) method to investigate the ligand-dependent structural dynamics of the M2 muscarinic GPCR. The GaMD simulations revealed distinct structural flexibility and free energy profiles that depict graded activation of the GPCR. Both dissociation and binding of an orthosteric ligand were captured in a single all-atom GPCR simulation(1). In addition, we have implemented a unique structure-based approach to design allosteric modulators as selective drug leads of the M2 muscarinic receptor. Through iterative molecular docking and experimental testing, half of our 38 computationally selected lead compounds were validated as effective allosteric modulators of the M2 receptor. Our method successfully identified both positive and negative allosteric modulators of the M2 muscarinic receptor with unprecedented chemical diversity and outstanding potential for further structure-activity relationship studies(2). This approach shall be of wide applicability for drug discovery of many other GPCRs(3).

3D descriptors calculation and conformational search to investigate potential bioactive conformations, with application in 3D-QSAR and virtual screening in drug design

The knowledge of the bioactive conformation for an active hit is relevant because of the easier interpretation and the general quality of the recognition models of protein and ligand. With the aim of investigating potential bioactive conformations without previous structural knowledge of the molecular target, we present herewith a ‘protocol’ that could be used which includes generation of low-energy conformations, calculations of tridimensional descriptors and investigation of structural similarity via principal component analysis. The protocol was used in the search for potential bioactive conformations. An initial selection of targets was made from a set of protein–ligand complexes with structure deposited in the Protein Data Bank, which was systematically filtered by lead-like rules, resulting in 45 ligands of 8 important therapeutic targets. After extensive optimization of the protocol and parameters of both OMEGA and Pentacle softwares, the best results were obtained for series of compounds such as the beta-trypsin and urokinase inhibitors, which are more structurally related among each other, inside the respective therapeutic class. Future improvements of the protocol, including a suitable choice and combination of robust 3D descriptors, could yield more reliable and less restrictive results, with general and diverse applications in drug design, in particular for improving the 3D-QSAR methodologies as well as virtual screening experiments for a more reliable selection of new lead compounds for different molecular targets.

The process of drug discovery and the Yin/Yang of small-molecule/biotech option

Until the mid 90ties of last century, talking about drug discovery and development was essentially referred to the process of discovery and development of small-molecule chemical entities. All the language, common procedures and technicalities were inherent to this setting; concepts such as drug design, hit and lead compounds, high-throughput screening were referring to the typical mode of investigating and selecting small-molecule candidates through pre-clinical development. At that time, the arrival of the first biotech drugs has markedly changed the landscape, requiring a completely new approach to pre-clinical development. Issues related to drug-receptor interaction or to the selection from a huge number of candidates were obviously simplified to a minimum compared to the small-molecule setting. Conversely, the pharmaceutical development of biotech drugs showed far higher complexity, and the overall high complexity of industrial production initially represented a major issue to justify the high costs of biotech drugs.Besides, the technical impossibility to make ‘generics’ of biotech drugs was another strong reason driving Pharma companies toward the development of biotech drugs. Thus, in the beginning the early development phases of small-molecules or biotech drugs really stood as two distant planets. However, by the time such initial distances have been progressively reduced. From the biotech site, the technique of phage-display library scanning and similar approaches made the selection of biotech lead compounds resembling more closely what happens with small molecules; also the complexities and high costs of pharmaceutical production have progressively reduced their impact. On the other site, the impressive progresses of basic knowledge on the human kinome and other relevant fields of molecular and cellular biology made possible that today we have small-molecule drugs targeting the same pathologies once specifically targeted by biotech drugs, MoAbs in particular.Another factor (albeit not related to drug development) with an important role in reducing distances between small-molecule and biotech drugs has been the novel approach to estimate the value of drugs, and therefore their prices. Since nowadays the major drive fixing the value (and costs) of new drugs is the added value for human health, any difference related to the costs of production has been greatly reduced, and we have today several high-cost small molecules along with their sibling biotech drugs.Last but not least, biotech drugs can be ‘copied’ as well, since we now have biosimilar drugs, although the pathway to develop copies of biotech drugs remains steeper compared to that leading to the equivalents of small-molecule drugs.

Abstract 4798: Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL

Abstract Interleukin-1 receptor associated kinases (IRAKs) are serine/threonine protein kinases belonging to the tyrosine-like kinase (TLK) family. IRAKs function as mediators of Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) signaling pathways and play an important role in innate immune signaling. TLR/IL-1R stimulation leads to recruitment of MYD88, an adaptor molecule, to the activated receptor complex, which then complexes with IRAK4 and activates IRAK1. TRAF6 is then activated by IRAK1 leading to NFkB activation. Recent studies have reported the occurrence of gain of function oncogenic mutation (L265P) in MYD88 in ∼30% of activated B cell diffuse large B-cell lymphoma(ABC DLBCL) and ∼90% of Waldenstrom's macroglobulinemia (WM) leading to constitutive activation of IRAK4 and NFkB pathway. Among the DLBCL subtypes (GCB, ABC DLBCL and PMBL), ABC DLBCL is the most refractory. Inhibition of constitutive IRAK4 signalling can be used as a therapeutic strategy to treat ABC DLBCL Small molecule inhibitors of IRAK4 were synthesized based on hits originating from Aurigene's compound library. Structure guided drug design approach was used to further improve the potency. Lead compounds demonstrated moderate to very high selectivity towardsIRAK4 (S35 score of 0.03) when screened against a large panel of 329 kinases. Aurigene's lead compounds have excellent PK profile and good oral bioavailability in mice, leading to good in-vivo activity in TLR4 induced cytokine release model. Selected lead compounds were tested in a OCI-Ly3 xenograft model, which has a MYD88(L265P) mutation leading to constitutive activation of IRAK4 signaling. An advanced lead compound has demonstrated excellent efficacy in OCI-Ly3 model, with tumor stasis at low doses and tumor regression at higher doses. The compound is well tolerated and has a good therapeutic window as determined in a 14 day rodent toxicity study. In summary, a selective IRAK4 inhibitor has been identified with excellent efficacy and good safety profile. Citation Format: Wesley Roy Balasubramanian, Venkateshwar Rao Gummadi, Kavitha Nellore, Subhendu Mukherjee, Sivapriya Marappan, Aravind Basavaraju, Bharathi Raja Ainan, Girish Daginakatte, Sreevalsam Gopinath, Sanjeev Giri, Thomas Antony, Shekar Chelur, Susanta Samajdar, Chetan Pandit, Murali Ramachandra. Efficacy and safety of highly selective novel IRAK4 inhibitors for treatment of ABC-DLBCL. [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 4798.

Abstract 4337: Novel potent and selective orally available CDK8/19 kinase inhibitors

Abstract CDK8 and its paralog CDK19 are cyclin-dependent kinases and together with CDK7 and CDK9 belongs to the group of C-terminal domain (CTD) kinases that phosphorylate the CTD of RNA polymerase II, thus regulating transcription. CDK8 together with its partner Cyclin C, MED12 and MED13 are components of multi-protein Mediator complex which couples action of transcription factors with the molecular machinery that carries out transcription, e.g. CDK8 couple basal transcriptional machinery to sequence-specific transcription factors such as Notch, p53, β-catenin, and also repress the transcription of other genes. As Mediator independent roles, CDK8 has been shown to act as part of a separate complex behaving as a histone kinase. Moreover, CDK8 has been identified as a major kinase in the response to IFN signaling mediated STAT1-S727 phosphorylation. Several studies indicated that high overexpression and activity of CDK8 could be a driver of malignant progression in colorectal cancer being a marker of poor prognosis. Moreover, in gastric cancer CDK8 expression and the delocalization of β-catenin expression showed a significant positive correlation with carcinogenesis and tumor progression, especially lymph node metastasis. Recently, gene amplification of CDK8, CDK19, CCNC and MED13 in breast cancers has been related with poor response to adjuvant therapy. These results suggest that CDK8 inhibitors may become a unique class of anticancer drugs that could increase the efficacy of cancer therapy by blocking chemotherapy-induced production of tumor-promoting secreted factors. We have carried out a medium throughput screening campaign which led to the discovery of several low nanomolar hits belonging to 3 different chemical series. Later on, we have performed a Hit Generation phase, designing novel inhibitors within a patentable chemical space. Here, we have taken into account information from the chemical structures of the screening hits, X-ray structure of CDK8 protein co-crystalized with sorafenib, docking studies, chemical feasibility and intellectual property. We have identified a novel chemical series of CDK8/19 inhibitors. After a HtL exploration we have reached lead compounds with potency in the picomolar range and high selectivity versus a panel of 468 protein kinases. The leads display cellular activity by blocking β−catenin reporter activity and STAT1 phoshorylation in the low nanomolar range. Lead compounds have been screened in a panel of tumoral cell lines, showing a defined profile in terms of sensitivity with GI50s which range from nanomolar to low micromolar values. These inhibitors induce cell death in a dose response manner. The novel chemical series show a drug-like profile in terms of solubility, permeability, CYP450 inhibition and hERG. The identified lead compounds are orally bioavailable, with good clearance, volume of distribution and exposure levels. A selected lead compound has been used in in vivo PK-PD studies showing positive results. Citation Format: Carmen Blanco Aparicio, Oliver Renner, Elena Gomez-Casero, Maria Isabel Albarran, Antonio Cebria, Borja Barrera, Enara Aguirre, Maria del Carmen Rodriguez de Miguel, Manuel Urbano, Ana Isabel Hernandez, Cristina Gomez de la Oliva, Virginia Rivero, Rosario Concepcion Riesco, Sonia Martinez Gonzalez, Joaquin Pastor. Novel potent and selective orally available CDK8/19 kinase inhibitors. [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 4337.

Abstract 4861: Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy

Abstract Recent successes in achieving highly durable clinical responses with antibodies to immune checkpoint receptors such as CTLA4 and PD1 have transformed the outlook for cancer therapy. While these antibody-based therapies show impressive clinical activity, they suffer from the shortcomings including the need to administer by intravenous injection, failure to show response in majority of patients and immune-related adverse events (irAEs) due to the breaking of immune self-tolerance. Sustained target inhibition as a result of a long half-life (>15-20 days) and >70% target occupancy for months may be factors contributing to irAEs observed. We sought to discover and develop small molecule immune checkpoint antagonists capable of targeting PD-L1 and another immune checkpoint pathway. We reasoned that such therapeutic agents will be amenable for oral dosing, likely show greater response rate due to dual antagonism and allow better management of irAEs due a shorter pharmacokinetic profile. A focused library of compounds mimicking the interaction of checkpoint proteins was designed and synthesized. Screening and analysis of the resulting library led to the identification of hits capable of functional disruption of the checkpoint protein(s) signaling depending upon the pockets of sequence similarity of interacting proteins. Further optimization resulted in compounds targeting PD-L1/VISTA or PD-L1/TIM-3 with desirable physico-chemical properties and exposure upon oral administration.. The ability of compounds to disrupt specific immune checkpoint pathways was confirmed though functional studies. Identified lead compounds exhibit potent activity when tested in assays to rescue lymphocyte proliferation and effector functions inhibited by respective ligands/proteins. In a panel of functional assays, the selected lead compounds showed selectivity against other immune checkpoint pathways including CTLA4, LAG3 and BTLA. Lead compounds exhibited sustained immune PD in vitro and in vivo suggesting that drug efficacy may extend beyond drug clearance. Lead compounds exhibited significant efficacy in syngeneic pre-clinical tumor models of melanoma, breast carcinoma and colon cancers upon once a day oral dosing. In repeated dose toxicity studies, the most advanced compound, AUPM-170, a dual antagonist of PD-L1 and VISTA, was well tolerated at >100x of the efficacious doses. The data demonstrating the inhibition of PD-L1 and another immune checkpoint pathway (VISTA or Tim3) resulting in activation of T cells and anti-tumor activities support further development of these orally bioavailable agents. IND-enabling studies with one of the lead compounds, AUPM-170, are underway towards advancing it to the clinic. Citation Format: Pottayil Sasikumar, N S Sudarshan, Nagaraj Gowda, D S Samiulla, Raghuveer Ramachandra, T Chandrasekhar, Sreenivas Adurthi, Jiju Mani, Rashmi Nair, Sharad Singh, Amit Dhudashia, Nagesh Gowda, Murali Ramachandra. Oral immune checkpoint antagonists targeting PD-L1/VISTA or PD-L1/Tim3 for cancer therapy. [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 4861.