Discovery of novel spiro compound as RAF kinase inhibitor with in vitro potency against KRAS mutant cancer.

The development of RAF inhibitors targeting cancers with wild type RAF kinase and/or RAS mutation has been challenging due to the paradoxical activation of the RAS-RAF-MEK-ERK cascade following RAF inhibitor treatment. Herein is the discovery and optimization of a series of RAF inhibitors with a novel spiro structure. The most potent spiro molecule 9 showed excellent in vitro potency against b/c RAF enzymes and RAS mutant H358 cancer cells with minimal paradoxical RAF signaling activation. Compound 9 also exhibited good drug-like properties as demonstrated by in vitro cytochrome P450 (CYP), liver microsome stability (LMS) data and moderate oral pharmacokinetics (PK) profiles in rat and mouse.

Discovery of EBI-1051: A novel and orally efficacious MEK inhibitor with benzofuran scaffold.

A novel series of benzodihydrofuran derivatives was developed as potent MEK inhibitors through scaffold hopping based on known clinical compounds. Further SAR exploration and optimization led to another benzofuran series with good oral bioavailability in rats. One of the compounds EBI-1051 (28d) demonstrated excellent in vivo efficacy in colo-205 tumor xenograft models in mouse and is suitable for pre-clinical development studies for the treatment of melanoma and MEK associated cancers. Compared to AZD6244, EBI-1051 showed superior potency in some cancer cell lines such as colon-205, A549 and MDA-MB-231.

WITHDRAWN: Discovery of SHR1977: A highly potent and selective ROMK inhibitor.

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Discovery of EBI-907: A highly potent and orally active B-Raf(V600E) inhibitor for the treatment of melanoma and associated cancers.

A novel series of pyrazolo[3,4-c]isoquinoline derivatives was discovered as B-Raf(V600E) inhibitors through scaffold hopping based on a literature lead PLX4720. Further SAR exploration and optimization led to the discovery of potent B-Raf(V600E) inhibitors with good oral bioavailability in rats and dogs. One of the compounds EBI-907 (13g) demonstrated excellent in vivo efficacy in B-Raf(V600E) dependent Colo-205 tumor xenograft models in mouse and is under preclinical studies for the treatment of melanoma and B-Raf(V600E) associated cancers.

Induction of apoptosis by an inhibitor of the mitotic kinesin KSP requires both activation of the spindle assembly checkpoint and mitotic slippage.

The inhibition of KSP causes mitotic arrest by activating the spindle assembly checkpoint. While transient inhibition of KSP leads to reversible mitotic arrest, prolonged exposure to a KSP inhibitor induces apoptosis. Induction of apoptosis by the KSP inhibitor couples with mitotic slippage. Slippage-refractory cells show resistance to KSP inhibitor-mediated lethality, whereas promotion of slippage after mitotic arrest enhances apoptosis. However, attenuation of the spindle checkpoint confers resistance to KSP inhibitor-induced apoptosis. Furthermore, sustained KSP inhibition activates the proapoptotic protein, Bax, and both activation of the spindle checkpoint and subsequent mitotic slippage are required for Bax activation. These studies indicate that in response to KSP inhibition, activation of the spindle checkpoint followed by mitotic slippage initiates apoptosis by activating Bax.

Pyridyl aminothiazoles as potent Chk1 inhibitors: optimization of cellular activity.

Translation of significant biochemical activity of pyridyl aminothiazole class of Chk1 inhibitors into functional CEA potency required analysis and adjustment of both physical properties and kinase selectivity profile of the series. The steps toward optimization of cellular potency included elimination of CDK7 activity, reduction of molecular weight and polar surface area and increase in lipophilicity of the molecules in the series.

Mechanistically probing lipid-siRNA nanoparticle-associated toxicities identifies Jak inhibitors effective in mitigating multifaceted toxic responses.

A major hurdle for harnessing small interfering RNA (siRNA) for therapeutic application is an effective and safe delivery of siRNA to target tissues and cells via systemic administration. While lipid nanoparticles (LNPs) composed of a cationic lipid, poly-(ethylene glycol) lipid and cholesterol, are effective in delivering siRNA to hepatocytes via systemic administration, they may induce multi-faceted toxicities in a dose-dependent manner, independently of target silencing. To understand the underlying mechanism of toxicities, pharmacological probes including anti-inflammation drugs and specific inhibitors blocking different pathways of innate immunity were evaluated for their abilities to mitigate LNP-siRNA-induced toxicities in rodents. Three categories of rescue effects were observed: (i) pretreatment with a Janus kinase (Jak) inhibitor or dexamethasone abrogated LNP-siRNA-mediated lethality and toxicities including cytokine induction, organ impairments, thrombocytopenia and coagulopathy without affecting siRNA-mediated gene silencing; (ii) inhibitors of PI3K, mammalian target of rapamycin (mTOR), p38 and IκB kinase (IKK)1/2 exhibited a partial alleviative effect; (iii) FK506 and etoricoxib displayed no protection. Furthermore, knockout of Jak3, tumor necrosis factor receptors (Tnfr)p55/p75, interleukin 6 (IL-6) or interferon (IFN)-γ alone was insufficient to alleviate LNP-siRNA-associated toxicities in mice. These indicate that activation of innate immune response is a primary trigger of systemic toxicities and that multiple innate immune pathways and cytokines can mediate toxic responses. Jak inhibitors are effective in mitigating LNP-siRNA-induced toxicities.

Discovery of SHR2415, a Novel Pyrrole-Fused Urea Scaffold ERK1/2 Inhibitor.

ERK1/2 kinase is a key downstream node of the RAS-RAF-MEK-ERK signaling pathway. A highly potent and selective ERK1/2 inhibitor is a promising option for cancer treatment that will provide a potential solution for overcoming drug resistance. Herein we designed and synthesized a novel scaffold featuring a pyrrole-fused urea template. The lead compound, SHR2415, was shown to be a highly potent ERK1/2 inhibitor that exhibited high cell potency based on the Colo205 assay. In addition, SHR2415 displayed favorable PK profiles across species as well as robust in vivo efficacy in a mouse Colo205 xenograft model.

Discovery of Isoindoline Amide Derivatives as Potent and Orally Bioavailable ADAMTS-4/5 Inhibitors for the Treatment of Osteoarthritis.

Osteoarthritis (OA) treatment is a highly unmet medical need. Development of a disease-modifying OA drug (DMOAD) is challenging with no approved drugs on the market. Inhibition of ADATMS-4/5 is a promising OA therapeutics to target cartilage degradation and potentially can reduce joint pain and restore its normal function. Starting from the reported ADAMTS-5 inhibitor GLPG1972, we applied a scaffold hopping strategy to generate a novel isoindoline amide scaffold. Representative compound 18 showed high potency in ADATMS-4/5 inhibition, as well as good selectivity over a panel of other metalloproteases. In addition, compound 18 exhibited excellent druglike properties and showed better pharmacokinetic (PK) profiles than GLPG1972 cross-species. Compound 18 demonstrated dose-dependent efficacy in two in vivo rat osteoarthritis models.

Discovery of SHR5133, a Highly Potent and Novel HBV Capsid Assembly Modulator.

Capsid assembly modulators (CpAMs) represent a new class of antivirals targeting hepatitis B virus (HBV) core protein to disrupt the assembly process. In this work, a novel chemotype featuring a fused heterocycle amide was discovered through pharmacophore exploration. Lead optimization resulted in compound 8 with an EC50 value of 511 nM, and then methyl substitution on the piperazine was found to improve the in vitro potency remarkably. Further SAR studies established the key compound SHR5133, which showed high in vitro antiviral potency, favorable pharmacokinetic profiles across species, and robust in vivo efficacy.

Novel Small Molecule Tyrosine Kinase 2 Pseudokinase Ligands Block Cytokine-Induced TYK2-Mediated Signaling Pathways.

A member of the Janus kinase (JAK) family, Tyrosine Kinase 2 (TYK2), is crucial in mediating various cytokine-signaling pathways such as interleukin-23 (IL23), interleukin-12 (IL12) and type I Interferons (IFN) which contribute to autoimmune disorders (e.g., psoriasis, lupus, and inflammatory bowel disease). Thus, TYK2 represents an attractive target to develop small-molecule therapeutics for the treatment of cytokine-driven inflammatory diseases. Selective inhibition of TYK2 over other JAK isoforms is critical to achieve a favorable therapeutic index in the development of TYK2 inhibitors. However, designing small molecule inhibitors to target the adenosine triphosphate (ATP) binding site of TYK2 kinase has been challenging due to the substantial structural homology of the JAK family catalytic domains. Here, we employed an approach to target the JAK homology 2 (JH2) pseudokinase regulatory domain of the TYK2 protein. We developed a series of small-molecule TYK2 pseudokinase ligands, which suppress the TYK2 catalytic activity through allosteric regulation. The TYK2 pseudokinase-binding small molecules in this study simultaneously achieve high affinity-binding for the TYK2 JH2 domain while also affording significantly reduced affinity for the TYK2 JAK homology 1 (JH1) kinase domain. These TYK2 JH2 selective molecules, although possessing little effect on suppressing the catalytic activity of the isolated TYK2 JH1 catalytic domain in the kinase assays, can still significantly block the TYK2-mediated receptor-stimulated pathways by binding to the TYK2 JH2 domain and allosterically regulating the TYK2 JH1 kinase. These compounds are potent towards human T-cell lines and primary immune cells as well as in human whole-blood specimens. Moreover, TYK2 JH2-binding ligands exhibit remarkable selectivity of TYK2 over JAK isoforms not only biochemically but also in a panel of receptor-stimulated JAK1/JAK2/JAK3-driven cellular functional assays. In addition, the TYK2 JH2-targeting ligands also demonstrate high selectivity in a multi-kinase screening panel. The data in the current study underscores that the TYK2 JH2 pseudokinase is a promising therapeutic target for achieving a high degree of biological selectivity. Meanwhile, targeting the JH2 domain represents an appealing strategy for the development of clinically well-tolerated TYK2 inhibitors that would have superior efficacy and a favorable safety profile compared to the existing Janus kinase inhibitors against autoimmune diseases.

SHR1032, a novel STING agonist, stimulates anti-tumor immunity and directly induces AML apoptosis.

Stimulator of interferon genes (STING) activation induces type I interferons and pro-inflammatory cytokines which stimulate tumor antigen cross presentation and the adaptive immune responses against tumor. The first-generation of STING agonists, cyclic di-nucleotide (CDN), mimicked the endogenous STING ligand cyclic guanosine monophosphate adenosine monophosphate, and displayed limited clinical efficacy. Here we report the discovery of SHR1032, a novel small molecule non-CDN STING agonist. Compared to the clinical CDN STING agonist ADU-S100, SHR1032 has much higher activity in human cells with different STING haplotypes and robustly induces interferon ß (IFNß) production. When dosed intratumorally, SHR1032 induced strong anti-tumor effects in the MC38 murine syngeneic tumor model. Pharmacodynamic studies showed induction of IFNß, tumor necrosis factor α (TNFα) and interleukin-6 (IL-6) in the tumors and, to a lower extent, in the plasma. More importantly, we found SHR1032 directly causes cell death in acute myeloid leukemia (AML) cells. In conclusion, our findings demonstrate that in addition to their established ability to boost anti-tumor immune responses, STING agonists can directly eradicate AML cells, and SHR1032 may present a new and promising therapeutic agent for cancer patients.

Discovery of spiro amide SHR902275: A potent, selective, and efficacious RAF inhibitor targeting RAS mutant cancers.

The RAS-RAF-MEK-ERK signaling pathway plays a key role to regulate multiple cellular functions. Acquired resistance to the first-generation RAF inhibitors that only targeted the bRAFV600E mutation prompted the need for a new generation of RAF inhibitors to target cancers bearing mutant RAS and wild type RAF activity by inhibition of paradoxical activation. Starting from the company's previously reported RAF inhibitor 1, extensive drug potency and drug-like properties optimizations led to the discovery of molecule 33 (SHR902275) with greatly improved in vitro potency and solubility. Molecule 33 exhibited good DMPK (Drug Metabolism and Pharmacokinetics) properties, excellent permeability, and outstanding mouse/rat oral PK. It was further evaluated in an in vivo RAS mutant Calu6 xenograft mouse model and demonstrated dose dependent efficacy. To achieve high exposure in a toxicity study, pro-drug 48 was also explored.

Noninvasive imaging of lipid nanoparticle-mediated systemic delivery of small-interfering RNA to the liver.

Mouse models with liver-specific expression of firefly luciferase were developed that enable a noninvasive and longitudinal assessment of small-interfering RNA (siRNA)-mediated gene silencing in hepatocytes of live animals via bioluminescence imaging. Using these models, a set of lipid nanoparticles (LNPs) with different compositions of cationic lipids, polyethylene glycol (PEG), and cholesterol, were tested for their abilities in delivering a luciferase siRNA to the liver via systemic administration. A dose-dependent luciferase knockdown by LNP/siRNA assemblies was measured by in vivo bioluminescence imaging, which correlated well with the results from parallel ex vivo analyses of luciferase mRNA and protein levels in the liver. RNA interference (RNAi)-mediated target silencing was further confirmed by the detection of RNAi-specific target mRNA cleavage. A single dose of LNP02L at 3 mg/kg (siRNA) caused 90% reduction of luciferase expression and the target repression lasted for at least 10 days. With identical components, LNPs containing 2% PEG are more potent than those with 5.4% PEG. Our results demonstrate that these liver-luciferase mouse models provide a powerful tool for a high-throughput evaluation of hepatic delivery platforms by noninvasive imaging and that the molar ratio of PEG lipid can affect the efficacy of LNPs in silencing liver targets via systemic administration.

A Novel CD73 Inhibitor SHR170008 Suppresses Adenosine in Tumor and Enhances Anti-Tumor Activity with PD-1 Blockade in a Mouse Model of Breast Cancer.

INTRODUCTION: CD73 and adenosine support growth-promoting neovascularization, metastasis, and survival in cells, and promote anti-PD-1 mAb therapy-induced immune escape. Consequently, developing a CD73 inhibitor as monotherapy and a potential beneficial combination partner with immune-checkpoint inhibitors needs investigation. METHODS: CD73 inhibitors were evaluated in vitro with soluble and membrane-bound CD73 enzymes, as well as its PD biomarker responses in human peripheral blood mononuclear cells (PBMC) by flow cytometry and ELISA. The binding modes of the molecules were analyzed via molecular modeling. The anti-tumor activity and synergistic effect of SHR170008 in combination with anti-PD-1 mAb were evaluated in a syngeneic mouse breast cancer model. RESULTS: SHR170008 was discovered during the initial structural modifications on the link between the ribose and the α-phosphate of AMPCP, which significantly improved the stability of the compound confirmed by the metabolite identification study. Further modifications on the adenine base of AMPCP improved the potency due to forming stronger interactions with CD73 protein. It exhibited potent inhibitory activities on soluble and endogenous membrane-bound CD73 enzymes, and induced IFNγ production, reversed AMP-suppressed CD25+ and CD8+/CD25+ expression, and enhanced granzyme B production on CD8+ T cells in human PBMC. SHR170008 showed dose-dependent anti-tumor efficacy with suppression of adenosine in the tumors in EMT6 mouse breast tumor model. The increase of adenosine in tumor tissue by anti-PD-1 mAb alone was suppressed by SHR170008 in the combination groups. Simultaneous inhibition of CD73 and PD-1 neutralization synergistically enhanced antitumor immunity and biomarkers in response, and exposures of SHR170008 were correlated with the efficacy readouts. CONCLUSION: Our findings suggest that CD73 may serve as an immune checkpoint by generating adenosine, which suppresses the antitumor activity of anti-PD-1 mAb, and inhibition of CD73 may be a potential beneficial combination partner with immune-checkpoint inhibitors to improve their therapeutic outcomes in general.

Discovery of a novel RORγ antagonist with skin-restricted exposure for topical treatment of mild to moderate psoriasis.

Clinical success of IL-17/IL-23 pathway biologics for the treatment of moderate to severe psoriasis suggests that targeting RORγt, a master regulator for the proliferation and function of Th17 cells, could be an effective alternative. However, oral RORγ antagonists (VTP43742, TAK828) with high systemic exposure showed toxicity in phase I/II clinical trials and terminated development. To alleviate the potential safety concerns, identifying compounds with skin-restricted exposure amenable for topical use is of great interest. Systematic structure activity relationship study and multi-parameter optimization led to the discovery of a novel RORγ antagonist (SHR168442) with desired properties for a topical drug. It suppressed the transcription of IL-17 gene, leading to reduction of IL-17 cytokine secretion. It showed high exposure in skin, but low in plasma. Topical application of SHR168442 in Vaseline exhibited excellent efficacy in the imiquimod-induced and IL-23-induced psoriasis-like skin inflammation mouse models and correlated with the reduction of Th17 pathway cytokines, IL-6, TNFα and IL-17A. This work demonstrated restricted skin exposure of RORγ antagonist may provide a new topical treatment option as targeted therapeutics for mild to moderate psoriasis patients and may be suitable for the treatment of any other inflammatory disorders that are accessible locally.

Discovery of Hydroxyamidine Derivatives as Highly Potent, Selective Indoleamine-2,3-dioxygenase 1 Inhibitors.

In this study, a series of novel hydroxyamidine derivatives were identified as potent and selective IDO1 inhibitors by structure-based drug design. Among them, compounds 13-15 and 18 exhibited favorable enzymatic and cellular activities. Compound 18 showed improved bioavailability in mouse, rat, and dog (F% = 44%, 58.8%, 102.1%, respectively). With reasonable in vivo pharmacokinetic properties, compound 18 was further evaluated in a transgenic MC38 xenograft mouse model. The combination of compound 18 with PD-1 monoclonal antibody showed a synergistic antitumor effect. These data indicated that compound 18 as a potential cancer immunotherapy agent should warrant further investigation.

Discovery of 2-(Ortho-Substituted Benzyl)-Indole Derivatives as Potent and Orally Bioavailable RORγ Agonists with Antitumor Activity.

RORγ is a dual-functional drug target, which involves not only induction of inflammation but also promotion of cancer immunity. The development of agonists of RORγ promoting Th17 cell differentiation could provide a novel mechanism of action (MOA) as an immune-activating anticancer agent. Herein, we describe new 2-(ortho-substituted benzyl)-indole derivatives as RORγ agonists by scaffold hopping based on clinical RORγ antagonist VTP-43742. Interestingly, subtle structural differences of the compounds led to the opposite biological MOA. After rational optimization for structure-activity relationship and pharmacokinetic profile, we identified a potent RORγ agonist compound 17 that was able to induce the production of IL-17 and IFNγ in tumor tissues and elicit antitumor efficacy in MC38 syngeneic mouse colorectal tumor model. This is the first comprehensive work to demonstrate the in vivo antitumor efficacy of an RORγ agonist.

An inhibitor of the kinesin spindle protein activates the intrinsic apoptotic pathway independently of p53 and de novo protein synthesis.

The kinesin spindle protein (KSP), a microtubule motor protein, is essential for the formation of bipolar spindles during mitosis. Inhibition of KSP activates the spindle checkpoint and causes apoptosis. It was shown that prolonged inhibition of KSP activates Bax and caspase-3, which requires a competent spindle checkpoint and couples with mitotic slippage. Here we investigated how Bax is activated by KSP inhibition and the roles of Bax and p53 in KSP inhibitor-induced apoptosis. We demonstrate that small interfering RNA-mediated knockdown of Bax greatly attenuates KSP inhibitor-induced apoptosis and that Bax activation is upstream of caspase activation. This indicates that Bax mediates the lethality of KSP inhibitors and that KSP inhibition provokes apoptosis via the intrinsic apoptotic pathway where Bax activation is prior to caspase activation. Although the BH3-only protein Puma is induced after mitotic slippage, suppression of de novo protein synthesis that abrogates Puma induction does not block activation of Bax or caspase-3, indicating that Bax activation is triggered by a posttranslational event. Comparison of KSP inhibitor-induced apoptosis between matched cell lines containing either functional or deficient p53 reveals that inhibition of KSP induces apoptosis independently of p53 and that p53 is dispensable for spindle checkpoint function. Thus, KSP inhibitors should be active in p53-deficient tumors.

Discovery of SHR0687, a Highly Potent and Peripheral Nervous System-Restricted KOR Agonist.

Analgesics with no abuse liability are highly demanded in the market. KOR agonists have been proved to be strong analgesics without MOR agonist-related side effects, such as respiratory depression, tolerance, and dependence liability; however, activation of KOR in the central nervous system (CNS) may cause sedation and anxiety. It has been reported that peripheral KOR activation produces comparable bioactivity without CNS-related side effects. Herein, we designed and synthesized a novel tetrapeptide (SHR0687), which was shown to be a highly potent KOR agonist with excellent selectivity over other opioid receptors, such as MOR and DOR. In addition, SHR0687 displayed favorable PK profiles across species, as well as robust in vivo efficacy in a rat carrageenan-induced pain model. Notably, SHR0687 exhibited negligible blood-brain barrier penetration, which was meaningful in minimizing CNS-related side effects.