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Currently, there is a significant unmet need for novel analgesics with fewer side effects. In this study, we carried out structural modification of a hit compound previously identified in an artificial-intelligence (AI) virtual screening and discovered the potent analgesic, benzo[b]thiophene-2-carboxamide analog (compound 25) with new structural scaffold. We investigated the signaling pathways of opioid receptors mediated by compound 25, and found this racemic compound activated mu-opioid receptor through the cyclic adenosine monophosphate (cAMP) and β-arrestin-2-mediated pathways with strong potency and efficacy, and accompanying nociceptin-orphanin FQ opioid peptide and delta-opioid receptors through the cAMP pathway with weak potencies. Compound 25 elicited potent antinociception in thermal-stimulated pain (ED

Morphine is widely used for the treatment of severe pain. This analgesic effect is mediated principally by the activation of μ-opioid receptors (MOR). However, prolonged activation of MOR also results in tolerance, dependence, addiction, constipation, nausea, sedation, and respiratory depression. To address this problem, we sought alternative ways to activate MOR – either by use of novel ligands, or via a novel activation mechanism. To this end, a series of compounds were screened using a sensitive CHO-K1/MOR/Gα15 cell-based FLIPR® calcium high-throughput screening (HTS) assay, and the bithiazole compound 5a was identified as being able activate MOR in combination with naloxone. Structural modifications of 5a resulted in the discovery of lead compound 5j, which could effectively activate MOR in combination with the MOR antagonist naloxone or naltrexone. In vivo, naloxone in combination with 100 mg/kg of compound 5j elicited antinociception in a mouse tail-flick model with an ED50 of 17.5 ± 4 mg/kg. These results strongly suggest that the mechanism by which the 5j/naloxone combination activates MOR is worthy of further study, as its discovery has the potential to yield an entirely novel class of analgesics.

Significance A highly selective, safe, and potent CXCR4 antagonist, BPRCX807, has been designed and experimentally validated in various hepatocellular carcinoma models. Through combination therapy, it can synergize with either a kinase (e.g., sorafenib) or checkpoint inhibitor (e.g. anti–PD-1) to augment effectiveness of current anticancer treatments. With its unique mode of action, a new anticancer strategy for preventing cell migration and metastasis is provided., The CXC chemokine receptor type 4 (CXCR4) receptor and its ligand, CXCL12, are overexpressed in various cancers and mediate tumor progression and hypoxia-mediated resistance to cancer therapy. While CXCR4 antagonists have potential anticancer effects when combined with conventional anticancer drugs, their poor potency against CXCL12/CXCR4 downstream signaling pathways and systemic toxicity had precluded clinical application. Herein, BPRCX807, known as a safe, selective, and potent CXCR4 antagonist, has been designed and experimentally realized. In in vitro and in vivo hepatocellular carcinoma mouse models it can significantly suppress primary tumor growth, prevent distant metastasis/cell migration, reduce angiogenesis, and normalize the immunosuppressive tumor microenvironment by reducing tumor-associated macrophages (TAMs) infiltration, reprogramming TAMs toward an immunostimulatory phenotype and promoting cytotoxic T cell infiltration into tumor. Although BPRCX807 treatment alone prolongs overall survival as effectively as both marketed sorafenib and anti–PD-1, it could synergize with either of them in combination therapy to further extend life expectancy and suppress distant metastasis more significantly.

Background New therapeutic options to address the ongoing COVID-19 pandemic are urgently needed. One possible strategy is the repurposing of existing drugs approved for other indications as antiviral agents for SARS-CoV-2. Due to the commercial unavailability of SARS-CoV-2 drugs for treating COVID-19, we screened approximately 250 existing drugs or pharmacologically active compounds for their inhibitory activities against feline infectious peritonitis coronavirus (FIPV) and human coronavirus OC43 (HCoV-OC43), a human coronavirus in the same genus (Betacoronavirus) as SARS-CoV-2. Methods FIPV was proliferated in feline Fcwf-4 cells and HCoV-OC43 in human HCT-8 cells. Viral proliferation was assayed by visualization of cytopathic effects on the infected Fcwf-4 cells and immunofluorescent assay for detection of the nucleocapsid proteins of HCoV-OC43 in the HCT-8 cells. The concentrations (EC50) of each drug necessary to diminish viral activity to 50% of that for the untreated controls were determined. The viabilities of Fcwf-4 and HCT-8 cells were measured by crystal violet staining and MTS/PMS assay, respectively. Results Fifteen out of the 252 drugs or pharmacologically active compounds screened were found to be active against both FIPV and HCoV-OC43, with EC50 values ranging from 11 nM to 75 μM. They are all old drugs as follows, anisomycin, antimycin A, atovaquone, chloroquine, conivaptan, emetine, gemcitabine, homoharringtonine, niclosamide, nitazoxanide, oligomycin, salinomycin, tilorone, valinomycin, vismodegib. Conclusion All of the old drugs identified as having activity against FIPV and HCoV-OC43 have seen clinical use in their respective indications and are associated with known dosing schedules and adverse effect or toxicity profiles in humans. Those, when later confirmed to have an anti-viral effect on SARS-CoV-2, should be considered for immediate uses in COVID-19 patients., Graphical abstract Image 1

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C‐like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26., The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C‐like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 μM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Twenty five novel chemical analogs of the previously reported Aurora kinase inhibitor BPR1K653 (1-(4-(2-((5-chloro-6-phenylfuro[2,3-d]pyrimidin-4-yl)amino)ethyl)phenyl)-3-(2-((dimethylamino)methyl)phenyl)urea) have been designed, synthesized, and evaluated by Aurora-A and Aurora-B enzymatic kinase activity assays. Similar to BPR1K653, analogs 3b-3h bear alkyl or tertiary amino group at the ortho position of the phenylurea, and showed equal or better inhibition activity for Aurora-B over Aurora-A. Conversely, preferential Aurora-A inhibition activity was observed when the same functional group was moved to the meta position of the phenylurea. Compounds 3m and 3n, both of which harbor a tertiary amino group at the meta position of the phenylurea, showed 10–16 fold inhibition selectivity for Aurora-A over Aurora-B. The in vitro kinase inhibition results were verified by Western blot analysis, and indicated that compounds 3m and 3n were more than 75-fold superior in inhibiting T-loop autophosphorylation of Aurora-A (Thr288), compared to Aurora-B (Thr232) in HCT116 colon carcinoma cells. The computational docking analysis suggested that the tertiary amine at the meta position of the phenylurea formed a more stable interaction with residues in the back pocket of Aurora-A than in Aurora-B, a possible explanation for the observed discrepancy in the selectivity. These results support an alternative small molecule design strategy targeting the back pocket of Aurora kinases for selective isoform inhibition.

CXCR4 antagonists (e.g., PlerixaforTM ) have been successfully validated as stem cell mobilizers for peripheral blood stem cell transplantation. Applications of the CXCR4 antagonists have heralded the era of cell-based therapy and opened a potential therapeutic horizon for many unmet medical needs such as kidney injury, ischemic stroke, cancer, and myocardial infarction. In this review, we first introduce the central role of CXCR4 in diverse cellular signaling pathways and discuss its involvement in several disease progressions. We then highlight the molecular design and optimization strategies for targeting CXCR4 from a large number of case studies, concluding that polyamines are the preferred CXCR4-binding ligands compared to other structural options, presumably by mimicking the highly positively charged natural ligand CXCL12. These results could be further justified with computer-aided docking into the CXCR4 crystal structure wherein both major and minor subpockets of the binding cavity are considered functionally important. Finally, from the clinical point of view, CXCR4 antagonists could mobilize hematopoietic stem/progenitor cells with long-term repopulating capacity to the peripheral blood, promising to replace surgically obtained bone marrow cells as a preferred source for stem cell transplantation.

Aurora kinases have emerged as important anticancer targets so that there are several inhibitors have advanced into clinical study. Herein, we identified novel indazole derivatives as potent Aurora kinases inhibitors by utilizing in silico fragment-based approach and knowledge-based drug design. After intensive hit-to-lead optimization, compounds 17 (dual Aurora A and B), 21 (Aurora B selective) and 30 (Aurora A selective) possessed indazole privileged scaffold with different substituents, which provide sub-type kinase selectivity. Computational modeling helps in understanding that the isoform selectivity could be targeted specific residue in the Aurora kinase binding pocket in particular targeting residues Arg220, Thr217 or Glu177.

It is now known that "gain of function" mutations of RET (REarranged during Transfection) kinase are specific and key oncogenic events in the onset of thyroid gland cancers such as the Medullary Thyroid Carcinoma (MTC). Although a number of RET inhibitors exist and are capable of inhibiting RET variants, in which mutations are outside the enzyme active site, the majority becomes dramatically ineffective when mutations are within the protein active site (V804L and V804M). Pursuing a receptor-based virtual screening against the kinase domain of RET, we found that compound 5 is able to inhibit efficiently both wild type and V804L mutant RET. Compound 5 was able to significantly reduce proliferation of both commercially available TT cell lines and surgical thyroid tissues obtained from patients with MTC and displayed a suitable drug-like profile, thus standing out as a promising candidate for further development towards the treatment of clinically unresponsive MTC.

Motivated by the pivotal role of CXCR4 as an HIV entry co-receptor, we herein report a de novo hit-to-lead effort on the identification of subnanomolar purine-based CXCR4 antagonists against HIV-1 infection. Compound 24, with an EC50 of 0.5 nM against HIV-1 entry into host cells and an IC50 of 16.4 nM for inhibition of radioligand stromal-derived factor-1α (SDF-1α) binding to CXCR4, was also found to be highly selective against closely related chemokine receptors. We rationalized that compound 24 complementarily interacted with the critical CXCR4 residues that are essential for binding to HIV-1 gp120 V3 loop and subsequent viral entry. Compound 24 showed a 130-fold increase in anti-HIV activity compared to that of the marketed CXCR4 antagonist, AMD3100 (Plerixafor), whereas both compounds exhibited similar potency in mobilization of CXCR4(+)/CD34(+) stem cells at a high dose. Our study offers insight into the design of anti-HIV therapeutics devoid of major interference with SDF-1α function.

Morphine antinociceptive tolerance is highly correlated with its poor ability to promote mu-opioid– receptor (MOR) endocytosis. Our objective was to discover a novel positive allosteric modulator of MOR to enhance morphine-induced MOR endocytosis. We used high-throughput screening to identify several cardiotonic steroids as positive allosteric modulators of morphine-induced MOR endocytosis having high potency and efficacy, independently of Na+/K+-ATPase inhibition. Convallatoxin was found to enhance morphine-induced MOR endocytosis through an adaptor protein 2/clathrin-dependent mechanism without regulating G protein- or β-arrestin-mediated pathways. Both F243 and I292 residues of MOR were essential to the effect of convallatoxin on MOR endocytosis. Co-treatment with chronic morphine and convallatoxin reduced morphine tolerance in animal models of acute thermal pain and chronic inflammatory pain. Acute convallatoxin administration reversed morphine tolerance in morphine-tolerant mice. These findings suggest that cardiotonic steroids are potentially therapeutic for morphine side effects and open a new avenue for the study of MOR trafficking.

Here we report for the first time the use of fit quality (FQ), a ligand efficiency (LE) based measure for virtual screening (VS) of compound libraries. The LE based VS protocol was used to screen an in-house database of 125,000 compounds to identify aurora kinase A inhibitors. First, 20 known aurora kinase inhibitors were docked to aurora kinase A crystal structure (PDB ID: 2W1C); and the conformations of docked ligand were used to create a pharmacophore (PH) model. The PH model was used to screen the database compounds, and rank (PH rank) them based on the predicted IC50 values. Next, LE_Scale, a weight-dependant LE function, was derived from 294 known aurora kinase inhibitors. Using the fit quality (FQ = LE/LE_Scale) score derived from the LE_Scale function, the database compounds were reranked (PH_FQ rank) and the top 151 (0.12% of database) compounds were assessed for aurora kinase A inhibition biochemically. This VS protocol led to the identification of 7 novel hits, with compound 5 showing aurora kinase A IC50 = 1.29 μM. Furthermore, testing of 5 against a panel of 31 kinase reveals that it is selective toward aurora kinase A & B, with

Computer-guided drug design is a powerful tool for drug discovery. Herein we disclose the use of this approach for the discovery of dual FMS-like receptor tyrosine kinase-3 (FLT3)-Aurora A inhibitors against cancer. An Aurora hit compound was selected as a starting point, from which 288 virtual molecules were screened. Subsequently, some of these were synthesized and evaluated for their capacity to inhibit FLT3 and Aurora kinase A. To further enhance FLT3 inhibition, structure-activity relationship studies of the lead compound were conducted through a simplification strategy and bioisosteric replacement, followed by the use of computer-guided drug design to prioritize molecules bearing a variety of different terminal groups in terms of favorable binding energy. Selected compounds were then synthesized, and their bioactivity was evaluated. Of these, one novel inhibitor was found to exhibit excellent inhibition of FLT3 and Aurora kinase A and exert a dramatic antiproliferative effect on MOLM-13 and MV4-11 cells, with an IC50 value of 7 nM. Accordingly, it is considered a highly promising candidate for further development.

μ-Opioid receptor (MOR) agonists are analgesics used clinically for the treatment of moderate to severe pain, but their use is associated with severe adverse effects such as respiratory depression, constipation, tolerance, dependence, and rewarding effects. In this study, we identified N-({2-[(4-bromo-2-trifluoromethoxyphenyl)sulfonyl]-1,2,3,4-tetrahydro-1-isoquinolinyl}methyl)cyclohexanecarboxamide (1) as a novel opioid receptor agonist by high-throughput screening. Structural modifications made to 1 to improve potency and blood-brain-barrier (BBB) penetration resulted in compounds 45 and 46. Compound 45 was a potent MOR/KOR (κ-opioid receptor) agonist, and compound 46 was a potent MOR and medium KOR agonist. Both 45 and 46 demonstrated a significant anti-nociceptive effect in a tail-flick test performed in wild type (WT) B6 mice. The ED50 value of 46 was 1.059 mg/kg, and the brain concentrations of 45 and 46 were 7424 and 11696 ng/g, respectively. Accordingly, compounds 45 and 46 are proposed for lead optimization and in vivo disease-related pain studies.

We describe the 3D-QSAR-assisted design of an Aurora kinase A inhibitor with improved physicochemical properties, in vitro activity, and in vivo pharmacokinetic profiles over those of the initial lead. Three different 3D-QSAR models were built and validated by using a set of 66 pyrazole (Model I) and furanopyrimidine (Model II) compounds with IC(50) values toward Aurora kinase A ranging from 33 nM to 10.5 μM. The best 3D-QSAR model, Model III, constructed with 24 training set compounds from both series, showed robustness (r(2) (CV) =0.54 and 0.52 for CoMFA and CoMSIA, respectively) and superior predictive capacity for 42 test set compounds (R(2) (pred) =0.52 and 0.67, CoMFA and CoMSIA). Superimposition of CoMFA and CoMSIA Model III over the crystal structure of Aurora kinase A suggests the potential to improve the activity of the ligands by decreasing the steric clash with Val147 and Leu139 and by increasing hydrophobic contact with Leu139 and Gly216 residues in the solvent-exposed region of the enzyme. Based on these suggestions, the rational redesign of furanopyrimidine 24 (clog P=7.41; Aurora A IC(50) =43 nM; HCT-116 IC(50) =400 nM) led to the identification of quinazoline 67 (clog P=5.28; Aurora A IC(50) =25 nM; HCT-116 IC(50) =23 nM). Rat in vivo pharmacokinetic studies showed that 67 has better systemic exposure after i.v. administration than 24, and holds potential for further development.

FMS-like receptor tyrosine kinase-3 (FLT3), a member of the class III tyrosine kinase receptor family, is expressed by immature hematopoietic cells, and highly associates with acute myeloid leukemia (AML). While acquired resistance to selective FLT3 inhibitors, the treatment became inefficient. Many literatures suggested that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML. Thus, utilizing dual FLT3-Aurora inhibitors may be the viable therapy for the treatment of AML. Recently, our group reported a novel AURKA inhibitor by using high-throughput parallel synthesis and structure-based drug design (SBDD). Based on this, we wanted to modify the selective AURKA inhibitor to maintain its Aurora A inhibition as well as improve its FLT3 activity to develop dual FLT3-Aurora kinase inhibitors. Through rapid construction of the platform of FLT3 virtual screening, the lead was quickly and efficiently identified. Proceeding investigation of structure-activity relationship could lead to 6-phenyl pyrrolopyrimidine analogue, showing FLT3 inhibition with an IC50 of 101 nM as well as Aurora kinase A inhibition with an IC50 of 33 nM. To further increase the level of inhibition of FLT3, binding energy calculations and docking study of FLT3 model were introduced to select terminal groups of the side chain with high priority. The selected compounds were synthesized and evaluated the bioactivity. The dramatic antiproliferation of the dual FLT3-AURKA inhibitor against MOLM-13 and MV4-11 (FLT3-ITD (+) human AML cell lines) was observed, and its biologic mechanism of inhibition of FLT3 and Aurora kinase A was proved the by significant evidence of biomarker. For this finding, this novel inhibitor holds potential for further development of preclinical investigations as an anti-AML drug candidate. Citation Format: Yung Chang Hsu, Hui-Yi Shiao, Yi-Yu Ke, John T. A. Hsu, Wen-Hsing Lin, Chun-Hwa Chen, Hsing-Pang Hsieh. Optimization of 5,6-fused pyrimidine-based kinase inhibitors by computer-aided drug design for the treatment of AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2530. doi:10.1158/1538-7445.AM2014-2530