Novel hematopoietic progenitor kinase 1 inhibitor KHK-6 enhances T-cell activation.

Inhibiting the functional role of negative regulators in immune cells is an effective approach for developing immunotherapies. The serine/threonine kinase hematopoietic progenitor kinase 1 (HPK1) involved in the T-cell receptor signaling pathway attenuates T-cell activation by inducing the degradation of SLP-76 through its phosphorylation at Ser-376, reducing the immune response. Interestingly, several studies have shown that the genetic ablation or pharmacological inhibition of HPK1 kinase activity improves the immune response to cancers by enhancing T-cell activation and cytokine production; therefore, HPK1 could be a promising druggable target for T-cell-based cancer immunotherapy. To increase the immune response against cancer cells, we designed and synthesized KHK-6 and evaluated its cellular activity to inhibit HPK1 and enhance T-cell activation. KHK-6 inhibited HPK1 kinase activity with an IC50 value of 20 nM and CD3/CD28-induced phosphorylation of SLP-76 at Ser-376 Moreover, KHK-6 significantly enhanced CD3/CD28-induced production of cytokines; proportion of CD4+ and CD8+ T cells that expressed CD69, CD25, and HLA-DR markers; and T-cell-mediated killing activity of SKOV3 and A549 cells. In conclusion, KHK-6 is a novel ATP-competitive HPK1 inhibitor that blocks the phosphorylation of HPK1 downstream of SLP-76, enhancing the functional activation of T cells. In summary, our study showed the usefulness of KHK-6 in the drug discovery for the HPK1-inhibiting immunotherapy.

Orally bioavailable BTK PROTAC active against wild-type and C481 mutant BTKs in human lymphoma CDX mouse models.

Bruton tyrosine kinase (BTK) is an important signaling hub that activates the B-cell receptor (BCR) signaling cascade. BCR activation can contribute to the growth and survival of B-cell lymphoma or leukemia. The inhibition of the BCR signaling pathway is critical for blocking downstream events and treating B-cell lymphomas. Herein, we report potent and orally available proteolysis-targeting chimeras (PROTACs) that target BTK to inactivate BCR signaling. Of the PROTACs tested, UBX-382 showed superior degradation activity for wild-type (WT) and mutant BTK proteins in a single-digit nanomolar range of half-maximal degradation concentration in diffuse large B-cell lymphoma cell line. UBX-382 was effective on 7 out of 8 known BTK mutants in in vitro experiments and was highly effective in inhibiting tumor growth in murine xenograft models harboring WT or C481S mutant BTK-expressing TMD-8 cells over ibrutinib, ARQ-531, and MT-802. Remarkably, oral dosing of UBX-382 for <2 weeks led to complete tumor regression in 3 and 10 mg/kg groups in murine xenograft models. UBX-382 also provoked the cell type-dependent and selective degradation of cereblon neosubstrates in various hematological cancer cells. These results suggest that UBX-382 treatment is a promising therapeutic strategy for B-cell-related blood cancers with improved efficacy and diverse applicability.

DCLK1 promotes colorectal cancer stemness and aggressiveness via the XRCC5/COX2 axis.

Rationale: Doublecortin-like kinase 1 (DCLK1) is a serine/threonine kinase that selectively marks cancer stem-like cells (CSCs) and promotes malignant progression in colorectal cancer (CRC). However, the exact molecular mechanism by which DCLK1 drives the aggressive phenotype of cancer cells is incompletely determined. Methods: Here, we performed comprehensive genomics and proteomics analyses to identify binding proteins of DCLK1 and discovered X-ray repair cross-complementing 5 (XRCC5). Thus, we explored the biological role and downstream events of the DCLK1/XRCC5 axis in human CRC cells and CRC mouse models. Results: The results of comprehensive bioinformatics analyses suggested that DCLK1-driven CRC aggressiveness is linked to inflammation. Mechanistically, DCLK1 bound and phosphorylated XRCC5, which in turn transcriptionally activated cyclooxygenase-2 expression and enhanced prostaglandin E2 production; these events collectively generated the inflammatory tumor microenvironment and enhanced the aggressive behavior of CRC cells. Consistent with the discovered mechanism, inhibition of DCLK1 kinase activity strongly impaired the tumor seeding and growth capabilities in CRC mouse models. Conclusion: Our study illuminates a novel mechanism that mediates the pro-inflammatory function of CSCs in driving the aggressive phenotype of CRC, broadening the biological function of DCLK1 in CRC.

Structure-activity relationship analysis of novel GSPT1 degraders based on benzotriazinone scaffold and its antitumor effect on xenograft mouse model.

Molecular glue degraders, such as lenalidomide and pomalidomide, bind to cereblon (CRBN) E3 ligase and subsequently recruit neosubstrate proteins, Ikaros (IKZF1) and Aiolos (IKZF3), for the ubiquitination-proteasomal degradation process. In this study, we explored structure-activity relationship analysis for novel GSPT1 degraders utilizing a benzotriazinone scaffold previously discovered as a novel CRBN binder. In particular, we focused on the position of the ureido group on the benzotriazinone scaffold, substituent effect on the phenylureido group, and methyl substitution on the benzylic position of benzotriazinone. As a result, we identified 34f (TD-522), which exhibits strong anti-proliferative effects in both KG-1 (EC50 = 0.5 nM) and TMD-8 (EC50 = 5.2 nM) cell lines. Compound 34f effectively induced GSPT1 degradation with a DC50 of 0.269 nM and Dmax of >95 % at 10 nM concentration in KG-1 cells. An in vivo xenograft study showed that compound 34f effectively suppressed TMD8-driven tumor growth, suggesting a potential role in the development of novel GSPT1 degraders.

A novel cereblon modulator for targeted protein degradation.

Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.

Grignard-mediated rearrangement of trifluoroacetyl from dihydroisoquinoline enamides to afford tertiary trifluoromethylcarbinols.

Treatment of the trifluoroacetyl enamides of dihydroisoquinolines 2 with diverse Grignard reagents afforded tertiary trifluoromethyl-carbinols 4 by facilitating the addition of tertiary carbinols to the ß-carbon of enamides 2. Based on the confirmed formation of vinylogous amides 3, the transformation likely proceeds via unique acyl group rearrangement to the ß-carbon of the enamide and subsequent nucleophilic addition of the Grignard reagent. Given the synthetic utility and novelty of this reaction, this work may open new avenues for the synthesis of pharmaceutically important tertiary trifluoromethylcarbinols on cyclic enamide systems.

Novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety for anaplastic lymphoma kinase (ALK) inhibitor.

In this study, a series of novel 2,4-diaminopyrimidines bearing fused tricyclic ring moiety was described for ALK inhibitor. The pyrazole, imidazole, 1,2,4-triazole, piperazine and phenanthridine moieties were employed at the 2-position of pyrimidine. Among the compounds synthesized, 28, 29, 36, and 42 showed promising anti-ALK activities in enzymatic- and cell-based assays. In vivo H3122 xenograft model study showed that compound 29 effectively suppressed ALK-driven tumor growth, similar to the extent of ceritinib, suggesting that it could be used for a novel ALK inhibitor development.

Replacing the terminal piperidine in ceritinib with aliphatic amines confers activities against crizotinib-resistant mutants including G1202R.

The piperidine fragment in ceritinib was replaced with diverse aliphatic amines to improve inherent resistance issues of ceritinib. While most of the prepared compounds exhibit as similar in vitro activities as ceritinib, compound 10 shows encouraging activities against wild-type ALK as well as crizotinib-resistant mutants including extremely resistant G1202R mutant with an IC50 of 1.8 nM. Furthermore, pharmacokinetic profiles of 10 is apparently better than that of ceritinib. In murine xenograft studies, compound 10 turns out to be as active as ceritinib, suggesting that further optimization of 10 may lead to clinical candidates overcoming ALK mutant issues.

Discovery of novel tetrahydroisoquinoline-containing pyrimidines as ALK inhibitors.

Exploration of the two-position side chain of pyrimidine in LDK378 with tetrahydroisoquinolines (THIQs) led to discovery of 8 and 17 as highly potent ALK inhibitors. THIQs 8 and 17 showed encouraging in vitro and in vivo xenograft efficacies, comparable with those of LDK378. Although THIQ analogs (8a-o and 17a-i) prepared were not as active as their parent compounds, both 8 and 17 have significant inhibitory activities against various ALK mutant enzymes including G1202R, indicating that this series of compounds could be further optimized as useful ALK inhibitors overcoming the resistance issues found from crizotinib and LDK378.

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor.

A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were synthesized and evaluated for their anti-ALK activities. The activities of these compounds were confirmed in both enzyme- and cell-based ALK assays. Amongst compounds synthesized, KRCA-0445 showed very promising results in pharmacokinetic study and in vivo efficacy study with H3122 xenograft mouse model.

Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins.

Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151_L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15-20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants.

Synthesis and anti-influenza virus activity of 4-oxo- or thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-ones.

A target-free approach was applied to discover anti-influenza viral compounds, where influenza infected Madin-Darby canine kidney cells were treated 7500 different small organic chemicals individually and reduction of virus-induced cytopathic effect was measured. One of the hit compounds was (Z)-1-((5-fluoro-1H-indol-3-yl)methylene)-6-methyl-4-thioxo-4,5-dihydrofuro[3,4-c]pyridin-3(1H)-one (15a) with half-maximal effective concentrations of 17.4-21.1µM against influenza A/H1N1, A/H3N2 and B viruses without any cellular toxicity at 900µM. To investigate the structure-activity relationships, two dozens of the hit analogs were synthesized. Among them, 15g, 15j, 15q, 15s, 15t and 15x had anti-influenza viral activity comparable or superior to that of the initial hit. The anti-influenza viral compounds efficiently suppressed not only viral protein level of the infected cells but also production of viral progeny in the culture supernatants in a dose-dependent manner. Based on a mode-of-action study, they did not affect virus entry or RNA replication. Instead, they suppressed viral neuraminidase activity. This study is the first to demonstrate that dihydrofuropyridinones could serve as lead compounds for the discovery of alternative influenza virus inhibitors.

Novel 2,4-dianilino-5-fluoropyrimidine derivatives possessing ALK inhibitory activities.

A new series of 2,4-dianilino-5-fluoropyrimidine derivatives were designed and synthesized and their anaplastic lymphoma kinase (ALK) inhibitory activities were evaluated by biochemical and cell-based assays in order to discover a new ALK inhibitor. Most compounds synthesized showed good inhibitory activities against ALK and good cytotoxic activities in H3122 cell line. The best compound 6f showed good activity against wild-type ALK along with crizotinib-resistant mutant ALK, and it showed 6 times better activity in cell-based assay than crizotinib. Some SAR studies were performed by the comparisons of the activities between 6 and the designed-synthesized compounds.

3D-QSAR and cell wall permeability of antitubercular nitroimidazoles against Mycobacterium tuberculosis.

Inhibitory activities of monocyclic nitroimidazoles against Mycobacterium tuberculosis (Mtb) deazaflavin-dependent nitroreductase (DDN) were modeled by using docking, pharmacophore alignment and comparative molecular similarity indices analysis (CoMSIA) methods. A statistically significant model obtained from CoMSIA was established based on a training set using pharmacophore-based molecular alignment. The leave-one out cross-validation correlation coefficients q2 (CoMSIA) were 0.681. The CoMSIA model had a good correlation (r2(pred)/CoMSIA = 0.611) between the predicted and experimental activities against excluded test sets. The generated model suggests that electrostatic, hydrophobic and hydrogen bonding interactions all play important roles for interaction between ligands and receptors. The predicted cell wall permeability (logP(app)) for substrates with high inhibitory activity against Mtb were investigated. The distribution coefficient (logD) range was 2.41 < logD < 2.89 for the Mtb cell wall membrane permeability. The larger the polar surface area is, the better the permeability is. A larger radius of gyration (rgry) and a small fraction of rotatable bonds (f(rtob)) of these molecules leads to higher cell wall penetration ability. The information obtained from the in silico tools might be useful in the design of more potent compounds that are active against Mtb.

Novel bis-ortho-alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines (KRCA-0008) as potent and selective ALK inhibitors for anticancer treatment.

The synthesis of bis-ortho-alkoxy-para-piperazinesubstituted-2,4-dianilinopyrimidines is described and their structure-activity-relationship to anaplastic lymphoma kinase (ALK) is presented. KRCA-0008 is selective and potent to ALK and Ack1, and displays drug-like properties without hERG liability. KRCA-0008 demonstrates in vivo efficacy comparable to Crizotinib in xenograft mice model.

Inhibition of influenza virus internalization by (-)-epigallocatechin-3-gallate.

(-)-Epigallocatechin-3-gallate (EGCG), one of the major flavonoid components of green tea, is known to have a broad antiviral activity against several enveloped viruses, including the influenza virus. However, its mode of action and the mechanism that allows it to target influenza virus molecules have not been fully elucidated. Thus, this study investigated the molecular mechanism by which EGCG suppresses influenza virus infections. EGCG was found to block an early step in the influenza viral life cycle, but it did not affect viral adsorption to target cells or viral RNA replication. However, EGCG inhibited hemifusion events between virus particles and the cellular membrane by reducing the viral membrane integrity, thereby resulting in the loss of the cell penetration capacity of the influenza virus. EGCG also marginally suppressed the viral and nonviral neuraminidase (NA) activity in an enzyme-based assay system. In conclusion, it is suggested that the anti-influenza viral efficacy of EGCG is attributable to damage to the physical properties of the viral envelope and partial inhibition of the NA surface glycoprotein. These results may facilitate future investigations of the antiviral activity of EGCG against other enveloped viruses as well as influenza virus.

Efficient synthesis of 3H,3'H-spiro[benzofuran-2,1'-isobenzofuran]-3,3'-dione as novel skeletons specifically for influenza virus type B inhibition.

An efficient and novel two step synthetic procedure to prepare various substituted 3H,3'H-spiro[benzofuran-2,1'-isobenzofuran]-3,3'-diones A, was established from very simple and easily available starting materials. The developed method is a robust and general approach for the synthesis of these structures. The prepared compounds were tested against influenza virus type A viz., A/Taiwan/1/86 (H1N1), A/Hong Kong/8/68 (H3N2) and type B viz., B/Panama/45/90, B/Taiwan/2/62, B/Lee/40, B/Brisbane/60/2008. Among 31 compounds tested, some of them showed good activity (selective index values >10) against these influenza viruses preferentially for type B. The most active compound 3b showed activity in 3.0-16.1 µM range with a selectivity index value between 30 and 166 against these type B viruses, in which it was comparable to the antiviral agent favipiravir. Also, 3b is found to be inactive against other enveloped viruses (viz., HIV and HSV) showing its specificity for influenza viruses.

Design, synthesis, and structure-activity relationship studies of tryptanthrins as antitubercular agents.

The natural product tryptanthrin (1a) represents a potential lead for new tuberculosis (TB) drugs since tryptanthrin and its synthetic analogues possess potent in vitro activity against Mycobacterium tuberculosis (Mtb). However, in spite of their in vitro activity, none of these agents have been shown to be efficacious in vivo against animal models of TB. Described herein are syntheses of new tryptanthrin analogues together with a systematic investigation of their in vitro antitubercular activity and ADME properties followed by pharmacokinetic characterization in rodents for the most promising compounds. Those with the best potency and oral bioavailability were progressed to evaluations of efficacy against acute murine TB. The work aimed to prove the concept that this compound class can limit growth of Mtb during infection as well as to establish the SAR for in vitro activity against Mtb and the range of in vitro ADME parameters for this class of natural products. Novel C-11-deoxy (5b) and A-ring-saturated (6) tryptanthrin analogues were discovered that maintained activity against Mtb and showed improved solubility compared to tryptanthrin as well as evidence of oral bioavailability in rodents. However, neither 5b nor 6 demonstrated efficacy against acute murine TB following administration at doses up to 400 mg/kg daily for 4 weeks. Although 5b and 6 failed to inhibit replication or kill Mtb in vivo, they illuminate a path to new structural variations of the tryptanthrin scaffold that may maximize the potential of this class of compounds against TB.

Thiolactomycin-based ß-ketoacyl-AcpM synthase A (KasA) inhibitors: fragment-based inhibitor discovery using transient one-dimensional nuclear overhauser effect NMR spectroscopy.

Thiolactomycin (TLM) is a natural product inhibitor of KasA, the ß-ketoacyl synthase A from Mycobacterium tuberculosis. To improve the affinity of TLM for KasA, a series of TLM analogs have been synthesized based on interligand NOEs between TLM and a pantetheine analog when both are bound simultaneously to the enzyme. Kinetic binding data reveal that position 3 of the thiolactone ring is a suitable position for elaboration of the TLM scaffold, and the structure-activity relationship studies provide information on the molecular features that govern time-dependent inhibition in this enzyme system. These experiments also exemplify the utility of transient one-dimensional NOE spectroscopy for obtaining interligand NOEs compared with traditional steady state two-dimensional NOESY spectroscopy.

Design and synthesis of 1H-1,2,3-triazoles derived from econazole as antitubercular agents.

Econazole has been known to be active against Mycobacterium tuberculosis. We have designed and synthesized 1H-1,2,3-triazoles derived from econazole as antitubercular agents. The majority of triazole derivatives have been prepared by microwave-assisted click chemistry. It turned out that all of the prepared triazoles had no antifungal activities. However, most of the hydroxy-triazoles (6a and 10) apparently turned out to have antitubercular activities. Overall, hydroxy-triazoles 10 were more active than their corresponding ether-triazoles 11. While the MIC value of hydroxy-triazole 10d was as good as econazole (16 µg/mL), the MIC value of 10a was two-fold more active than econazole, suggesting that this 1H-1,2,3-triazole scaffold (3) could be further optimized to develop Mtb specific agents.