An Antiherpesviral Host-Directed Strategy Based on CDK7 Covalently Binding Drugs: Target-Selective, Picomolar-Dose, Cross-Virus Reactivity.

The repertoire of currently available antiviral drugs spans therapeutic applications against a number of important human pathogens distributed worldwide. These include cases of the pandemic severe acute respiratory coronavirus type 2 (SARS-CoV-2 or COVID-19), human immunodeficiency virus type 1 (HIV-1 or AIDS), and the pregnancy- and posttransplant-relevant human cytomegalovirus (HCMV). In almost all cases, approved therapies are based on direct-acting antivirals (DAAs), but their benefit, particularly in long-term applications, is often limited by the induction of viral drug resistance or side effects. These issues might be addressed by the additional use of host-directed antivirals (HDAs). As a strong input from long-term experiences with cancer therapies, host protein kinases may serve as HDA targets of mechanistically new antiviral drugs. The study demonstrates such a novel antiviral strategy by targeting the major virus-supportive host kinase CDK7. Importantly, this strategy focuses on highly selective, 3D structure-derived CDK7 inhibitors carrying a warhead moiety that mediates covalent target binding. In summary, the main experimental findings of this study are as follows: (1) the in vitro verification of CDK7 inhibition and selectivity that confirms the warhead covalent-binding principle (by CDK-specific kinase assays), (2) the highly pronounced antiviral efficacies of the hit compounds (in cultured cell-based infection models) with half-maximal effective concentrations that reach down to picomolar levels, (3) a particularly strong potency of compounds against strains and reporter-expressing recombinants of HCMV (using infection assays in primary human fibroblasts), (4) additional activity against further herpesviruses such as animal CMVs and VZV, (5) unique mechanistic properties that include an immediate block of HCMV replication directed early (determined by Western blot detection of viral marker proteins), (6) a substantial drug synergism in combination with MBV (measured by a Loewe additivity fixed-dose assay), and (7) a strong sensitivity of clinically relevant HCMV mutants carrying MBV or ganciclovir resistance markers. Combined, the data highlight the huge developmental potential of this host-directed antiviral targeting concept utilizing covalently binding CDK7 inhibitors.

Safety, Tolerability, Pharmacokinetics, and Metabolism of Telacebec (Q203) for the Treatment of Tuberculosis: a Randomized, Placebo-Controlled, Multiple Ascending Dose Phase 1B Trial.

A phase 1b, randomized, placebo-controlled, double-blind, multiple ascending dose study (NCT02858973) was conducted to assess the safety, tolerability, and pharmacokinetics of the new antituberculosis agent telacebec (Q203). A total of 47 healthy adult subjects entered the study; 36 received telacebec, and 11 received placebo. Telacebec at doses of 20, 50, 100, 160, 250, and 320 mg was orally administered once daily with a standard meal for 14 days. Multiple oral doses of telacebec up to 320 mg daily for 14 days appeared to be safe and well tolerated by healthy adult subjects in this study. There were no deaths, serious adverse events, or subject discontinuations due to adverse events. Following oral doses of telacebec, the overall extent (AUCτ) and peak (Cmax) exposures of telacebec increased from 538.94 to 10,098.47 ng·h/mL and from 76.43 to 1502.33 ng/mL, respectively, with increasing telacebec doses from 20 mg to 320 mg. A steady state was achieved for plasma telacebec by day 12, and there was 1.9- to 3.1-fold accumulation in the extent of telacebec exposure after daily doses for 14 days. Analysis of plasma samples from the participants indicated that telacebec was the primary circulating entity with no significant metabolites. Three potential metabolites of telacebec have been identified, which may be relatively minimal compared to the parent drug. Consistent with findings from preclinical and previous single-dose clinical studies, these results also support the potential of telacebec for further development as a safe and effective agent for the treatment of tuberculosis.

A Novel Selective Axl/Mer/CSF1R Kinase Inhibitor as a Cancer Immunotherapeutic Agent Targeting Both Immune and Tumor Cells in the Tumor Microenvironment.

Although immune checkpoint blockade (ICB) represents a major breakthrough in cancer immunotherapy, only a limited number of patients with cancer benefit from ICB-based immunotherapy because most immune checkpoint inhibitors (ICIs) target only T cell activation. Therefore, targeting non-T cell components in the tumor microenvironment (TME) can help subvert resistance and increase the applications of ICB-based therapy. Axl and Mer are involved in the carcinogenesis of multiple types of cancer by modulating immune and biological behaviors within tumors. Colony stimulating factor 1 receptor (CSF1R) mediates tumorigenesis in the TME by enhancing tumor associated macrophage (TAM) and myeloid-derived suppressor cell (MDSC) infiltration, facilitating immune escape. Therefore, the simultaneous inhibition of Axl, Mer, and CSF1R kinases may improve therapeutic efficacy by targeting non-T cell components in the TME. Here, we present Q702, a selective, potent small molecule inhibitor targeting Axl, Mer, and CSF1R, for oral administration. Q702 induced antitumor activity in syngeneic tumor mouse models by: remodeling the TME toward immune stimulation; expanding M1 macrophage and CD8 T cell populations and decreasing M2 macrophage and MDSC populations in the TME; and increasing MHC class I and E-cadherin expression in tumor cells. Thus, Q702 may have great potential to broaden the coverage of populations benefiting from ICB-based immunotherapy.

Safety, Tolerability, and Pharmacokinetics of Telacebec (Q203), a New Antituberculosis Agent, in Healthy Subjects.

Telacebec (Q203) is a potent drug candidate under clinical development for the treatment of drug-naïve and drug-resistant tuberculosis. The first-in-human randomized, placebo-controlled, double-blind, dose-escalation Phase 1A trial (Q203-TB-PI-US001) was conducted to evaluate the safety, tolerability, and pharmacokinetics of telacebec. A total of 56 normal, healthy, male and female subjects (42 active and 14 placebo) were enrolled in the study. The doses of telacebec were 10 mg (Cohort 1), 30 mg (Cohort 2), 50 mg (Cohort 3), 100 mg (Cohort 4), 200 mg (Cohort 5), 400 mg (Cohort 6), and 800 mg (Cohort 7) in a fasted state. Subjects participating in Cohort 4 were also enrolled in Cohort 8 to investigate the food effect on the pharmacokinetics of telacebec after a high-fat meal. In all subjects dosed with telacebec (10 to 800 mg), telacebec was well tolerated and did not lead to any significant or serious adverse events. Following a single oral administration of telacebec (10 to 800 mg), telacebec plasma concentration reached the maximal plasma concentration (Cmax) in average 2.0 to 3.5 h and showed multi-exponential decline thereafter. The area under the plasma concentration versus time curve (AUC) was approximately dose-proportional. A significant increase in plasma concentrations was observed in the fed condition compared with the fasted condition with the geometric mean ratio of 3.93 for Cmax. Moderate delay in Tmax (4.5 h) was also observed in the fed condition. These results, combined with the demonstrated activity against drug-sensitive and multidrug-resistant Mycobacterium tuberculosis, support further investigation of telacebec for the treatment of tuberculosis.

Involvement of small GTPase RhoA in the regulation of superoxide production in BV2 cells in response to fibrillar Aß peptides.

Fibrillar amyloid-beta (fAß) peptide causes neuronal cell death, which is known as Alzheimer's disease. One of the mechanisms for neuronal cell death is the activation of microglia which releases toxic compounds like reactive oxygen species (ROS) in response to fAß. We observed that fAß rather than soluble form blocked BV2 cell proliferation of microglial cell line BV2, while N-acetyl-l-cysteine (NAC), a scavenger of superoxide, prevented the cells from death, suggesting that cell death is induced by ROS. Indeed, both fAß1-42 and fAß25-35 induced superoxide production in BV2 cells. fAß25-35 produced superoxide, although fAß25-35 is not phagocytosed into BV2 cells. Thus, superoxide production by fAß does not seem to be dependent on phagocytosis of fAß. Herein we studied how fAß produces superoxide in BV2. Transfection of dominant negative (DN) RhoA (N19) cDNA plasmid, small hairpin (sh)-RhoA forming plasmid, and Y27632, an inhibitor of Rho-kinase, abrogated the superoxide formation in BV2 cells stimulated by fAß. Furthermore, fAß elevated GTP-RhoA level as well as Rac1 and Cdc42. Tat-C3 toxin, sh-RhoA, and Y27632 inhibited the phosphorylation of p47(PHOX). Moreover, peritoneal macrophages from p47(PHOX) (-/-) knockout mouse could not produce superoxide in response to fAß. These results suggest that RhoA closely engages in the regulation of superoxide production induced by fAß through phosphorylation of p47(PHOX) in microglial BV2 cells.

Interaction of microglia and amyloid-beta through beta2-integrin is regulated by RhoA.

Amyloid-beta (Abeta) is one of the main factors to cause Alzheimer's disease. Although fibrillar Abeta (fAbeta) activates microglial cells that release toxic compounds to induce partial neuronal death, the mechanism of interaction between Abeta and microglia remains unclear. Therefore, we examined the interaction of microglial cells (BV2) and fAbeta on a gelatin-precoated plate. The binding was markedly enhanced by RhoA inactivation using Tat-C3, dominant negative RhoA, and si-RhoA. To identify the receptor for fAbeta, we tested various antibodies to mask receptors. Among them, anti-beta2-integrin antibody mostly suppressed cell binding to fAbeta. The incremental binding of cells induced by RhoA inhibition was also blocked by addition of anti-beta2-integrin antibody. These results suggest that RhoA inhibition stimulates beta2-integrin-mediated cell interaction to fAbeta.

Transforming growth factor-beta1 regulates macrophage migration via RhoA.

Brief treatment with transforming growth factor (TGF)-beta1 stimulated the migration of macrophages, whereas long-term exposure decreased their migration. Cell migration stimulated by TGF-beta1 was markedly inhibited by 10 mug/mL Tat-C3 exoenzyme. TGF-beta1 increased mRNA and protein levels of macrophage inflammatory protein (MIP)-1alpha in the initial period, and these effects also were inhibited by 10 mug/mL Tat-C3 and a dominant-negative (DN)-RhoA (N19RhoA). Cycloheximide, actinomycin D, and antibodies against MIP-1alpha and monocyte chemoattractant protein-1 (MCP-1) abolished the stimulation of cell migration by TGF-beta1. These findings suggest that migration of these cells is regulated directly and indirectly via the expression of chemokines such as MIP-1alpha and MCP-1 mediated by RhoA in response to TGF-beta1. TGF-beta1 activated RhoA in the initial period, and thereafter inactivated them, suggesting that the inactivation of RhoA may be the cause of the reduced cell migration in response to TGF-beta1 at later times. We therefore attempted to elucidate the molecular mechanism of the inactivation of RhoA by TGF-beta1. First, TGF-beta1 phosphorylated RhoA via protein kinase A, leading to inactivation of RhoA. Second, wild-type p190 Rho GTPase activating protein (p190RhoGAP) reduced and DN-p190RhoGAP reversed the reduction of cell migration induced by TGF-beta, suggesting that it inactivated RhoA via p190 Rho GAP.