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.

Novel modeling approach integrating population pharmacokinetics and interspecies scaling to predict human pharmacokinetics of the new anti-tuberculosis agent telacebec (Q203).

Telacebec is a new anti-tuberculosis agent with promising therapeutic activity and a favorable safety profile. This study aimed to characterize the pharmacokinetics of telacebec via interspecies scaling and population pharmacokinetic modeling for the prediction of human pharmacokinetics. Preclinical pharmacokinetic data were obtained from mice, rats, and dogs following intravenous and oral doses of telacebec. A population pharmacokinetic model was developed to describe the pharmacokinetic data from all three species. The disposition parameters were well correlated with the body weight for all species using an allometric equation. Thus, the allometric scaling was incorporated into the population pharmacokinetic model, which could simultaneously describe the plasma concentration vs. time data from all preclinical studies as well as the Phase 1A clinical study. The developed model was used to predict the pharmacokinetics of telacebec after IV injection, including the clearance (CL) of 168.58 [118.86 - 238.73] mL/min and volume of distribution (Vss) of 968.84 [396.87 - 2831.31] L for 80-kg human. The absolute bioavailability of telacebec in humans in the fed state was estimated as 70.34 ± 9.91%. Finally, the population pharmacokinetic model with allometric scaling was utilized to simulate the plasma concentration vs. time profiles of telacebec after multiple oral doses in humans. The model-predicted profiles well agreed with the observed data in Phase 1B clinical trial. The present pharmacokinetic model may help better understand the activity of telacebec, leading to the design of optimal dosing regimens and new formulation development.

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.

Synthesis and structure-activity relationships of novel fused ring analogues of Q203 as antitubercular agents.

A set of fused ring analogues of a new antitubercular agent, Q203, was designed and synthesized. To reduce the lipophilicity of Q203 caused by linearly extended side chains, shorter and heteroatoms containing fused rings were introduced into the side chain region. Antitubercular activity was tested against H37Rv-GFP replicating in liquid broth culture medium (extracellular) and within macrophages (intracellular). Many analogues showed potent extracellular activities as well as intracellular activities without cytotoxicity. Among them, compounds 18-21 displayed significant antitubercular activities with favorable metabolic stabilities. Representative compound 21 exhibited excellent in vivo pharmacokinetic values at high drug exposure levels in the plasma, which makes this compound promising candidate for a new antitubercular drug.

Synthesis and structure-activity studies of side chain analogues of the anti-tubercular agent, Q203.

The anti-tubercular activity of 6-chloro-2-ethyl-N-(4-(4-(4-(trifluoromethoxy)phenyl)piperidin-1-yl)benzyl)imidazo [1,2-a]pyridine-3-carboxamide (Q203) is modified by varying its side chain. In this study, we synthesized Q203 analogues with different side chains and studied their effects on anti-tubercular activity. Many analogues showed good potency against M. tuberculosis replicating in liquid broth culture medium (extracellular activity) regardless of chain length and conformational changes. However, a polar character in the side chain region was unfavorable for anti-tubercular activity. The analogues, 25, 28, 35, and 36, displayed excellent activity against M. tuberculosis replicating inside macrophages (intracellular activity) and promising pharmacokinetic (PK) properties with high drug exposure level and long half-life.

Lead optimization of a novel series of imidazo[1,2-a]pyridine amides leading to a clinical candidate (Q203) as a multi- and extensively-drug-resistant anti-tuberculosis agent.

A critical unmet clinical need to combat the global tuberculosis epidemic is the development of potent agents capable of reducing the time of multi-drug-resistant (MDR) and extensively-drug-resistant (XDR) tuberculosis therapy. In this paper, we report on the optimization of imidazo[1,2-a]pyridine amide (IPA) lead compound 1, which led to the design and synthesis of Q203 (50). We found that the amide linker with IPA core is very important for activity against Mycobacterium tuberculosis H37Rv. Linearity and lipophilicity of the amine part in the IPA series play a critical role in improving in vitro and in vivo efficacy and pharmacokinetic profile. The optimized IPAs 49 and 50 showed not only excellent oral bioavailability (80.2% and 90.7%, respectively) with high exposure of the area under curve (AUC) but also displayed significant colony-forming unit (CFU) reduction (1.52 and 3.13 log10 reduction at 10 mg/kg dosing level, respectively) in mouse lung.

Steroid hormone binding receptors: application of homology modeling, induced fit docking, and molecular dynamics to study structure-function relationships.

Steroid nuclear hormone binding receptors (SHRs) are ligand activated transcription factors involved in the regulation of target genes associated with key physiological and developmental processes. As such they are important targets for drug discovery. Crystal structures are now available for all members of the SHR family, however, earlier studies carried out using homology models proved to be quite valuable for understanding the binding of natural ligands and for designing novel therapeutic agents. The maleability of the binding pocket means that the crystal structure of an SHR in complex with one ligand may not suffice to explain the binding interactions of that same target with a different ligand. Consequently, induced fit docking and molecular dynamics are shown to be useful and necessary tools for understanding these receptors.

QSAR models for predicting the similarity in binding profiles for pairs of protein kinases and the variation of models between experimental data sets.

We propose a direct QSAR methodology to predict how similar the inhibitor-binding profiles of two protein kinases are likely to be, based on the properties of the residues surrounding the ATP-binding site. We produce a random forest model for each of five data sets (one in-house, four from the literature) where multiple compounds are tested on many kinases. Each model is self-consistent by cross-validation, and all models point to only a few residues in the active site controlling the binding profiles. While all models include the "gatekeeper" as one of the important residues, consistent with previous literature, some models suggest other residues as being more important. We apply each model to predict the similarity in binding profile to all pairs in a set of 411 kinases from the human genome and get very different predictions from each model. This turns out not to be an issue with model-building but with the fact that the experimental data sets disagree about which kinases are similar to which others. It is possible to build a model combining all the data from the five data sets that is reasonably self-consistent but not surprisingly, given the disagreement between data sets, less self-consistent than the individual models.

p38 MAP kinase inhibitors. Part 6: 2-arylpyridazin-3-ones as templates for inhibitor design.

p38 inhibitors based on 3,4-dihydropyrido[4,3-d]pyrimidazin-2-one template were synthesized and their SAR explored. Benchmark compounds 30, 35, and 36 were found to be potent against the enzyme. Crystal structure of p38 in complex with 30 indicated a key pi-stacking interaction with the pendant tyrosine residue-35 in the glycine-rich loop.

Dynamic control of allosteric antagonism of leukocyte function antigen-1 and intercellular adhesion molecule-1 interaction.

Leukocyte function associated antigen-1 (LFA-1) plays a critical role in T cell migration and has been recognized as a therapeutic target for immune disorders. Several classes of small molecule antagonists have been developed to block LFA-1 interaction with intercellular adhesion molecule-1 (ICAM-1). Recent structural studies show that the antagonists bind to an allosteric site in the I-domain of LFA-1. However, it is not yet clear how these small molecules work as antagonists since no significant conformational change is observed in the I-domain-antagonist complex structures. Here we present a computational study suggesting how these allosteric antagonists affect the dynamics of the I-domain. The lowest frequency vibrational mode calculated from an LFA-1 I-domain structure shows large scale "coil-down" motion of the C-terminal alpha7 helix, which may lead to the open form of the I-domain. The presence of an allosteric antagonist greatly reduces this motion of the alpha7 helix as well as other parts of the I-domain. Thus, our study suggests that allosteric antagonists work by eliminating breathing motion that leads to the open conformation of the I-domain.

Structural components of SCAN-domain dimerizations.

The SCAN or leucine-rich domain has been characterized as a highly conserved sequence in zinc finger transcription factors that mediates selective dimer formation between SCAN-domain-containing proteins. In order to accommodate various SCAN-domain sequence features, a minimal functional folding unit was defined on the premise of proper structural folding and biochemical binding. The 58-amino acid minimal functional units derived from each of four SCAN-domain protein families were subjected to a three-dimensional position-specific scoring matrix (3D-PSSM) and ungapped threading analysis. The resulting fold prediction represented the SCAN-domain's minimal functional unit as a bundle of three alpha helices folded to a core structure. In addition, the minimal functional folding unit biochemically retained the selective dimerization properties of the native proteins. In order to elucidate the structural components within the SCAN-domain that engage in binding interactions, we attempted to correlate the physicochemical helix properties, as represented by a hydropathy profile, with the experimental dimerization selectivities. The amino-terminal helix revealed the highest diversity measure among the three helices of the minimal functional unit and is therefore likely to offer critical surface-exposed binding residues. Indeed, by interchanging the amino-terminal helix between SCAN-domains without alteration of their structural frames consisting of conserved hydrophobic residues, a modulation of binding preferences was demonstrated. The minimal functional folding unit of SCAN-domains may therefore contain within the amino-terminal alpha helix structural components that determine selective dimerization patterns and combinatorial control of transcription factors.

Glucocorticoid receptor antagonism by cyproterone acetate and RU486.

The steroid compound cyproterone acetate was identified in a high-throughput screen for glucocorticoid receptor (GR) binding compounds. Cyproterone (Schering AG) is clinically used as an antiandrogen for inoperable prostate cancer, virilizing syndromes in women, and the inhibition of sex drive in men. Despite its progestin properties, cyproterone shares a similar pharmacological profile with the antiprogestin mifepristone (RU486; Roussel Uclaf SA). The binding affinities of cyproterone and RU486 for the GR and progesterone receptor were similar (K(d), 15-70 nM). Both compounds were characterized as competitive antagonists of dexamethasone without intrinsic transactivating properties in rat hepatocytes (K(i), 10-30 nM). In osteosarcoma cells, RU486 revealed a higher potency than cyproterone acetate to prevent responses to dexamethasone-induced GR transactivation and NF kappa B transrepression. Upon administration to Sprague-Dawley rats, both compounds were found to be orally bioavailable and to inhibit transactivation of liver GR. Molecular docking of cyproterone acetate and RU486 into the homology model for the GR ligand binding domain illustrated overlapping steroid scaffolds in the binding pocket. However, in contrast to RU486, cyproterone lacks a bulky side chain at position C11 beta that has been proposed to trigger active antagonism of nuclear receptors by displacing the C-terminal helix of the ligand-binding domain, thereby affecting activation function 2. Cyproterone may therefore inhibit transactivation of the GR by a molecular mechanism recently described as passive antagonism. New therapeutic profiles may result from compounds designed to selectively stabilize the inactive and active conformations of certain nuclear receptors.

Simulation of the different biological activities of diethylstilbestrol (DES) on estrogen receptor alpha and estrogen-related receptor gamma.

We describe the application of the molecular dynamics (MD) and molecular mechanics-generalized Born/surface area (MM-GB/SA) approaches to the simulation of the different biological activity of diethylstilbestrol (DES) on two highly homologous nuclear receptors-estrogen receptor alpha (ER-alpha) and estrogen-related receptor gamma (ERR-gamma). DES exerts an agonistic effect against ER-alpha and an antagonistic effect against ERR-gamma. Using the x-ray crystal structures of ER-alpha in the canonical agonist bound form (PDB code: 3ERD) and antagonist bound form (PDB code: 3ERT), ERR-gamma homology models have been constructed for the receptor in two different conformations. MM-GB/SA binding free energy calculations of DES in the ER-alpha and ERR-gamma structures suggest that DES exhibits a greater free energy of binding in the agonist bound conformation of ER-alpha, while the antagonist bound conformation is preferred for ERR-gamma. Further dissection of the free energy contributions coupled with calculation of the ligand binding pocket volume suggests that the van der Waals interactions for DES within the smaller binding pocket volume of ERR-gamma are less favorable and this is the main factor for DES antagonism in ERR-gamma. This approach has potential general applicability to the prediction of the biological activity of nuclear receptor ligands.