Non-Covalent Inhibitors of SARS-CoV-2 Papain-Like Protease (PLpro): In Vitro and In Vivo Antiviral Activity.

The SARS-CoV-2 papain-like protease (PLpro), essential for viral processing and immune response disruption, is a promising target for treating acute infection of SARS-CoV-2. To date, there have been no reports of PLpro inhibitors with both submicromolar potency and animal model efficacy. To address the challenge of PLpro's featureless active site, a noncovalent inhibitor library with over 50 new analogs was developed, targeting the PLpro active site by modulating the BL2-loop and engaging the BL2-groove. Notably, compounds 42 and 10 exhibited strong antiviral effects and were further analyzed pharmacokinetically. 10, in particular, showed a significant lung accumulation, up to 12.9-fold greater than plasma exposure, and was effective in a mouse model of SARS-CoV-2 infection, as well as against several SARS-CoV-2 variants. These findings highlight the potential of 10 as an in vivo chemical probe for studying PLpro inhibition in SARS-CoV-2 infection.

JOTROL, a Novel Formulation of Resveratrol, Shows Beneficial Effects in the 3xTg-AD Mouse Model.

BACKGROUND: Alzheimer's disease (AD) has minimally effective treatments currently. High concentrations of resveratrol, a polyphenol antioxidant found in plants, have been reported to affect several AD-related and neuroprotective genes. To address the low bioavailability of resveratrol, we investigated a novel oral formulation of resveratrol, JOTROL™, that has shown increased pharmacokinetic properties compared to non-formulated resveratrol in animals and in humans. OBJECTIVE: We hypothesized that equivalent doses of JOTROL, compared to non-formulated resveratrol, would result in greater brain exposure to resveratrol, and more efficacious responses on AD biomarkers. METHODS: For sub-chronic reversal studies, 15-month-old male triple transgenic (APPSW/PS1M146V/TauP301L; 3xTg-AD) AD mice were treated orally with vehicle or 50 mg/kg JOTROL for 36 days. For prophylactic studies, male and female 3xTg-AD mice were similarly administered vehicle, 50 mg/kg JOTROL, or 50 mg/kg resveratrol for 9 months starting at 4 months of age. A behavioral battery was run, and mRNA and protein from brain and blood were analyzed for changes in AD-related gene and protein expression. RESULTS: JOTROL displays significantly increased bioavailability over non-formulated resveratrol. Treatment with JOTROL resulted in AD-related gene expression changes (Adam10, Bace1, Bdnf, Psen1) some of which were brain region-dependent and sex-specific, as well as changes in inflammatory gene and cytokine levels. CONCLUSION: JOTROL may be effective as a prophylaxis and/or treatment for AD through increased expression and/or activation of neuroprotective genes, suppression of pro-inflammatory genes, and regulation of central and peripheral cytokine levels.

RP-3500: A Novel, Potent, and Selective ATR Inhibitor that is Effective in Preclinical Models as a Monotherapy and in Combination with PARP Inhibitors.

Ataxia telangiectasia and Rad3-related (ATR) kinase protects genome integrity during DNA replication. RP-3500 is a novel, orally bioavailable clinical-stage ATR kinase inhibitor (NCT04497116). RP-3500 is highly potent with IC50 values of 1.0 and 0.33 nmol/L in biochemical and cell-based assays, respectively. RP-3500 is highly selective for ATR with 30-fold selectivity over mammalian target of rapamycin (mTOR) and more than 2,000-fold selectivity over ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and phosphatidylinositol 3-kinase alpha (PI3Kα) kinases. In vivo, RP-3500 treatment results in potent single-agent efficacy and/or tumor regression in multiple xenograft models at minimum effective doses (MED) of 5 to 7 mg/kg once daily. Pharmacodynamic assessments validate target engagement, with dose-proportional tumor inhibition of phosphorylated checkpoint kinase 1 (pCHK1) (IC80 = 18.6 nmol/L) and induction of phosphorylated H2A.X variant histone (γH2AX), phosphorylated DNA-PK catalytic subunit (pDNA-PKcs), and phosphorylated KRAB-associated protein 1 (pKAP1). RP-3500 exposure at MED indicates that circulating free plasma levels above the in vivo tumor IC80 for 10 to 12 hours are sufficient for efficacy on a continuous schedule. However, short-duration intermittent (weekly 3 days on/4 days off) dosing schedules as monotherapy or given concomitantly with reduced doses of olaparib or niraparib, maximize tumor growth inhibition while minimizing the impact on red blood cell depletion, emphasizing the reversible nature of erythroid toxicity with RP-3500 and demonstrating superior efficacy compared with sequential treatment. These results provide a strong preclinical rationale to support ongoing clinical investigation of the novel ATR inhibitor, RP-3500, on an intermittent schedule as a monotherapy and in combination with PARP inhibitors as a potential means of maximizing clinical benefit.

Discovery of bicyclic pyrazoles as class III histone deacetylase SIRT1 and SIRT2 inhibitors.

A series of bicyclic pyrazole carboxamides was synthesized and tested for inhibitory activity against the class III deacetylase sirtuin enzymes. Moderate to low micromolar inhibitory activities were obtained against SIRT1 and SIRT2. These bicyclic pyrazole compounds represent a new class of sirtuin inhibitors with a preference for SIRT1 over SIRT2.

Immunostimulatory activity and structure of polysaccharide from Streptococcus equi subsp. zooepidemicus.

In the present study, crude capsule polysaccharides from Streptococcus equi subsp. zooepidemicus C55129 (CP) were purified by DEAE cellulose-52 column and Sephadex G-100 column chromatography to afford purified fractions of CP-1, CP-2 and CP-3. The relative molecular weights of CP-1, CP-2 and CP-3 were 29.0, 891.0 and 1338.0 kDa, respectively. CP-1 was composed of mannose, glucose, arabinose and galactose in a percentage ratio of 66.15:28.97:2.43:2.45. CP-2 was composed of mannose, glucose, galactose and glucuronic acid in a percentage ratio of 40.94:27.71:5.96:25.39. In CP-1, there were pyranose rings, α-configuration glycosidic bond residues, one backbone chain (1→6 glycosidic bonds) and two branch chains (1→3 and 1→4 glycosidic bonds) in one repeating unit. In vitro immunostimulatory assay, it was found that CP could promote the splenocyte proliferation and increase the activity of acid phosphatase in peritoneal macrophages. The immunostimulatory activity of CP-1 might be related to its monosaccharide composition, molecular weight and α-configuration glycosidic bond.

Adva-27a, a novel podophyllotoxin derivative found to be effective against multidrug resistant human cancer cells.

BACKGROUND/AIM: Multidrug resistance poses a serious challenge in cancer therapy. To address this problem, we designed and synthesized Adva-27a, a novel non-ester GEM-difluorinated C-glycoside derivative of podophyllotoxin. MATERIALS AND METHODS: Adva-27a activity was evaluated in a variety of assays including inhibition of topoisomerase IIα, cytotoxic activity in drug-sensitive and drug-resistant cancer cell lines, metabolic stability in human liver microsomes and pharmacokinetic properties in rats. RESULTS: Adva-27a exhibited dose-dependent human topoisomerase IIα inhibitory activity and dose-dependent growth inhibitory activity in several drug-sensitive and two multidrug-resistant cancer cell lines. In the multidrug-resistant cell lines, MCF-7/MDR (breast cancer) and H69AR (small-cell lung cancer), Adva-27a was significantly more potent than etoposide. The metabolic stability of Adva-27a in human liver microsomes and its pharmacokinetic properties in rats were better than those of etoposide. CONCLUSION: Our studies have identified Adva-27a as a novel topoisomerase II inhibitor with superior cytotoxic activity against multidrug-resistant human cancer cells and more desirable pharmacokinetic properties than etoposide.

Mocetinostat for relapsed classical Hodgkin's lymphoma: an open-label, single-arm, phase 2 trial.

BACKGROUND: The prognosis of patients with relapsed Hodgkin's lymphoma, especially those who relapse after stem-cell transplantation, is poor, and the development of new agents for this patient population is an unmet medical need. We tested the safety and efficacy of mocetinostat, an oral isotype-selective histone deacetylase inhibitor, in patients with relapsed classical Hodgkin's lymphoma. METHODS: Patients with relapsed or refractory classical Hodgkin's lymphoma aged 18 years or older were treated with mocetinostat administered orally three times per week, in 28-day cycles. Two doses were assessed (85 mg and 110 mg). Patients were treated until disease progression or prohibitive toxicity. The primary outcome was disease control rate, defined as complete response, partial response, or stable disease (for at least six cycles), analysed by intention to treat. This trial has been completed and is registered with ClinicalTrials.gov, number NCT00358982. FINDINGS: 51 patients were enrolled. Initially, 23 patients were enrolled in the 110 mg cohort. Subsequently, because toxicity-related dose reductions were necessary in the 110 mg cohort, we treated 28 additional patients with a dose of 85 mg. On the basis of intent-to-treat analysis, the disease control rate was 35% (eight of 23 patients) in the 110 mg group and 25% (seven of 28) in the 85 mg group. 12 patients (24%) discontinued treatment because of adverse events, nine (32%) in the 85 mg cohort and three (13%) in the 110 mg cohort. The most frequent treatment-related grade 3 and 4 adverse events were neutropenia (four patients [17%] in the 110 mg group, three [11%] in the 85 mg group); fatigue (five patients [22%] in the 110 mg group, three [11%] in the 85 mg group); and pneumonia (four patients [17%] in the 110 mg group, two [7%] in the 85 mg group). Four patients, all in the 110 mg cohort, died during the study, of which two might have been related to treatment. INTERPRETATION: Mocetinostat, 85 mg three times per week, has promising single-agent clinical activity with manageable toxicity in patients with relapsed classical Hodgkin's lymphoma. FUNDING: MethylGene Inc, Montreal, Canada; Celgene Corporation, Summit, NJ, USA; Tufts Medical Center, Boston, MA, USA.

The class I HDAC inhibitor MGCD0103 induces cell cycle arrest and apoptosis in colon cancer initiating cells by upregulating Dickkopf-1 and non-canonical Wnt signaling.

Colorectal cancer metastatic recurrence and chemoresistance are major causes of morbidity and mortality. Colon cancer initiating cells (CCIC) are thought to contribute to both these processes. To identify drugs with anti-CCIC activity we screened a number of FDA approved and investigational compounds. We found that the class I selective histone deacetylase inhibitor (HDACi) MGCD0103 has significant activity against CCIC, and also significantly inhibits non-CCIC CRC cell xenograft formation. Both MGCD0103 and the pan-HDAC inhibitor Trichostatin impairs CCIC clonogenicity and cause cell cycle arrest and cell death. Gene expression profiling revealed that the canonical WNT ligand DKK-1 is a highly upregulated target of HDAC inhibitors. Despite the presence of APC mutations and constitutive WNT signaling in CCIC, both transfected and recombinant DKK-1 dramatically inhibit CCIC proliferation and clonogenicity. Overall, these data show that inhibition of class I HDACs is a promising novel approach to target both CCIC and non-CCIC CRC cells. Our studies also provide novel insights into roles for DKK1 in addition to canonical WNT signaling and the mechanism of CCIC tumor formation.

Diphenylmethylene hydroxamic acids as selective class IIa histone deacetylase inhibitors.

We have identified a series of diphenylmethylene hydroxamic acids as novel and selective HDAC class IIa inhibitors. The original hit, N-hydroxy-2,2-diphenylacetamide (6), has sub-micromolar class IIa HDAC inhibitory activity, while the rigidified oxygen analogue, N-hydroxy-9H-xanthene-9-carboxamide (13), is slightly more selective for HDAC7 with an IC(50) of 0.05muM. Substitution of 6 allows for the modulation of selectivity and potency amongst the class IIa HDAC isotypes.

Synthesis and evaluation of lysine derived sulfamides as histone deacetylase inhibitors.

We have recently reported on a novel class of histone deacetylase (HDAC) inhibitors bearing a sulfamide group as the zinc-binding unit. Herein, we report on the synthesis of sulfamide based inhibitors designed around a lysine scaffold and their structure-activity relationships against HDAC1 and HDAC6 isotypes as well as 293T cells. Our efforts led us to an improvement of the originally disclosed lysine-based sulfamide, 2a to compound 12h which has equal potency in enzyme and cell-based assays as well as enhanced metabolic stability and PK profile.

Novel HDAC6 isoform selective chiral small molecule histone deacetylase inhibitors.

In an effort to identify HDAC isoform selective inhibitors, we designed and synthesized novel, chiral 3,4-dihydroquinoxalin-2(1H)-one and piperazine-2,5-dione aryl hydroxamates showing selectivity (up to 40-fold) for human HDAC6 over other class I/IIa HDACs. The observed selectivity and potency (IC(50) values 10-200 nM against HDAC6) is markedly dependent on the absolute configuration of the chiral moiety, and suggests new possibilities for use of chiral compounds in selective HDAC isoform inhibition.

SAR and biological evaluation of analogues of a small molecule histone deacetylase inhibitor N-(2-aminophenyl)-4-((4-(pyridin-3-yl)pyrimidin-2-ylamino)methyl)benzamide (MGCD0103).

Analogues of the clinical compound MGCD0103 (A) were designed and synthesized. These compounds inhibit recombinant human HDAC1 with IC(50) values in the sub-micromolar range. In human cancer cells growing in culture these compounds induce hyperacetylation of histones, cause expression of the tumor suppressor protein p21(WAF1/CIP1), and inhibit cellular proliferation. Lead molecule of the series, compound 25 is metabolically stable, possesses favorable pharmacokinetic characteristics and is orally active in vivo in different mouse tumor xenograft models.

Sulfamides as novel histone deacetylase inhibitors.

The sulfamide moiety has been utilized to design novel HDAC inhibitors. The potency and selectivity of these inhibitors were influenced both by the nature of the scaffold, and the capping group. Linear long-chain-based analogs were primarily HDAC6-selective, while analogs based on the lysine scaffold resulted in potent HDAC1 and HDAC6 inhibitors.

Evaluation of the pharmacodynamic effects of MGCD0103 from preclinical models to human using a novel HDAC enzyme assay.

PURPOSE: The pharmacodynamic properties of MGCD0103, an isotype-selective inhibitor of histone deacetylase (HDAC), were evaluated in preclinical models and patients with a novel whole-cell HDAC enzyme assay. EXPERIMENTAL DESIGN: Boc-Lys(epsilon-Ac)-AMC, a HDAC substrate with fluorescent readout, was found to be cell permeable and was used to monitor MGCD0103-mediated HDAC inhibition in cultured cancer cells in vitro, in peripheral WBC ex vivo, in mice in vivo, and in human patients. RESULTS: MGCD0103 inhibited HDAC activity in several human cancer cell lines in vitro and in human peripheral WBC ex vivo in a dose-dependent manner. Unlike suberoylanilide hydroxamic acid, the HDAC inhibitory activity of MGCD0103 was time dependent and sustained for at least 24 hours following drug removal in peripheral WBC ex vivo. Inhibitory activity of MGCD0103 was sustained for at least 8 hours in vivo in mice and 48 hours in patients with solid tumors. HDAC inhibitory activity of MGCD0103 in peripheral WBC correlated with induction of histone acetylation in blood and in implanted tumors in mice. In cancer patients, sustained pharmacodynamic effect of MGCD0103 was visualized only by dose-dependent enzyme inhibition in peripheral WBC but not by histone acetylation analysis. CONCLUSIONS: This study shows that MGCD0103 has sustained pharmacodynamic effects that can be monitored both in vitro and in vivo with a cell-based HDAC enzyme assay.

Discovery of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide (MGCD0103), an orally active histone deacetylase inhibitor.

The design, synthesis, and biological evaluation of N-(2-aminophenyl)-4-[(4-pyridin-3-ylpyrimidin-2-ylamino)methyl]benzamide 8 (MGCD0103) is described. Compound 8 is an isotype-selective small molecule histone deacetylase (HDAC) inhibitor that selectively inhibits HDACs 1-3 and 11 at submicromolar concentrations in vitro. 8 blocks cancer cell proliferation and induces histone acetylation, p21 (cip/waf1) protein expression, cell-cycle arrest, and apoptosis. 8 is orally bioavailable, has significant antitumor activity in vivo, has entered clinical trials, and shows promise as an anticancer drug.

Phase 1 study of the oral isotype specific histone deacetylase inhibitor MGCD0103 in leukemia.

MGCD0103 is an isotype-selective inhibitor of histone deacetylases (HDACs) targeted to isoforms 1, 2, 3, and 11. In a phase 1 study in patients with leukemia or myelodysplastic syndromes (MDS), MGCD0103 was administered orally 3 times weekly without interruption. Twenty-nine patients with a median age of 62 years (range, 32-84 years) were enrolled at planned dose levels (20, 40, and 80 mg/m(2)). The majority of patients (76%) had acute myelogenous leukemia (AML). In all, 24 (83%) of 29 patients had received 1 or more prior chemotherapies (range, 0-5), and 18 (62%) of 29 patients had abnormal cytogenetics. The maximum tolerated dose was determined to be 60 mg/m(2), with dose-limiting toxicities (DLTs) of fatigue, nausea, vomiting, and diarrhea observed at higher doses. Three patients achieved a complete bone marrow response (blasts

Phase I study of MGCD0103 given as a three-times-per-week oral dose in patients with advanced solid tumors.

PURPOSE: MGCD0103 is a novel isotype-selective inhibitor of human histone deaceylases (HDACs) with the potential to regulate aberrant gene expression and restore normal growth control in malignancies. PATIENTS AND METHODS: A phase I trial of MGCD0103, given as a three-times-per-week oral dose for 2 of every 3 weeks, was performed in patients with advanced solid tumors. Primary end points were safety, tolerability, pharmacokinetics (PK), pharmacodynamic (PD) assessments of HDAC activity, and histone acetylation status in peripheral WBCs. RESULTS: Six dose levels ranging from 12.5 to 56 mg/m(2)/d were evaluated in 38 patients over 99 cycles (median, 2; range, 1 to 11). The recommended phase II dose was 45 mg/m(2)/d. Dose-limiting toxicities consisting of fatigue, nausea, vomiting, anorexia, and dehydration were observed in three (27%) of 11 and two (67%) of three patients treated at the 45 and 56 mg/m(2)/d dose levels, respectively. Disease stabilization for four or more cycles was observed in five (16%) of 32 patients assessable for efficacy. PK analyses demonstrated interpatient variability which was improved by coadministration with low pH beverages. Elimination half-life ranged from 6.7 to 12.2 hours, and no accumulation was observed with repeated dosing. PD evaluations confirmed inhibition of HDAC activity and induction of acetylation of H3 histones in peripheral WBCs from patients by MGCD0103. CONCLUSION: At doses evaluated, MGCD0103 appears tolerable and exhibits favorable PK and PD profiles with evidence of target inhibition in surrogate tissues.

MGCD0103, a novel isotype-selective histone deacetylase inhibitor, has broad spectrum antitumor activity in vitro and in vivo.

Nonselective inhibitors of human histone deacetylases (HDAC) are known to have antitumor activity in mice in vivo, and several of them are under clinical investigation. The first of these, Vorinostat (SAHA), has been approved for treatment of cutaneous T-cell lymphoma. Questions remain concerning which HDAC isotype(s) are the best to target for anticancer activity and whether increased efficacy and safety will result with an isotype-selective HDAC inhibitor. We have developed an isotype-selective HDAC inhibitor, MGCD0103, which potently targets human HDAC1 but also has inhibitory activity against HDAC2, HDAC3, and HDAC11 in vitro. In intact cells, MGCD0103 inhibited only a fraction of the total HDAC activity and showed long-lasting inhibitory activity even upon drug removal. MGCD0103 induced hyperacetylation of histones, selectively induced apoptosis, and caused cell cycle blockade in various human cancer cell lines in a dose-dependent manner. MGCD0103 exhibited potent and selective antiproliferative activities against a broad spectrum of human cancer cell lines in vitro, and HDAC inhibitory activity was required for these effects. In vivo, MGCD0103 significantly inhibited growth of human tumor xenografts in nude mice in a dose-dependent manner and the antitumor activity correlated with induction of histone acetylation in tumors. Our findings suggest that the isotype-selective HDAC inhibition by MGCD0103 is sufficient for antitumor activity in vivo and that further clinical investigation is warranted.

4-(Heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides and their analogs as a novel class of histone deacetylase inhibitors.

The synthesis and biological evaluation of a variety of 4-(heteroarylaminomethyl)-N-(2-aminophenyl)-benzamides and their analogs is described. Some of these compounds were shown to inhibit HDAC1 with IC(50) values below the micromolar range, induce hyperacetylation of histones, upregulate expression of the tumor suppressor p21(WAF1/Cip1), and inhibit proliferation of human cancer cells. In addition, certain compounds of this class were active in several human tumor xenograft models in vivo.