Potent Phosphodiesterase Inhibitor Research Articles

Drug repurposing and structure-based discovery of new PDE4 and PDE5 inhibitors

Phosphodiesterase-4 (PDE4) and PDE5 responsible for the hydrolysis of intracellular cAMP and cGMP, respectively, are promising targets for therapeutic intervention in a wide variety of diseases. Here, we report the discovery of novel, drug-like PDE4 inhibitors by performing a high-throughput drug repurposing screening of 2560 approved drugs and drug candidates in clinical trial studies. It allowed us to identify eight potent PDE4 inhibitors with IC50 values ranging from 0.41 to 2.46 μM. Crystal structures of PDE4 in complex with four compounds, namely ethaverine hydrochloride (EH), benzbromarone (BBR), CX-4945, and CVT-313, were further solved to elucidate molecular mechanisms of action of these new inhibitors, providing a solid foundation for optimizing the inhibitors to improve their potency as well as selectivity. Unexpectedly, selectivity profiling of other PDE subfamilies followed by crystal structure determination revealed that CVT-313 was also a potent PDE5 inhibitor with a binding mode similar to that of tadalafil, a marketed PDE5 inhibitor, but distinctively different from the binding mode of CVT-313 with PDE4. Structure-guided modification of CVT-313 led to the discovery of a new inhibitor, compound 2, with significantly improved inhibitory activity as well as selectivity towards PDE5 over PDE4. Together, these results highlight the utility of the drug repurposing in combination with structure-based drug design in identifying novel inhibitors of PDE4 and PDE5, which provides a prime example for efficient discovery of drug-like hits towards a given target protein.

PDE5 inhibitor drugs for use in dementia.

Potent phosphodiesterase-5 (PDE5) inhibitors are in clinical use as vasodilators.In animals PDE5 inhibitors enhance synaptic function and cognitive ability.In humans the PDE5 inhibitor sildenafil is associated with reduced risk of Alzheimer's disease.Licensed PDE5 inhibitors have potential for repurposing in dementia.Prospective clinical trials of PDE5 inhibitors are warranted.

Ensifentrine as a Novel, Inhaled Treatment for Patients with COPD.

Ensifentrine is a novel, potent, and selective dual inhibitor of phosphodiesterase (PDE)3 and PDE4 designed for delivery by inhalation that combines effects on airway inflammation, bronchodilation and ciliary function in bronchial epithelia. In Phase 2 studies in subjects with COPD, ensifentrine demonstrated clinically meaningful bronchodilation and improvements in symptoms and health-related quality of life when administered alone or in combination with current standard of care therapies. Ensifentrine is currently in late-stage clinical development for the maintenance treatment of patients with COPD. This review summarizes non-clinical data as well as Phase 1 and Phase 2 efficacy and safety results of nebulized ensifentrine relevant to the maintenance treatment of patients with COPD.

Pharmacology of orismilast, a potent and selective PDE4 inhibitor.

There remains an unmet need for oral medications that are safe and efficacious for long-term management of chronic inflammatory skin diseases (CISD). Inhibition of phosphodiesterase 4 (PDE4) can modulate a broad range of pro-inflammatory cytokines that play a major role in CISD pathogenesis. Orismilast is a second generation PDE4 inhibitor in clinical development for CISD treatment. The objective of this study was to examine the PDE4 enzymatic activity and anti-inflammatory effects of orismilast in vitro, ex vivo, and in vivo. The PDE1-11 enzymatic activity of orismilast was tested in vitro using a single concentration of 308 nM orismilast. The PDE4 selectivity and inhibitory potency was further examined in a radiometric assay. Orismilast was tested on human whole blood and human peripheral blood mononuclear cells (PBMC) to determine effects on its cytokine secretion and inhibition profile ex vivo. Orismilast was orally administered in a murine model of chronic oxazolone-induced ear skin inflammation. Ear thickness, a marker of inflammation, and inflammatory cytokines were analysed. Orismilast selectively inhibited PDE4 and demonstrated potent inhibition of PDE4B and PDE4D subtype splice variants in vitro. Orismilast inhibited whole blood and PBMC production of tumour necrosis factor α (TNFα), and the secretion of T-helper (Th)1 (TNFα and IFNγ), Th17 (IL-22 and IL-23), and Th2 (IL-4, IL-5, and IL-13) related cytokines in PBMC. In vivo, 10 and 30 mg/kg doses of orismilast significantly reduced ear thickness and inflammation markers (p < 0.0001, respectively). Orismilast displayed selective and potent PDE4 inhibition and broad-spectrum anti-inflammatory activity in several pre-clinical models. The results of the study support clinical development of oral orismilast as a novel treatment option for CISD including psoriasis, atopic dermatitis, and hidradenitis suppurativa.

33468 Efficacy and safety of once-daily roflumilast cream 0.3% in patients with knee/elbow involvement: Pooled results from phase 3 trials (DERMIS-1 and DERMIS-2)

Roflumilast cream 0.3% is a selective and potent phosphodiesterase-4 inhibitor under investigation as a once-daily treatment for psoriasis. Psoriatic plaques on the knees/elbows can be challenging to treat due to thicker stratum corneum, which impedes topical absorption. We present pooled efficacy results from a post hoc analysis of the subgroup of patients with knee and/or elbow involvement from two identical phase 3 randomized controlled trials of roflumilast (DERMIS-1: NCT04211363 and DERMIS-2: NCT04211389).

33463 Pooled efficacy and safety results from the DERMIS-1 and DERMIS-2 phase 3 trials of once-daily roflumilast cream 0.3% by baseline body surface area

Roflumilast cream 0.3% is a selective and potent phosphodiesterase-4 inhibitor under investigation as a once-daily treatment for psoriasis. Here we describe pooled efficacy and safety results from 2 identical phase 3 randomized controlled trials of roflumilast cream (DERMIS-1: NCT04211363 and DERMIS-2: NCT04211389) analyzed by baseline body surface area (BSA) affected ( 10%). Patients (≥2 years) with psoriasis involving 2-20% BSA were randomized to roflumilast (n = 576) or vehicle (n = 305) for 8 weeks.

Structure-based optimization of Toddacoumalone as highly potent and selective PDE4 inhibitors with anti-inflammatory effects

Phosphodiesterase-4 (PDE4) is an important drug target for inflammatory diseases. Previously, we identified a series of novel PDE4 inhibitors derived from the natural Toddacoumalone, among which the hit compound 2 with a naphthyridine scaffold showed moderate potency with the IC50 value of 400 nM. Based on the co-crystal structure of PDE4D-2, further structural optimizations and structure–activity relationship studies led to a highly potent PDE4 inhibitor 23a with the IC50 value of 0.25 nM and excellent selectivity profiles over other PDEs (>4000-fold). The co-crystal structure of PDE4D-23a elucidated that 23a has strong interactions with the M and Q pocket of PDE4D. Importantly, compound 23a significantly inhibits the release of inflammatory cytokines TNF-α and IL-6 in lipopolysaccharide-stimulated RAW264.7 cells. Thus, compound 23a with a naphthyridine scaffold is a promising PDE4 inhibitor for the treatment of inflammatory diseases.

E2027 Cardiac Safety Evaluation With Concentration-Response Modeling of ECG Data to Inform Dose Selection in Studies in Patients With Dementia With Lewy Bodies.

E2027 is a novel, highly selective and potent inhibitor of phosphodiesterase 9 in development for dementia with Lewy bodies. Cardiac safety assessments for emerging agents are essential to avoid drug-induced QT interval prolongation, which may predispose individuals to potentially fatal ventricular arrhythmias. To evaluate the cardiac safety of E2027 and to inform dose selection for the phase 2 study of E2027 in dementia with Lewy bodies, we evaluated concentration-response modeling of pooled electrocardiogram data. A post hoc concentration-QTc analysis evaluated potential QT effects using data from 2 randomized, double-blind studies in healthy subjects: a single ascending dose (SAD) study and a multiple ascending dose (MAD) study. Daily E2027 doses ranged from 5 to 1200 mg. A linear mixed-effects model was used to establish the relationship between plasma concentrations of E2027 and change from the baseline of QTcF (ΔQTcF). A significant but shallow relationship was observed in the estimated slope of the concentration-ΔQTcF: 0.002 ms/ng/mL (90% confidence interval: 0.0007-0.0031) with a small, nonsignificant treatment effect-specific intercept of -0.6 ms. Based on this pooled concentration-QTc analysis, an effect on the QTcF interval >10 ms can be excluded up to E2027 plasma concentrations of ∼3579 ng/mL, corresponding to a dose at least 4-fold larger than the 50 mg phase 2 dose. This pooled post hoc analysis evaluating cardiac safety of E2027 demonstrated that clinically concerning QTcF prolongation and related cardiac complications are highly unlikely with proposed E2027 doses planned for phase 2.

Discovery and Structural Optimization of Toddacoumalone Derivatives as Novel PDE4 Inhibitors for the Topical Treatment of Psoriasis.

Psoriasis is a common immune-mediated skin disorder manifesting in abnormal skin plaques, and phosphodiesterase 4 (PDE4) is an effective target for the treatment of inflammatory diseases such as psoriasis. Toddacoumalone is a natural PDE4 inhibitor with moderate potency and imperfect drug-like properties. To discover novel and potent PDE4 inhibitors with considerable druggability, a series of toddacoumalone derivatives were designed and synthesized, leading to the compound (2R,4S)-6-ethyl-2-(2-hydroxyethyl)-2,8-dimethyl-4-(2-methylprop-1-en-1-yl)-2,3,4,6-tetrahydro-5H-pyrano[3,2-c][1,8]naphthyridin-5-one (33a) with high inhibitory potency (IC50 = 3.1 nM), satisfactory selectivity, favorable skin permeability, and a well-characterized binding mechanism. Encouragingly, topical administration of 33a exhibited remarkable therapeutic effects in an imiquimod-induced psoriasis mouse model.

Once-Daily Roflumilast Cream 0.3%, a Potent Phosphodiesterase-4 Inhibitor, Provided Safe and Effective Treatment of Psoriasis in the DERMIS-1 and DERMIS-2 Phase 3 Trials

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The Potential Role of Sildenafil in Cancer Management through EPR Augmentation.

Enhanced permeation retention (EPR) was a significant milestone discovery by Maeda et al. paving the path for the emerging field of nanomedicine to become a powerful tool in the fight against cancer. Sildenafil is a potent inhibitor of phosphodiesterase 5 (PDE-5) used for the treatment of erectile dysfunction (ED) through the relaxation of smooth muscles and the modulation of vascular endothelial permeability. Overexpression of PDE-5 has been reported in lung, colon, metastatic breast cancers, and bladder squamous carcinoma. Moreover, sildenafil has been reported to increase the sensitivity of tumor cells of different origins to the cytotoxic effect of chemotherapeutic agents with augmented apoptosis mediated through inducing the downregulation of Bcl-xL and FAP-1 expression, enhancing reactive oxygen species (ROS) generation, phosphorylating BAD and Bcl-2, upregulating caspase-3,8,9 activities, and blocking cells at G0/G1 cell cycle phase. Sildenafil has also demonstrated inhibitory effects on the efflux activity of ATP-binding cassette (ABC) transporters such as ABCC4, ABCC5, ABCB1, and ABCG2, ultimately reversing multidrug resistance. Accordingly, there has been a growing interest in using sildenafil as monotherapy or chemoadjuvant in EPR augmentation and management of different types of cancer. In this review, we critically examine the basic molecular mechanism of sildenafil related to cancer biology and discuss the overall potential of sildenafil in enhancing EPR-based anticancer drug delivery, pointing to the outcomes of the most important related preclinical and clinical studies.

Repurposing of sildenafil as antitumour; induction of cyclic guanosine monophosphate/protein kinase G pathway, caspase-dependent apoptosis and pivotal reduction of Nuclear factor kappa light chain enhancer of activated B cells in lung cancer

Lung cancer is one of the most frequent types of cancers that lead to death. Sildenafil is a potent inhibitor of phosphodiesterase-5 and showed potential anticancer effects, which has not yet been fully evaluated. Thus, this study aims to investigate the potential anticancer effect of sildenafil in urethane-induced lung cancer in BALB/c mice. Five-week-old male BALB/c mice were treated with either (i) normal saline only, (ii) sildenafil only 50 mg kg-1/ P.O every other day for the last four successive weeks, (iii) urethane 1.5 gm kg-1 i.p (at day 1 and day 60), (iv) carboplatin after urethane induction, or (v) sildenafil after urethane induction. It was shown that sildenafil significantly increased the levels of cGMP and Caspase-3 with a reduction of NF-κB, Bcl-2, Cyclin D1, intercellular adhesion molecule 1, matrix metalloproteinase-2 levels and normalisation of Nrf2 along with pronounced improvement in the histological patterns. These results indicated that sildenafil markedly induces cell cycle arrest, apoptosis and inhibits the metastatic activity through activation of cyclic guanosine monophosphate/protein kinase G pathway and down-regulation of cyclin D1 and nuclear factor kappa light chain enhancer of activated B cells with downstream anti-apoptotic gene Bcl-2, which underscores the critical importance of future using sildenafil in the treatment of lung cancer.

Human Aldehyde Oxidase 1-Mediated Carbazeran Oxidation in Chimeric TK-NOG Mice Transplanted with Human Hepatocytes.

Carbazeran is a potent phosphodiesterase inhibitor with species-dependent metabolic profiles in rats, dogs, and humans. In this study, we investigated the aldehyde oxidase (AOX)-mediated oxidation of carbazeran to 4-oxo derivatives in chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene with humanized livers (humanized-liver mice). Liver cytosolic fractions from humanized-liver mouse effectively catalyzed carbazeran 4-oxidation with high affinity for the substrate, similar to those of the human liver cytosolic fractions and recombinant human AOX1 protein. Furthermore, hepatocytes prepared from humanized-liver mice and humans also exhibited substantial metabolism via carbazeran 4-oxidation. After a single oral administration of carbazeran (10 mg/kg), plasma levels of 4-oxo-carbazeran, N-desethyl-4-oxo-carbazeran, and 6,7-dimethoxy-1-[4-(hydroxy)-piperidino]-4-phthalazinone (three human metabolites formed via 4-oxidation) were higher in humanized-liver mice than in the control mice. In contrast, plasma levels of O-desmethylcarbazeran (a major metabolite in dogs) in control mice were higher than those in the humanized-liver mice. Relative excreted amounts of the three 4-oxidation-derived human-specific metabolites in the urine and feces were greater for humanized-liver mice than control mice, whereas the relative excreted amounts of O-desmethylcarbazeran were predominant in the urine and feces of control mice. Thus, the production of carbazeran 4-oxo derivatives was elevated in humanized-liver mice compared with control mice, in agreement with our in vitro enzyme-mediated oxidation data. These results suggest that hepatic human AOX1 functions in humanized-liver mice at the in vivo level and that humanized-liver mice may be useful for predicting drug metabolism in humans, at least with regard to human AOX1-dependent metabolism. SIGNIFICANCE STATEMENT: We found that the production of carbazeran 4-oxo derivatives was higher in humanized-liver mice than in control mice. These results were supported by the fact that carbazeran was rapidly metabolized to 4-oxo-carbazeran in humanized-liver mouse hepatocytes expressing human aldehyde oxidase 1. These results suggest that human aldehyde oxidase 1 is functional in humanized-liver mice in vivo and that chimeric NOD/Shi-scid IL2 receptor gamma-null mice expressing a herpes simplex virus type 1 thymidine kinase transgene transplanted with human hepatocytes may be a suitable model animal for predicting aldehyde oxidase-dependent biotransformation of drugs in humans.

CHF6001 Inhibits NF-κB Activation and Neutrophilic Recruitment in LPS-Induced Lung Inflammation in Mice.

Inhibitors of phosphodiesterase 4 (PDE4) are potent anti-inflammatory agents, inhibiting the production of inflammatory mediators through the elevation of intracellular cAMP concentrations. We studied the activity of a novel PDE4 inhibitor, CHF6001, both in vitro in human cells and in vivo, using bioluminescence imaging (BLI) in mice lung inflammation. Mice transiently transfected with the luciferase gene under the control of an NF-κB responsive element (NF-κB-luc) have been used to assess the in vivo anti-inflammatory activity of CHF6001 in lipopolysaccharide (LPS)-induced lung inflammation. BLI as well as inflammatory cells and the concentrations of pro-inflammatory cytokines were monitored in bronchoalveolar lavage fluids (BALF) while testing in vitro its ability to affect the production of leukotriene B4 (LTB4), measured by LC/MS/MS, by LPS/LPS/N-formyl--methionyl--leucyl-phenylalanine (fMLP)-activated human blood. CHF6001 inhibited the production of LTB4 in LPS/fMLP-activated human blood at sub-nanomolar concentrations. LPS-induced an increase of BLI signal in NF-κB-luc mice, and CHF6001 administered by dry powder inhalation decreased in parallel luciferase signal, cell airway infiltration, and pro-inflammatory cytokine concentrations in BALF. The results obtained provide in vitro and in vivo evidence of the anti-inflammatory activity of the potent PDE4 inhibitor CHF6001, showing that with a topical administration that closely mimics inhalation in humans, it efficiently disrupts the NF-κB activation associated with LPS challenge, an effect that may be relevant for the prevention of exacerbation episodes in chronic obstructive pulmonary disease subjects.

Exploration of icariin analog structure space reveals key features driving potent inhibition of human phosphodiesterase-5.

The natural product icariin inhibits human phosphodiesterase-5 (PDE5) and represents a unique pharmacophore for treating erectile dysfunction, pulmonary hypertension, and other diseases. In this study, we explore the available icariin-derived chemical scaffolds through medicinal chemistry to develop novel icariin PDE5 inhibitors with improved potency and specificity. We synthesized six novel semi-synthetic icariin analogs as well as three naturally occurring icariin analogs, and characterized the structure-activity relationship in the context of human PDE5 inhibition using in vitro enzyme inhibition and kinetics assays and molecular modeling. Mammalian-cell-based assays and in vitro enzyme inhibition assays against human PDE6C further helped to identify the most potent and selective icariin analogs. Our results reveal the synergistic contribution of functional groups at the C3 and C7 positions of the icariin backbone towards PDE5 inhibition. Whereas a hydrophobic and flexible alkanol group at the C7 position is sufficient to enhance icariin analog potency, combining this group with a hydrophilic sugar group at the C3 position leads to further enhancement of potency and promotes specificity towards PDE5 versus PDE6C. In particular, compounds 3 and 7 exhibit Ki values of 0.036 ± 0.005 μM and 0.036 ± 0.007 μM towards PDE5 respectively, which are approaching those of commercial PDE5 inhibitors, and can effectively reduce GMP levels in cultured human BJ-hTERT cells. This study identifies novel icariin analogs as potent and selective PDE5 inhibitors poised to become lead compounds for further pharmaceutical development.

Inhibition of phosphodiesterase‐4D reverses memory deficits and depression‐like effects via cAMP signaling in mouse models of Alzheimer's disease

Depression is highly correlated with Alzheimer's disease (AD), but treatment for the comorbidity of both is lacking. Phosphodiesterase‐4 (PDE4), an enzyme that catalyzes the hydrolysis of cyclic AMP (cAMP), has been considered as a promising target for treatment of both memory loss in AD and depression. Using mouse models of AD and potent PDE4 inhibitors and PDE4‐subtype knockout mice, we demonstrated that PDE4D plays a role in the comorbidity of memory loss and depression in AD. Treatment with the PDE4 inhibitor rolipram or roflumilast reversed memory deficits in novel object recognition and Morris water‐maze tests in APP/PS1 double transgenic and 3xTg‐AD mice, which are widely used models for AD. Similarly, in the tail‐suspension and forced‐swimming tests, the PDE4 inhibitors reversed the decreased immobility in AD mice, suggesting antidepressant‐like effects. The effects of PDE4 inhibitors were mimicked in the tests using mice deficient in PDE4D, which displayed memory‐enhancing and antidepressant‐like effects, relative to the WT mice microinfused with beta‐amyloid peptide 1–42 into the hippocampus. In addition, the PDE4 inhibitor treatment reversed the decreased ratio of Bcl‐2/BAX and inhibited the increased expression of PDE4D in the cerebral cortex and hippocampus of AD mice. Further, the PDE4 inhibitors reversed the decreased levels of cAMP and expression of phosphorylated cAMP response element‐binding protein (CREB) and brain derived neurotrophic factor (BDNF) in AD mice. These were mimicked by PDE4D deficiency and treatment with a PDE4D relatively selective inhibitor. In conclusions, these results suggest that PDE4 is an important target for the comorbidity of memory loss and depression in AD, which appears to be mediated by PDE4D‐cAMP signaling.Support or Funding InformationThis work was supported by research grants from NIH/NIA AG031687 and NIH/NIAAA HHSN275201700001C (both to HTZ) and the National Natural Science Foundation of China (81441111 to HW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

3,7-Bis(2-hydroxyethyl)icaritin, a potent inhibitor of phosphodiesterase-5, prevents monocrotaline-induced pulmonary arterial hypertension via NO/cGMP activation in rats

Pulmonary arterial hypertension (PAH) is a chronic progressive disease which leads to elevated pulmonary arterial pressure and right heart failure. 3,7-Bis(2-hydroxyethyl)icaritin (ICT), an icariin derivatives, was reported to have potent inhibitory activity on phosphodiesterase type 5 (PDE5) which plays a crucial role in the pathogenesis of PAH. The present study was designed to investigate the effects of ICT on monocrotaline (MCT)-induced PAH rat model and reveal the underlying mechanism. MCT-induced PAH rat models were established with intragastric administration of ICT (10, 20, 40 mg/kg/d), Icariin (ICA) (40 mg/kg/d) and Sildenafil (25 mg/kg/d). The mean pulmonary arterial pressure (mPAP) and right ventricle hypertrophy index (RVHI) were measured. Pulmonary artery remodeling was assessed by H&E staining. Blood and lung tissue were collected to evaluate the level of endothelin 1 (ET-1), nitric oxide (NO), and cyclic guanosine monophosphate (cGMP). The expressions endothelial nitric oxide synthase (eNOS) and PDE5A in lung tissues were determined by Western blot analysis. The results showed that ICT reduced RVHI and mPAP, and reversed lung vascular remodeling in rats with MCT-induced PAH. ICT also reversed MCT-induced ET-1 elevation, NO and cGMP reduction in serum or lung tissue. Moreover, ICT administration significantly induced eNOS activation and PDE5A inhibition. ICT with lower dose had better effects than ICA. In summary, ICT is more effective in preventing MCT-induced PAH in rats via NO/cGMP activation compared with ICA. These findings demonstrate a novel mechanism of the action of ICT that may have value in prevention of PAH.

Design, synthesis, and molecular modeling of heterocyclic bioisostere as potent PDE4 inhibitors.

A new hybrid template was designed by combining the structural features of phosphodiesterase 4 (PDE4) inhibitors with several heterocyclic moieties which present an integral part in the skeleton of many apoptotic agents. Thirteen compounds of the synthesized hybrids displayed higher inhibitory activity against PDE4B than the reference drug, roflumilast. Further investigation indicated that compounds 13b and 20 arrested the cell cycle at the G2/M phase and the pre-G1 phase, and induced cell death by apoptosis of A549 cells in a caspase-dependent manner.

Concise synthesis of polymethoxyflavone sudachitin and its derivatives, and biological evaluations

We accomplished a divergent synthesis of sudachitin (2), a polymethoxyflavone isolated from citrus fruits, and six derivatives from acetophenone 9, which was an intermediate in our previous synthesis of nobiletin (1). Compound 2 enhanced glucose-induced insulin secretion in INS-1D cells, but was less potent than 1. Compared with 1, compound 2 and two derivatives were more potent inhibitors of cAMP-specific phosphodiesterase.

Synthesis, docking studies andin vitroevaluation of novel chalcones as potent inhibitors of phosphodiesterase 5 from human platelets and 5A from bovine recombinant

Chalcones with a nitric oxide (NO) donating scaffold and a variety of substituents were synthesized. A docking study was performed and molecules were evaluated forin vitrophosphodiesterase 5 and 5A inhibitory potency.