Development of PDE6D and CK1α Degraders through Chemical Derivatization of FPFT-2216.

Immunomodulatory drugs are a class of drugs approved for the treatment of multiple myeloma. These compounds exert their clinical effects by inducing interactions between the CRL4CRBN E3 ubiquitin ligase and a C2H2 zinc finger degron motif, resulting in degradation of degron-containing targets. However, although many cellular proteins feature the degron motif, only a subset of those are degradable via this strategy. Here, we demonstrated that FPFT-2216, a previously reported "molecular glue" compound, degrades PDE6D, in addition to IKZF1, IKZF3, and CK1α. We used FPFT-2216 as a starting point for a focused medicinal chemistry campaign and developed TMX-4100 and TMX-4116, which exhibit greater selectivity for degrading PDE6D and CK1α, respectively. We also showed that the region in PDE6D that interacts with the FPFT-2216 derivatives is not the previously pursued prenyl-binding pocket. Moreover, we found that PDE6D depletion by FPFT-2216 does not impede the growth of KRASG12C-dependent MIA PaCa-2 cells, highlighting the challenges of drugging PDE6D-KRAS. Taken together, the approach we described here represents a general scheme to rapidly develop selective degraders by reprogramming E3 ubiquitin ligase substrate specificity.

New Hybrid Compounds Combining Fragments of Usnic Acid and Monoterpenoids for Effective Tyrosyl-DNA Phosphodiesterase 1 Inhibition.

Usnic acid (UA) is a secondary metabolite of lichens that exhibits a wide range of biological activities. Previously, we found that UA derivatives are effective inhibitors of tyrosyl-DNA phosphodiesterase 1 (TDP1). It can remove covalent complex DNA-topoisomerase 1 (TOP1) stabilized by the TOP1 inhibitor topotecan, neutralizing the effect of the drugs. TDP1 removes damage at the 3' end of DNA caused by other anticancer agents. Thus, TDP1 is a promising therapeutic target for the development of drug combinations with topotecan, as well as other drugs for cancer treatment. Ten new UA enamino derivatives with variation in the terpene fragment and substituent of the UA backbone were synthesized and tested as TDP1 inhibitors. Four compounds, 11a-d, had IC50 values in the 0.23-0.40 µM range. Molecular modelling showed that 11a-d, with relatively short aliphatic chains, fit to the important binding domains. The intrinsic cytotoxicity of 11a-d was tested on two human cell lines. The compounds had low cytotoxicity with CC50 ≥ 60 µM for both cell lines. 11a and 11c had high inhibition efficacy and low cytotoxicity, and they enhanced topotecan's cytotoxicity in cancerous HeLa cells but reduced it in the non-cancerous HEK293A cells. This "protective" effect from topotecan on non-cancerous cells requires further investigation.

Discovery of Potent Phosphodiesterase-9 Inhibitors for the Treatment of Hepatic Fibrosis.

Hepatic fibrosis commonly exists in chronic liver disease and would eventually develop to cirrhosis and liver cancer with high fatality. Phosphodiesterase-9 (PDE9) has attracted profound attention as a drug target because of its highest binding affinity among phosphodiesterases (PDEs) with cyclic guanosine monophosphate. However, no published study has reported PDE9 inhibitors as potential agents against hepatic fibrosis yet. Herein, structural modification from a starting hit LL01 led to lead 4a, which exhibited an IC50 value of 7.3 nM against PDE9, excellent selectivity against other PDE subfamilies, and remarkable microsomal stability. The cocrystal structure of PDE9 with 4a revealed an important residue, Phe441, capable of improving the selectivity of PDE9 inhibitors. Administration of 4a exerted a significant antifibrotic effect in bile duct-ligation-induced rats with hepatic fibrosis and transforming growth factor-ß-induced fibrogenesis. This therapeutic effect was indeed achieved by selectively inhibiting PDE9 rather than other PDE isoforms, identifying PDE9 inhibitors as potential agents against hepatic fibrosis.

Synthesis of Methoxy-, Methylenedioxy-, Hydroxy-, and Halo-Substituted Benzophenanthridinone Derivatives as DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Inhibitors and Their Biological Activity for Drug-Resistant Cancer.

As a recently discovered DNA repair enzyme, tyrosyl-DNA phosphodiesterase 1 (TDP1) removes topoisomerase IB (TOP1)-mediated DNA protein cross-links. Inhibiting TDP1 can potentiate the cytotoxicity of TOP1 inhibitors and overcome cancer cell resistance to TOP1 inhibitors. On the basis of our previous study, herein we report the synthesis of benzophenanthridinone derivatives as TOP1 and TDP1 inhibitors. Seven compounds (C2, C4, C5, C7, C8, C12, and C14) showed a robust TOP1 inhibitory activity (+++ or ++++), and four compounds (A13, C12, C13, and C26) showed a TDP1 inhibition (half-maximal inhibitory concentration values of 15 or 19 µM). We also show that the dual TOP1 and TDP1 inhibitor C12 induces both cellular TOP1cc, TDP1cc formation and DNA damage, resulting in cancer cell apoptosis at a sub-micromolar concentration. In addition, C12 showed an enhanced activity in drug-resistant MCF-7/TDP1 cancer cells and was synergistic with topotecan in both MCF-7 and MCF-7/TDP1 cells.

The long and winding road of designing phosphodiesterase inhibitors for the treatment of heart failure.

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes known to play a critical role in the indirect regulation of several intracellular metabolism pathways through the selective hydrolysis of the phosphodiester bonds of specific second messenger substrates such as cAMP (3',5'-cyclic adenosine monophosphate) and cGMP (3',5'-cyclic guanosine monophosphate), influencing the hypertrophy, contractility, apoptosis and fibroses in the cardiovascular system. The expression and/or activity of multiple PDEs is altered during heart failure (HF), which leads to changes in levels of cyclic nucleotides and function of cardiac muscle. Within the cardiovascular system, PDEs 1-5, 8 and 9 are expressed and are interesting targets for the HF treatment. In this comprehensive review we will present a briefly description of the biochemical importance of each cardiovascular related PDE to the HF, and cover almost all the "long and winding road" of designing and discovering ligands, hits, lead compounds, clinical candidates and drugs as PDE inhibitors in the last decade.

New Deoxycholic Acid Derived Tyrosyl-DNA Phosphodiesterase 1 Inhibitors Also Inhibit Tyrosyl-DNA Phosphodiesterase 2.

A series of deoxycholic acid (DCA) amides containing benzyl ether groups on the steroid core were tested against the tyrosyl-DNA phosphodiesterase 1 (TDP1) and 2 (TDP2) enzymes. In addition, 1,2,4- and 1,3,4-oxadiazole derivatives were synthesized to study the linker influence between a para-bromophenyl moiety and the steroid scaffold. The DCA derivatives demonstrated promising inhibitory activity against TDP1 with IC50 in the submicromolar range. Furthermore, the amides and the 1,3,4-oxadiazole derivatives inhibited the TDP2 enzyme but at substantially higher concentration. Tryptamide 5 and para-bromoanilide 8 derivatives containing benzyloxy substituent at the C-3 position and non-substituted hydroxy group at C-12 on the DCA scaffold inhibited both TDP1 and TDP2 as well as enhanced the cytotoxicity of topotecan in non-toxic concentration in vitro. According to molecular modeling, ligand 5 is anchored into the catalytic pocket of TDP1 by one hydrogen bond to the backbone of Gly458 as well as by π-π stacking between the indolyl rings of the ligand and Tyr590, resulting in excellent activity. It can therefore be concluded that these derivatives contribute to the development of specific TDP1 and TDP2 inhibitors for adjuvant therapy against cancer in combination with topoisomerase poisons.

The First Berberine-Based Inhibitors of Tyrosyl-DNA Phosphodiesterase 1 (Tdp1), an Important DNA Repair Enzyme.

A series of berberine and tetrahydroberberine sulfonate derivatives were prepared and tested against the tyrosyl-DNA phosphodiesterase 1 (Tdp1) DNA-repair enzyme. The berberine derivatives inhibit the Tdp1 enzyme in the low micromolar range; this is the first reported berberine based Tdp1 inhibitor. A structure-activity relationship analysis revealed the importance of bromine substitution in the 12-position on the tetrahydroberberine scaffold. Furthermore, it was shown that the addition of a sulfonate group containing a polyfluoroaromatic moiety at position 9 leads to increased potency, while most of the derivatives containing an alkyl fragment at the same position were not active. According to the molecular modeling, the bromine atom in position 12 forms a hydrogen bond to histidine 493, a key catalytic residue. The cytotoxic effect of topotecan, a clinically important topoisomerase 1 inhibitor, was doubled in the cervical cancer HeLa cell line by derivatives 11g and 12g; both displayed low toxicity without topotecan. Derivatives 11g and 12g can therefore be used for further development to sensitize the action of clinically relevant Topo1 inhibitors.

Synthesis of biphenyl oxazole derivatives via Suzuki coupling and biological evaluations as nucleotide pyrophosphatase/phosphodiesterase-1 and -3 inhibitors.

Oxazole derivatives are important medicinal compounds which are inhibitors of various enzymes such as NPP1, NPP2, NPP3, tyrosine kinase, dipeptidyl-peptidase IV, cyclooxygenase-2, and protein tyrosine phosphatase. In this study, an extensive range of new biologically active biphenyl oxazole derivatives was synthesized in high to excellent yields (57-93%) through Suzuki-Miyaura cross-coupling of bromophenyloxazole with different boronic acids. The reaction was carried out in wet toluene under mild conditions. Overexpression of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and NPP3 has been associated with various health disorders including chondrocalcinosis, cancer, osteoarthritis, and type 2 diabetes. We evaluated the inhibitory potential and selectivity of the synthesized compounds (3a-3q) towards NPP1 and NPP3 at 100 µM concentrations. We found two compounds that were selective and potent inhibitors of these two enzymes on the artificial substrate thymidine 5'-monophosphate para-nitrophenyl ester: compound 3n inhibited NPP1 with an IC50 of 0.15 µM, and compound 3f inhibited NPP3 with an IC50 value of 0.17 µM. The compounds with promising inhibitory potential were docked inside the proteins of NPP1 and NPP3 isozymes to get insight into the plausible binding interactions with active site residues.

Molecular modelling guided design, synthesis and QSAR analysis of new small molecule non-lipid autotaxin inhibitors.

The lysophospholipase D autotaxin (ATX) generates lysophosphatidic acid (LPA) that activates six cognate G-protein coupled receptors (GPCR) in cancerous cells, promoting their motility and invasion. Four novel compounds were generated aided by molecular docking guided design and synthesis techniques to obtain new dual inhibitors of ATX and the lysophosphatidic acid receptor subtype 1 (LPAR1). Biological evaluation of these compounds revealed two compounds, 10 and 11, as new ATX enzyme inhibitors with potencies in the range of 218-220 nM and water solubility (>100 µg/mL), but with no LPAR1 inhibitory activity. A QSAR model was generated that included four newly designed compounds and twenty-one additional compounds that we have reported previously. The QSAR model provided excellent predictability of the pharmacological activity and potency among structurally related drug candidates. This model will be highly useful in guiding the synthesis of new ATX inhibitors in the future.

Design, Synthesis, and Biological Investigation of Novel Classes of 3-Carene-Derived Potent Inhibitors of TDP1.

Two novel structural types of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors with hexahydroisobenzofuran 11 and 3-oxabicyclo [3.3.1]nonane 12 scaffolds were discovered. These monoterpene-derived compounds were synthesized through preliminary isomerization of (+)-3-carene to (+)-2-carene followed by reaction with heteroaromatic aldehydes. All the compounds inhibit the TDP1 enzyme at micro- and submicromolar levels, with the most potent compound having an IC50 value of 0.65 µM. TDP1 is an important DNA repair enzyme and a promising target for the development of new chemosensitizing agents. A panel of isogenic clones of the HEK293FT cell line knockout for the TDP1 gene was created using the CRISPR-Cas9 system. Cytotoxic effects of topotecan (Tpc) and non-cytotoxic compounds of the new structures were investigated separately and jointly in the TDP1 gene knockout cells. For two TDP1 inhibitors, 11h and 12k, a synergistic effect was observed with Tpc in the HEK293FT cells but was not found in TDP1 -/- cells. Thus, it is likely that the synergistic effect is caused by inhibition of TDP1. Synergy was also found for 11h in other cancer cell lines. Thus, sensitizing cancer cells using a non-cytotoxic drug can enhance the efficacy of currently used pharmaceuticals and, concomitantly, reduce toxic side effects.

Discovery of Novel Selective and Orally Bioavailable Phosphodiesterase-1 Inhibitors for the Efficient Treatment of Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and devastating lung disease lacking effective therapy. To identify whether phosphodiesterase-1 (PDE1) inhibition could act as a novel target for the treatment of IPF, hit-to-lead structural optimizations were performed on the PDE9/PDE1 dual inhibitor (R)-C33, leading to compound 3m with an IC50 of 2.9 nM against PDE1C, excellent selectivity across PDE subfamilies, reasonable drug-like properties, and remarkable pharmacodynamic effects as an anti-IPF agent. Oral administration of compound 3m (10 mg/kg) exerted more significant anti-pulmonary fibrosis effects than pirfenidone (150 mg/kg) in a bleomycin-induced IPF rat model and prevented transforming growth factor-ß-induced fibroblast-to-myofibroblast conversion in vitro, indicating that PDE1 inhibition could serve as a novel target for the efficient treatment of IPF.

Discovery of Novel PDEδ Degraders for the Treatment of KRAS Mutant Colorectal Cancer.

KRAS-PDEδ protein-protein interaction represents an appealing target for cancer therapy. However, fast release of high-affinity inhibitors from PDEδ hampered drug binding affinity and antiproliferative activity. To overcome the limitations, the first proteolysis-targeting chimeric (PROTAC) small molecules targeting PDEδ were designed. By employment of PDEδ inhibitor deltazinone (2) and cereblon ligand pomalidomide (6), a series of potent PROTAC PDEδ degraders were obtained. The most promising compound 17f efficiently induced PDEδ degradation and demonstrated significantly improved antiproliferative potency in KRAS mutant SW480 cells. Compound 17f also achieved significant tumor growth inhibition in the SW480 colorectal cancer xenograft model. This proof-of-concept study provided a new strategy to validate the druggability of KRAS-PDEδ interaction and offered an effective lead compound for the treatment of KRAS mutant cancer.

Novel Autotaxin Inhibitor for the Treatment of Idiopathic Pulmonary Fibrosis: A Clinical Candidate Discovered Using DNA-Encoded Chemistry.

The activity of the secreted phosphodiesterase autotaxin produces the inflammatory signaling molecule LPA and has been associated with a number of human diseases including idiopathic pulmonary fibrosis (IPF). We screened a single DNA-encoded chemical library (DECL) of 225 million compounds and identified a series of potent inhibitors. Optimization of this series led to the discovery of compound 1 (X-165), a highly potent, selective, and bioavailable small molecule. Cocrystallization of compound 1 with human autotaxin demonstrated that it has a novel binding mode occupying both the hydrophobic pocket and a channel near the autotaxin active site. Compound 1 inhibited the production of LPA in human and mouse plasma at nanomolar levels and showed efficacy in a mouse model of human lung fibrosis. After successfully completing IND-enabling studies, compound 1 was approved by the FDA for a Phase I clinical trial. These results demonstrate that DECL hits can be readily optimized into clinical candidates.

Structure guided design of potent indole-based ATX inhibitors bearing hydrazone moiety with tumor suppression effects.

ATX was capable of catalyzing the hydrolysis of LPC to the lipid mediator LPA which attracted considerable attention on the development of potent ATX inhibitors. Herein, driven by the HTS product indole-based lead 1, a hybridization strategy was utilized to construct the trifluoroacetyl hydrazone moiety through assembling the phenyl thiazole fragment to the indole skeleton of lead 1. After a systematic structure guided optimization, by cycling the phenyl thiazole to the compacted benzothiazole or decreasing the lipophilicity, two promising ATX inhibitors (9j and 25a) were identified with IC50 values of 2.1 nM and 19.0 nM, respectively. All compounds were tested a panel of cancer cell lines and a preliminary affinity on breast cancer cell lines (SI > 16.5) were observed which shed a light on their potential application of breast cancer relevant cases. Through a dedicated docking study, the intramolecular pseudo-ring within the trifluoroacetylhydrazone moiety played a significant role in constraining the binding poses of 9j and 25a. Finally, a binding free energy calculation was conducted to examine the contribution of different interactions in binding affinity.

Identification of Potent In Vivo Autotaxin Inhibitors that Bind to Both Hydrophobic Pockets and Channels in the Catalytic Domain.

Autotaxin (ATX, also known as ENPP2) is a predominant lysophosphatidic acid (LPA)-producing enzyme in the body, and LPA regulates various physiological functions, such as angiogenesis and wound healing, as well as pathological functions, including proliferation, metastasis, and fibrosis, via specific LPA receptors. Therefore, the ATX-LPA axis is a promising therapeutic target for dozens of diseases, including cancers, pulmonary and liver fibroses, and neuropathic pain. Previous structural studies revealed that the catalytic domain of ATX has a hydrophobic pocket and a hydrophobic channel; these serve to recognize the substrate, lysophosphatidylcholine (LPC), and deliver generated LPA to LPA receptors on the plasma membrane. Most reported ATX inhibitors bind to either the hydrophobic pocket or the hydrophobic channel. Herein, we present a unique ATX inhibitor that binds mainly to the hydrophobic pocket and also partly to the hydrophobic channel, inhibiting ATX activity with high potency and selectivity in vitro and in vivo. Notably, our inhibitor can rescue the cardia bifida (two hearts) phenotype in ATX-overexpressing zebrafish embryos.

Huntington's Disease: A Review of the Known PET Imaging Biomarkers and Targeting Radiotracers.

Huntington's disease (HD) is a fatal neurodegenerative disease caused by a CAG expansion mutation in the huntingtin gene. As a result, intranuclear inclusions of mutant huntingtin protein are formed, which damage striatal medium spiny neurons (MSNs). A review of Positron Emission Tomography (PET) studies relating to HD was performed, including clinical and preclinical data. PET is a powerful tool for visualisation of the HD pathology by non-invasive imaging of specific radiopharmaceuticals, which provide a detailed molecular snapshot of complex mechanistic pathways within the brain. Nowadays, radiochemists are equipped with an impressive arsenal of radioligands to accurately recognise particular receptors of interest. These include key biomarkers of HD: adenosine, cannabinoid, dopaminergic and glutamateric receptors, microglial activation, phosphodiesterase 10 A and synaptic vesicle proteins. This review aims to provide a radiochemical picture of the recent developments in the field of HD PET, with significant attention devoted to radiosynthetic routes towards the tracers relevant to this disease.

Novel deazaflavin tyrosyl-DNA phosphodiesterase 2 (TDP2) inhibitors.

Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a DNA repair enzyme that removes 5'-phosphotyrosyl blockages resulting from topoisomerase II (TOP2)-DNA cleavage complexes trapped by TOP2 inhibitors. TDP2 is a logical target for the development of therapeutics to complement existing treatments based on inhibition of TOP2. There is, however, no TDP2 inhibitor in clinical development at present. Of the reported TDP2 inhibitors, the deazaflavins are the most promising chemical class centered around the lead compound SV-5-153. Recently we reported new subtypes derived within the deazaflavin family with improved membrane permeability properties. In this work we characterize two representative analogues from two new deazaflavin subtypes based on their biochemical TDP2 inhibitory potency and drug-likeness. We demonstrate that the ZW-1288 derivative represents a promising direction for the development of deazaflavins as therapeutic agents. ZW-1288 exhibits potent inhibitory activity at low nanomolar concentrations against recombinant and cellular human TDP2 with profile similar to that of the parent analog SV-5-153 based on high resistance against murine TDP2 and human TDP2 mutated at residue L313H. While expressing weak cytotoxicity on its own, ZW-1288 potentiates the clinical TOP2 inhibitors etoposide (ETP) and mitoxantrone in human prostate DU145 and CCRF-CEM leukemia and chicken lymphoma DT40 cells while not impacting the activity of the topoisomerase I (TOP1) inhibitor camptothecin or the PARP inhibitor olaparib. ZW-1288 increases the uptake of ETP to a lesser extent than SV-5-153 and remained active in TDP2 knockout cells indicating that the deazaflavin TDP2 inhibitors have additional cellular effects that will have to be taken into account for their further development as TDP2 inhibitors.

Promising New Inhibitors of Tyrosyl-DNA Phosphodiesterase I (Tdp 1) Combining 4-Arylcoumarin and Monoterpenoid Moieties as Components of Complex Antitumor Therapy.

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) is an important DNA repair enzyme in humans, and a current and promising inhibition target for the development of new chemosensitizing agents due to its ability to remove DNA damage caused by topoisomerase 1 (Top1) poisons such as topotecan and irinotecan. Herein, we report our work on the synthesis and characterization of new Tdp1 inhibitors that combine the arylcoumarin (neoflavonoid) and monoterpenoid moieties. Our results showed that they are potent Tdp1 inhibitors with IC50 values in the submicromolar range. In vivo experiments with mice revealed that compound 3ba (IC50 0.62 µM) induced a significant increase in the antitumor effect of topotecan on the Krebs-2 ascites tumor model. Our results further strengthen the argument that Tdp1 is a druggable target with the potential to be developed into a clinically-potent adjunct therapy in conjunction with Top1 poisons.

Synthesis, Anti-inflammatory Activity and Docking Studies of Some Newer 1,3-Thiazolidine-2,4-dione Derivatives as Dual Inhibitors of PDE4 and PDE7.

BACKGROUND: Phosphodiesterase 4 (PDE4) and phosphodiesterase 7 (PDE7), PDE superfamily members, increase inflammatory processes in immunomodulatory as well as pro-inflammatory cells via breakdown of cyclic adenosine monophosphate. Dual inhibitors of PDE4 and PDE7 are a novel class of drug candidates which can regulate pro-inflammatory as well as T-cell function and can be particularly advantageous in the treatment of a wide-ranging disorders associated with the immune system as well as inflammatory diseases with fewer unwanted adverse effects. OBJECTIVE: The current research work was planned to design and synthesize some newer substituted 1,3- thiazolidine-2,4-dione derivatives as dual inhibitors of PDE4 and PDE7 followed by evaluation of their anti-inflammatory activity and in silico docking studies. METHODS: A new series of substituted 1,3-thiazolidine-2,4-dione derivatives was synthesized followed by evaluation of their anti-inflammatory activity in animal models. In silico docking studies were performed for the evaluation of the binding pattern of synthesized derivatives in the binding site of both PDE4 and PDE7 proteins. RESULTS: Amongst the newly synthesized derivatives, compounds 5 and 12 showed higher antiinflammatory activity in the animal model. The results of in vivo animal studies were found to be in concordance with the results of molecular docking studies. CONCLUSION: These newly synthesized derivatives can act as the lead molecules for the design of safe and therapeutically effective agents for various inflammatory diseases acting via inhibition of both PDE4 and PDE7.

Discovery, Synthesis, and Evaluation of Oxynitidine Derivatives as Dual Inhibitors of DNA Topoisomerase IB (TOP1) and Tyrosyl-DNA Phosphodiesterase 1 (TDP1), and Potential Antitumor Agents.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a recently discovered enzyme repairing DNA lesions resulting from stalled topoisomerase IB (TOP1)-DNA covalent complex. Inhibiting TDP1 in conjunction with TOP1 inhibitors can boost the action of the latter. Herein, we report the discovery of the natural product oxynitidine scaffold as a novel chemotype for the development of TOP1 and TDP1 inhibitors. Three kinds of analogues, benzophenanthridinone, dihydrobenzophenanthridine, and benzophenanthridine derivatives, were synthesized and evaluated for both TOP1 and TDP1 inhibition and cytotoxicity. Analogue 19a showed high TOP1 inhibition (+++) and induced the formation of cellular TOP1cc and DNA damage, resulting in cancer cells apoptosis at nanomolar concentration range. In vivo studies indicated that 19a exhibits antitumor efficiency in HCT116 xenograft model. 41a exhibited additional TDP1 inhibition with IC50 value of 7 µM and synergistic effect with camptothecin in MCF-7 cells. This work will facilitate future efforts for the discovery of natural product-based TOP1 and TDP1 inhibitors.