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The WNT signaling system governs critical processes during embryonic development and tissue homeostasis, and its dysfunction can lead to cancer. Details concerning selectivity and differences in relative binding affinities of 19 mammalian WNTs to the cysteine-rich domain (CRD) of their receptors���the ten mammalian Frizzleds (FZDs)���remain unclear. Here, we used eGFP-tagged mouse WNT-3A for a systematic analysis of WNT interaction with every human FZD paralogue in HEK293A cells. Employing HiBiT-tagged full-length FZDs, we studied eGFP-WNT-3A binding kinetics, saturation binding, and competition binding with commercially available WNTs in live HEK293A cells using a NanoBiT/BRET-based assay. Further, we generated receptor chimeras to dissect the contribution of the transmembrane core to WNT-CRD binding. Our data pinpoint distinct WNT-FZD selectivity and shed light on the complex WNT-FZD binding mechanism. The methodological development described herein reveals yet unappreciated details of the complexity of WNT signaling and WNT-FZD interactions, providing further details with respect to WNT-FZD selectivity.

WNT binding to Frizzleds (FZD) is a crucial step that leads to the initiation of signalling cascades governing multiple processes during embryonic development, stem cell regulation and adult tissue homeostasis. Recent efforts have enabled us to shed light on WNT-FZD pharmacology in overexpressed HEK293 cell systems. However, it is important to assess ligand binding at endogenous receptor levels as there might be differential binding behaviour in a native environment. Here, we focus on one FZD paralogue: FZD7, and study its interactions with WNT-3A in a CRISPR-Cas9-edited SW480 colorectal cancer model. SW480 cells were CRISPR-Cas9-edited to insert a HiBiT-tag on the N-terminus of FZD7, preserving the native signal peptide. Subsequently, these cells were used to study eGFP-WNT-3A association to endogenous and overexpressed HiBiT-FZD7 using NanoBiT/BRET to measure ligand binding and quantification of NanoBiT-emitted luminescence to assess receptor internalization. eGFP-WNT-3A bound to endogenous HiBiT-FZD7 with significantly higher kon and with lower Kd than to overexpressed receptors. Importantly, as the fluorescent probe is an agonist, experiments performed in cell lysates demonstrated that eGFP-WNT-3A/HiBiT-FZD7 binding assessment is not altered by receptor internalization. In conclusion, binding affinities of eGFP-WNT-3A to HiBiT-FZD7 decreased with increasing receptor concentrations suggesting that HiBiT-FZD7 overexpression fails to recapitulate ligand binding behaviour in a (patho-)physiologically relevant context where endogenous receptor expression levels are lower.

Pain associated with musculoskeletal system disorders is often the reason why patients seek medical advice. This is also the case for osteoarthritis where the pain is the first and dominant symptom which reduces the quality of life. Therefore, pain reduction and improvement in joint mobility should be the objectives of therapy. Pharmacotherapy in osteoarthritis is based on paracetamol, NSAIDS, SYSADOA or opioids. Additionally, injections with corticosteroids, platelet-rich plasma or hyaluronic acid, as well as sesamin- and polyphenol-rich diet, have proven effective. It needs to be noted that optimal therapy in osteoarthritis should consist of pharmacotherapy combined with non-pharmacological methods, such as physiotherapy, bodyweight monitoring, well-balanced diet and education. Pain associated with musculoskeletal system disorders is often the reason why patients seek medical advice. This is also the case for osteoarthritis where the pain is the first and dominant symptom which reduces the quality of life. Therefore, pain reduction and improvement in joint mobility should be the objectives of therapy. Pharmacotherapy in osteoarthritis is based on paracetamol, NSAIDS, SYSADOA or opioids. Additionally, injections with corticosteroids, platelet-rich plasma or hyaluronic acid, as well as sesamin- and polyphenol-rich diet, have proven effective. It needs to be noted that optimal therapy in osteoarthritis should consist of pharmacotherapy combined with non-pharmacological methods, such as physiotherapy, bodyweight monitoring, well-balanced diet and education. Pain associated with musculoskeletal system disorders is often the reason why patients seek medical advice. This is also the case for osteoarthritis where the pain is the first and dominant symptom which reduces the quality of life. Therefore, pain reduction and improvement in joint mobility should be the objectives of therapy. Pharmacotherapy in osteoarthritis is based on paracetamol, NSAIDS, SYSADOA or opioids. Additionally, injections with corticosteroids, platelet-rich plasma or hyaluronic acid, as well as sesamin- and polyphenol-rich diet, have proven effective. It needs to be noted that optimal therapy in osteoarthritis should consist of pharmacotherapy combined with non-pharmacological methods, such as physiotherapy, bodyweight monitoring, well-balanced diet and education.

Smoothened (SMO) is a GPCR that mediates hedgehog signaling. Hedgehog binds the transmembrane protein Patched, which in turn regulates SMO activation. Overactive SMO signaling is oncogenic and is therefore a clinically established drug target. Here we establish a nanoluciferase bioluminescence resonance energy transfer (NanoBRET)-based ligand binding assay for SMO providing a sensitive and high throughput-compatible addition to the toolbox of GPCR pharmacologists. In the NanoBRET-based binding assay, SMO is N terminally tagged with nanoluciferase (Nluc) and binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between receptor and ligand. The assay allowed kinetic analysis of ligand-receptor binding in living HEK293 cells, competition binding experiments using commercially available SMO ligands (SANT-1, cyclopamine-KAAD, SAG1.3 and purmorphamine), and pharmacological dissection of two BODIPY-cyclopamine binding sites. This high throughput-compatible assay is superior to commonly used SMO ligand binding assays in the separation of specific from non-specific ligand binding and, provides a suitable complement to chemical biology strategies for the discovery of novel SMO-targeting drugs. SIGNIFICANCE STATEMENT: We established a NanoBRET-based binding assay for SMO with superior sensitivity compared to fluorescence-based assays. This assay allows distinction of two separate binding sites for BODIPY-cyclopamine on the SMO transmembrane core in live cells in real time. The assay is a valuable complement for drug discovery efforts and will support a better understanding of Class F GPCR pharmacology.

The Concise Guide to PHARMACOLOGY 2021/22 is the fifth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of nearly 1900 human drug targets with an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes over 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/bph.15538. G protein-coupled receptors are one of the six major pharmacological targets into which the Guide is divided, with the others being: ion channels, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2021, and supersedes data presented in the 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Smoothened (SMO) is a G protein-coupled receptor (GPCR) that mediates Hedgehog (Hh) signaling. SMO activity is regulated following the binding of Hh to the transmembrane protein Patched. Overactive SMO signaling is oncogenic, and hence this receptor is a target for several marketed drugs. However, development of new SMO ligands has been hampered by the fact that current radioligand and fluorescence-based binding assays are not high-throughput scalable. Here, we demonstrate two Nanoluciferase (Nluc) bioluminescence resonance energy transfer-based ligand binding assays (NanoBRET and NanoBiT/BRET) which provide a sensitive and high-throughput-compatible tool in drug screening efforts. In the described assays, SMO is N-terminally tagged either with full-length nanoluciferase or the partial HiBiT sequence, and subsequently binding of BODIPY-cyclopamine is assessed by quantifying resonance energy transfer between the receptor and the fluorescent ligand. Additionally, the assay allows performing competition binding experiments using commercially available SMO ligands, such as the SMO agonist SAG1.3.

The class Frizzled of G protein-coupled receptors (GPCRs), consisting of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO), remains one of the most enigmatic GPCR families. While SMO relies on cholesterol binding to the 7TM core of the receptor to activate downstream signaling, underlying details of receptor activation remain obscure for FZDs. Here, we aimed to investigate the activation mechanisms of class F receptors utilizing a computational biology approach and mutational analysis of receptor function in combination with ligand binding and downstream signaling assays in living cells. Our results indicate that FZDs differ substantially from SMO in receptor activation-associated conformational changes. SMO manifests a preference for a straight TM6 in both ligand binding and functional readouts. Similar to the majority of GPCRs, FZDs present with a kinked TM6 upon activation owing to the presence of residue P6.43. Functional comparison of FZD and FZD P6.43F mutants in different assay formats monitoring ligand binding, G protein activation, DVL2 recruitment and TOPflash activity, however, underlines further the functional diversity among FZDs and not only between FZDs and SMO., The class Frizzled of G protein-coupled receptors (GPCRs) consist of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO). Here the Schulte laboratory demonstrates that FZDs differ substantially from SMO in receptor activation-associated conformational changes, while SMO manifests a preference for a straight TM6, the TM6 of FZDs is kinked upon activation.

Class F receptors, including the ten Frizzleds (FZD1-10 ) and SMO, mediate the effects of WNTs and hedgehog proteins and belong to the superfamily of G protein-coupled receptors (GPCRs). While the recent, high-resolution insight into mechanisms of GPCR activation provides a better understanding of receptor activation in Class A, B and C GPCRs, it remains unclear how Class F receptors bind their ligands, how ligand binding is translated to receptor activation and how signal initiation and specification are achieved. Here, we summarize recent efforts in elucidating Class F receptor structure and activation mechanisms and critically discuss the progress made in this area. A better understanding of the activation mechanisms of Class F receptors is required to engage in mechanism-based and structure-guided drug discovery to exploit the large therapeutic potential of targeting these receptors pharmacologically.

Class F receptors are considered valuable therapeutic targets due to their role in human disease, but structural changes accompanying receptor activation remain unexplored. Employing population and cancer genomics data, structural analyses, molecular dynamics simulations, resonance energy transfer-based approaches and mutagenesis, we identify a conserved basic amino acid in TM6 in Class F receptors that acts as a molecular switch to mediate receptor activation. Across all tested Class F receptors (FZD4,5,6,7, SMO), mutation of the molecular switch confers an increased potency of agonists by stabilizing an active conformation as assessed by engineered mini G proteins as conformational sensors. Disruption of the switch abrogates the functional interaction between FZDs and the phosphoprotein Dishevelled, supporting conformational selection as a prerequisite for functional selectivity. Our studies reveal the molecular basis of a common activation mechanism conserved in all Class F receptors, which facilitates assay development and future discovery of Class F receptor-targeting drugs., Class F receptors are therapeutic targets in human disease and understanding their structural changes during receptor activation may provide important pharmacological insight. Here, the authors combine computational and experimental methods to identify a molecular switch in TM6/7 of Class F receptors that mediates receptor activation.

GPR61 is an orphan receptor that belongs to Class A of G-protein-coupled receptors. It has been reported that GPR61 has a constitutive activity and couples to Gαs. In the present study, we characterized GPR61 function and ligand binding by experimental and molecular docking studies. We demonstrated that heterologous expression of GPR61 in HEK293 cells enhanced the cAMP synthesis response to forskolin, whereas the basal cAMP synthesis was unaffected. 5-(Nonyloxy)tryptamine inhibited forskolin-stimulated cAMP production in GPR61-expressing HEK293 cells. These studies highlight that the intrinsic activity of this receptor is only measurable following its synergy with Gαs.

The Frizzled (FZD) family of WNT receptors consists of ten paralogues in mammals. They belong to the superfamily of G protein-coupled receptors and regulate crucial processes during embryonic development. Dysregulated FZD signaling leads to disease, most prominently to diverse forms of cancer, which renders these receptors attractive for drug discovery. Recent advances in assay development and the design of genetically encoded biosensors monitoring ligand-receptor interaction, conformational dynamics, and protein-protein interaction have allowed for a better pharmacological understanding of WNT/FZD signal transduction and open novel avenues for mechanism-based drug discovery and screening. In this chapter, we summarize the recent progress in the molecular dissection of FZD activation based on advanced biosensors.

Synapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an essential pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial protein biogenesis takes place locally to maintain functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain. Importantly, in a mouse model of fragile X syndrome, Fmr1 KO mice, a common disease associated with dysregulation of synaptic protein synthesis, we observed altered morphology and respiration rates of synaptic mitochondria. That indicates that the local production of mitochondrial proteins plays an essential role in synaptic functions., Using global proteomic analysis and a mouse model of fragile X syndrome, Fmr1 KO mice, this study reveals that the local production of mitochondrial proteins plays an essential role in synaptic functions.

The Wingless/Int1 (Wnt) signaling system plays multiple, essential roles in embryonic development, tissue homeostasis, and human diseases. Although many of the underlying signaling mechanisms are becoming clearer, the binding mode, kinetics, and selectivity of 19 mammalian WNTs to their receptors of the class Frizzled (FZD1–10) remain obscure. Attempts to investigate Wnt-FZD interactions are hampered by the difficulties in working with Wnt proteins and their recalcitrance to epitope tagging. Here, we used a fluorescently tagged version of mouse Wnt-3a for studying Wnt-FZD interactions. We observed that the enhanced GFP (eGFP)-tagged Wnt-3a maintains properties akin to wild-type (WT) Wnt-3a in several biologically relevant contexts. The eGFP-tagged Wnt-3a was secreted in an evenness interrupted (EVI)/Wntless-dependent manner, activated Wnt/β-catenin signaling in 2D and 3D cell culture experiments, promoted axis duplication in Xenopus embryos, stimulated low-density lipoprotein receptor-related protein 6 (LRP6) phosphorylation in cells, and associated with exosomes. Further, we used conditioned medium containing eGFP-Wnt-3a to visualize its binding to FZD and to quantify Wnt-FZD interactions in real time in live cells, utilizing a recently established NanoBRET-based ligand binding assay. In summary, the development of a biologically active, fluorescent Wnt-3a reported here opens up the technical possibilities to unravel the intricate biology of Wnt signaling and Wnt-receptor selectivity.

2,2′-dihydroxybenzophenones and their oxime and N-acyl hydrazone derivatives have been studied via the DFT/B3LYP-6-311++G** methodology. An almost coplanar bifurcated six-membered H bridge is found in ketones. A similar H bridge, accompanied by a seven-membered one in oximes and a nine-membered-like one in hydrazones, is also formed. While the closed (two pseudo rings) conformer is the lowest energy one in 2,2′-dihydroxybenzophenones and their oximes, the semi-closed conformer (one pseudo ring) corresponds to the lowest energy one in N-acyl hydrazones. The ΔHf of the closed conformer compared to its open counterpart is ca. 17 kcal/mol in 2,2′-dihydroxybenzophenones while that in oximes is ca. 11 kcal/mol. The energy barrier in changing from the closed to the open (no pseudo ring) conformation is

Calcitriol (1,25-dihydroxyvitamin D3, 1,25D3) and vitamin D side-chain modified analogs (VDAs) have gained considerable attention as potential drugs in the treatment of acute myeloid leukemia (AML), yet studies of the impact of 1,25D3 and VDAs upon other haematological malignancies are more limited. To address this gap in knowledge, we have examined the action of 1,25D3 and VDAs on a human cell line (DOHH2, K422) typifying diffuse large B-cell lymphoma (DLBCL) and also peripheral blood B-cells isolated from healthy donors. 1,25D3 and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. 1,25D3 and VDAs (100nM) caused the death of approximately 40% DOHH2 cells after 24h stimulation, similar to their impact on HL-60 cells (acute myeloid leukaemia cell line). In addition, 1,25D3 and VDAs displayed concentration and time-dependent anti-proliferative actions upon stimulated B-cells from healthy donors. The VDAs inhibited proliferation by approximately 30%. Hence VDAs may offer therapeutic potential for the treatment of DLBCL or conditions benefitted by B-cell depletion.

SummarySynapses are the regions of the neuron that enable the transmission and propagation of action potentials on the cost of high energy consumption and elevated demand for mitochondrial ATP production. The rapid changes in local energetic requirements at dendritic spines imply the role of mitochondria in the maintenance of their homeostasis. Using global proteomic analysis supported with complementary experimental approaches, we show that an important pool of mitochondrial proteins is locally produced at the synapse indicating that mitochondrial biogenesis takes place locally to maintain the pool of functional mitochondria in axons and dendrites. Furthermore, we show that stimulation of synaptoneurosomes induces the local synthesis of mitochondrial proteins that are transported to the mitochondria and incorporated into the protein supercomplexes of the respiratory chain.SynopsisMitochondria biogenesis in the synapse is supported by local translationMitochondrial proteins represent a large pool of proteins synthesized locally at the synapseNewly synthesized mitochondrial proteins are imported into the mitochondria and incorporated into the respiratory chain complexesUncoupling of mitochondria and blocking mitochondrial import inhibits incorporation of de novo synthesized proteins into the mitochondrial protein complexes

2,2’-dihydroxybenzophenones and derivatives have been synthesized directly or by oxidation of their incipiently obtained benzylic alcohols by diverse efficient methods. Oxime and N-acyl hydrazone derivatives have also been prepared. Their structure profile has been scrutinized by DFT/B3LYP-6-311++G** methodology, NMR spectroscopy and dihedral angle grid scan analysis. Energetically favorable conformations pointed to (i) an almost coplanar bifurcated 6-membered H bridge in ketones, (ii) a single 6-membered H bridge, accompanied by a 7-membered H bonding interaction in oximes and (iii) a single 6-membered H-bridge in hydrazones. In the latter case, a stable conformation with an additional 9-membered pseudo ring was also found. Highly deshielded protons in the NMR spectra are in accordance with the theoretically obtained findings on the H-bonded conformers. Significant anti-inflammatory activity of the compounds has been found by in vivo tests with their oxime and hydrazone derivatives showing the highest activity, hydrazone 11, in partucular, competing with marketed drugs. In silico docking studies point to the perspective potency of these structures as COX-1/COX-2 inhibitors.

Geissospermumspecies are medically important plants due to their health-promoting effects. The objective of this study was to determine the antioxidant ability and antiproliferative and cytotoxic effects of infusions, tinctures, and ethanolic extracts ofGeissospermum reticulatumbarks in relation to the contents of total phenolics and flavonoids. Seven samples of barks were collected in various regions of Peruvian Amazonia. We found that the amount of total phenolics in the studied products varied from 212.40±0.69 to 1253.92±11.20 mg GAE/kg. In our study there is a correlation (R2=0.7947) between the results of antioxidants assays: FRAP and ORAC for tinctures, infusions, and ethanolic extracts ofG. reticulatumbarks. We have also observed antiproliferative activities of the ethanolic extracts on normal T-cells. These extracts have caused death on malignant cell lines (THP-1 and HL-60) and this data correlates well with their antioxidant capacity measured by ORAC method. Interestingly, the highest concentration of the ethanolic extract was not toxic in the zebrafish embryo developmental assay. Our results indicate thatG. reticulatumis rich in antioxidants and have cytotoxic and antiproliferative properties. The data suggests potential immunosuppressive role of the extracts. This is the first study presenting the results of chemical and biological analysis of multiple preparations fromG. reticulatum.

Oxidatively generated β-nitroso-o-quinone methides undergo an o- and/or peri-intramolecular cyclization to arene-fused 1,2-oxazoles, 1,2-oxazines or indoles. The reaction, found to be an innate process, has been scrutinized by DFT/B3LYP and MP2 calculations. Due to its rapidity, the process has been termed a ‘reclusive’ one. Competing o-(1,5)- and peri-(1,6)- or (1,5)-cyclizations advance via successive transition states. Activation barriers are drastically lowered in AcOH, probably through H hopping or tunnelling whereas they are barely reduced in other solvents. Aromaticity indices, such as HOMA, IA and ABO, have been used to assess the stability of the end-heterocycles and the preponderance of any one of them. Thus, the preferred cyclization mode, that is, the prevalence or exclusive formation of one of the heterocycles, appears to be oxidant-directed rather than determined by the quinone methide geometry. The question of the peri-cyclization, being a primary or a secondary process, has been tackled.

DFT calculations have shown that the N–O dipole of benzene- and naphthalene-fused 1,2-oxazole N-oxides causes a distortion of their σ and π frame, concentrated on the 1,2-oxazole ring, such that it increases its susceptibility to opening. The distortion forces the benzene ring into some diene geometry, thus, reducing π delocalization over the bi- or tricyclic structure and ultimately their aromatic character. C-3 substitution has a marked influence mainly on the naphthalene-fused N-oxides. C-5 and particularly C-6 substitution, as the position of most extended interaction with the N–O dipole through the π ring density, contribute to the distortion of the 1,2-oxazole geometry and thereby to the decrease of aromaticity of the structure. Bond uniformity (I A), average bond order (ABO) and Harmonic Oscillator Model of Aromaticity (HOMA) indices have been recruited to measure aromaticity changes. I A and ABO appear to be more credible to 1,2-benzoxazole N-oxides and 1,2-naphthoxazole N-oxides, respectively, while HOMA has been found equally reliable to both. Hardness and dipole moments follow similar trends. Energies, localization and separation of the four frontiers orbitals, i.e. HO, HO−1, and LU, LU+1, indicate a rather notable aromatic character of the N-oxides. Their reactivity profile, portrayed by descriptors such as Fukui and electro(nucleo)philicity Parr functions, shows good agreement with experimental outcomes towards electrophiles but succumbs to discrepancies towards nucleophiles due to the susceptibility of the hetero-ring to opening. The “push–pull” character of the N–O dipole and more importantly the extent of its double bonding direct site selectivity.

The structure and reactivity profiles of prototype o-quinone methides 1, 2 and their β-nitroso analogues 6–9 have been investigated by means of DFT and MP2 calculations. These highly reactive unstable species are generated by oxidative dearomatization of their precursor oximes. The destabilization of their structure is more pronounced in the β-nitroso congeners 7–9. There is only a weak π conjugation across the nitrosoalkene arm. The latter gives rise to E and Z conformations and causes some distortion on the ring σ-frame, while the π-frame is weakly perturbed. The Z conformation is the most stable in all structures. Their geometry is also affected by the o-quinone ring and the 1,2-(7 and 8) and 2,3-(9) isomer pattern. The stability of these conformations is rationalized in terms of ortho- or peri- ring formations. The impact of their geometry profile on their reactivity pattern has been studied by means of reactivity descriptors such as Fukui and Parr functions, chemical potential and hardness, HOMO and LUMO energies and their separation (HOMO–LUMO gap) as well as aromaticity indices such as HOMA and out-of-plane deformability. All descriptors consistently demonstrate that the reactivity is dominated by an E/Z-controlled intramolecular ortho- or peri-cyclization mode to fused 1,2-oxazoles or 1,2-oxazines vs indoles, respectively. Intermolecular primary reactions can occur at the quinone alkene bond or that of the nitrosoalkene arm.

Alkaloid-rich extract from Uncaria tomentosa (cat’s claw) has been reported to cause apoptosis in cancer lines. Oxindole pentacyclic alkaloids of the plant are responsible for this effect, yet their biological mechanism of action is not fully understood. In this work the set of these alkaloids underwent an extensive theoretical study with reverse virtual screening and molecular docking methods implemented in AutoDock, AutoDock Vina, and Molegro Virtual Docker. The results from these computational methods indicate that inhibition of several important targets including dihydrofolate reductase and mouse double minute 2 homolog (MDM2) may be responsible for the biological activity of the alkaloids. The docking results also show that the alkaloids can interact with Dvl-2, Akt-2, and leukotriene A4 hydrolase. Reverse virtual screening and molecular docking are valuable tools to aid identification of protein targets for bioactive hit molecules and could guide the design of in-depth biochemical activity tests and utilization of these alkaloids in anticancer drug development. .