Plant carbonic anhydrase-like enzymes in neuroactive alkaloid biosynthesis.

Plants synthesize numerous alkaloids that mimic animal neurotransmitters1. The diversity of alkaloid structures is achieved through the generation and tailoring of unique carbon scaffolds2,3, yet many neuroactive alkaloids belong to a scaffold class for which no biosynthetic route or enzyme catalyst is known. By studying highly coordinated, tissue-specific gene expression in plants that produce neuroactive Lycopodium alkaloids4, we identified an unexpected enzyme class for alkaloid biosynthesis: neofunctionalized α-carbonic anhydrases (CAHs). We show that three CAH-like (CAL) proteins are required in the biosynthetic route to a key precursor of the Lycopodium alkaloids by catalysing a stereospecific Mannich-like condensation and subsequent bicyclic scaffold generation. Also, we describe a series of scaffold tailoring steps that generate the optimized acetylcholinesterase inhibition activity of huperzine A5. Our findings suggest a broader involvement of CAH-like enzymes in specialized metabolism and demonstrate how successive scaffold tailoring can drive potency against a neurological protein target.

Connectivity between surface and interior in catalytic subunits of acetylcholinesterases inferred from their X-ray structures.

Catalytic activity and function of acetylcholinesterase (AChE; EC 3.1.1.7) have been recognized and studied for over a century and its quaternary and primary structures for about half a century, and its tertiary structure has been known for about 33 years. Clear understanding of relationships between the structure and the function is still pending for this enzyme. Hundreds of crystallographic, static snapshots of AChEs from different sources reveal largely one general backbone conformation with narrow entry into the active center gorge, tightly fit to accept one acetylcholine (ACh) molecule, in contrast to its high catalytic turnover. This short review of available X-ray structures of AChEs from electric ray Torpedo californica, mouse and human, finds some limited, yet consistent deviations in conformations of selected secondary structure elements of AChE relevant for its function. The observed conformational diversity of the acyl pocket loop of AChE, unlike the large Ω-loop, appears consistent with structurally dynamic INS data and solution-based SAXS experiments to explain its dominant role in controlling the size of the active center gorge opening, as well as connectivity between the immediate surroundings of the buried active Ser, and catalytically relevant sites on the AChE surface.

A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

Recent advances in the total synthesis of galantamine, a natural medicine for Alzheimer's disease.

Covering: 2006 to 2023(-)-Galantamine is a natural product with distinctive structural features and potent inhibitory activity against acetylcholine esterase (AChE). It is clinically approved for the treatment of Alzheimer's disease. The clinical significance and scarcity of this natural product have prompted extensive and ongoing efforts towards the chemical synthesis of this challenging tetracyclic structure. The objective of this review is to summarize and discuss recent progress in the total synthesis of galantamine from 2006 to 2023. The contents are organized according to the synthetic strategies for the construction of the quaternary center. Key features of each synthesis have been highlighted, followed by a summary and outlook at the end.

Recent development and strategies towards target interactions: Synthesis, characterization and in silico analysis of benzimidazole based thiadiazole as potential anti-Alzheimer agents.

In the current research study, we aim to design and synthesize highly potent hybrid analogs of benzimidazole derived thiadiazole based Schiff base derivatives which can combat the cholinesterase enzymes (acetylcholinesterase and butyrylcholinesterase) accountable for developing Alzheimer's disease. In this context, we have synthesized 15 analogs of benzimidazole based thiadiazole derivatives, which were subsequently confirmed through spectroscopic techniques including 1H NMR, 13C NMR and HREI-MS. Biological investigation of all the analogs revealed their varied acetylcholinesterase inhibitory potency covering a range between 3.20 ± 0.10 µM to 20.50 ± 0.20 µM as well as butyrylcholinesterase inhibitory potential with a range of 4.30 ± 0.50 µM to 20.70 ± 0.50 µM when compared with the standard drug Donepezil having IC50 = 6.70 ± 0.20 µM for AChE and 7.90 ± 0.10 µM for BuChE. The promising inhibition by the analogs was evaluated in SAR analysis, where analog-1 (IC50 = 3.20 ± 0.10 µM for AChE and 4.30 ± 0.50 µM for BuChE), analog-4 (IC50 = 4.30 ± 0.30 µM for AChE and 5.50 ± 0.20 µM for BuChE) and analog-5 (IC50 = 4.10 ± 0.30 µM for AChE and 4.60 ± 0.40 µM for BuChE) were found as the lead candidates. Moreover, molecular docking and ADME analysis were conducted to explore the better binding interactions and drugs likeness respectively.

Pnictogen bond-driven control of the molecular interaction between organophosphorus and acetylcholinesterase enzyme.

This study addresses a comprehensive assessment of the interaction between chemical warfare agents (CWA) and acetylcholinesterase (AChE) systems, focus on the intriguing pnictogen-bond interaction (PnB). Utilizing the crystallographic data from the Protein Data Bank pertaining to the AChE-CWA complex involving Sarin (GB), Cyclosarin (GF), 2-[fluoro(methyl)phosphoryl]oxy-1,1-dimethylcyclopentane (GP) and venomous agent X (VX) agents, the CWA is systematically displaced by increments of 0.1 Å along the PO bond axis, extending its distance by 4 Å from the original position. The AIM analysis was carried out and consistently revealed the presence of a significant interaction along the PO bond. Investigating the intrinsic nature of the PnB, the NBO and the EDA analysis unearthed the contribution of orbital factors to the overall energy of the system. Strikingly, this observation challenges the conventional σ-hole explanation commonly associated with such interactions. This finding adds a layer of complexity to understanding of PnB, encouraging further exploration into the underlying mechanisms governing these intriguing chemical phenomena.

Rationally Designed Functionalization of Single-Walled Carbon Nanotubes for Real-Time Monitoring of Cholinesterase Activity and Inhibition in Plasma.

Enzymes play a pivotal role in regulating numerous bodily functions. Thus, there is a growing need for developing sensors enabling real-time monitoring of enzymatic activity and inhibition. The activity and inhibition of cholinesterase (CHE) enzymes in blood plasma are fluorometrically monitored using near-infrared (NIR) fluorescent single-walled carbon nanotubes (SWCNTs) as probes, strategically functionalized with myristoylcholine (MC)- the substrate of CHE. A significant decrease in the fluorescence intensity of MC-suspended SWCNTs upon interaction with CHE is observed, attributed to the hydrolysis of the MC corona phase of the SWCNTs by CHE. Complementary measurements for quantifying choline, the product of MC hydrolysis, reveal a correlation between the fluorescence intensity decrease and the amount of released choline, rendering the SWCNTs optical sensors with real-time feedback in the NIR biologically transparent spectral range. Moreover, when synthetic and naturally abundant inhibitors inhibit the CHE enzymes present in blood plasma, no significant modulations of the MC-SWCNT fluorescence are observed, allowing effective detection of CHE inhibition. The rationally designed SWCNT sensors platform for monitoring of enzymatic activity and inhibition in clinically relevant samples is envisioned to not only advance the field of clinical diagnostics but also deepen further understanding of enzyme-related processes in complex biological fluids.

Treatment of Organophosphorus Poisoning with 6-Alkoxypyridin-3-ol Quinone Methide Precursors: Resurrection of Methylphosphonate-Aged Acetylcholinesterase.

Organophosphorus (OP) nerve agents inhibit acetylcholinesterase (AChE), creating a cholinergic crisis in which death can occur. The phosphylated serine residue spontaneously dealkylates to the OP-aged form, which current therapeutics cannot reverse. Soman's aging half-life is 4.2 min, so immediate recovery (resurrection) of OP-aged AChE is needed. In 2018, we showed pyridin-3-ol-based quinone methide precursors (QMPs) can resurrect OP-aged electric eel AChE in vitro, achieving 2% resurrection after 24 h of incubation (pH 7, 4 mM). We prepared 50 unique 6-alkoxypyridin-3-ol QMPs with 10 alkoxy groups and five amine leaving groups to improve AChE resurrection. These compounds are predicted in silico to cross the blood-brain barrier and treat AChE in the central nervous system. This library resurrected 7.9% activity of OP-aged recombinant human AChE after 24 h at 250 µM, a 4-fold increase from our 2018 report. The best QMP (1b), with a 6-methoxypyridin-3-ol core and a diethylamine leaving group, recovered 20.8% (1 mM), 34% (4 mM), and 42.5% (predicted maximum) of methylphosphonate-aged AChE activity over 24 h. Seven QMPs recovered activity from AChE aged with Soman and a VX degradation product (EA-2192). We hypothesize that QMPs form the quinone methide (QM) to realkylate the phosphylated serine residue as the first step of resurrection. We calculated thermodynamic energetics for QM formation, but there was no trend with the experimental biochemical data. Molecular docking studies revealed that QMP binding to OP-aged AChE is not the determining factor for the observed biochemical trends; thus, QM formation may be enzyme-mediated.

Sulphated Fucooligosaccharide from Sargassum Horneri: Structural Analysis and Anti-Alzheimer Activity.

In the present study, sulfated polysaccharides were obtained by digestion of Sargassum horneri and preparation with enzyme-assisted extraction using three food-grade enzymes, and their anti- Alzheimer's activities were investigated. The results demonstrated that the crude sulfated polysaccharides extracted using AMGSP, CSP and VSP dose-dependently (25-100 µg·mL- 1) raised the spontaneous alternating manner (%) in the Y maze experiment of mice and reduced the escape latency time in Morris maze test. AMGSP, CSP and VSP also exhibited good anti-AChE and moderate anti-BuChE activities. CSP displayed the best inhibitory efficacy against AChE. with IC50 values of 9.77 µM. And, CSP also exhibited good inhibitory selectivity of AChE over BuChE. Next, CSP of the best active crude extract was separated by the preparation type high performance liquid phase to obtain the sulphated fucooligosaccharide section: SFcup (→3-α-L-fucp(2-SO3-)-1→4-α-L-fucp(2,3-SO3-)-1→section), SFcup showed a best inhibitory efficacy against AChE with IC50 values of 4.03 µM. The kinetic research showed that SFcup inhibited AChE through dual binding sites. Moreover, the molecular docking of SFcup at the AChE active site was in accordance with the acquired pharmacological results.

Fabrication of Hybrid Coated Microneedles with Donepezil Utilizing Digital Light Processing and Semisolid Extrusion Printing for the Management of Alzheimer's Disease.

Microneedle (MN) patches are gaining increasing attention as a cost-effective technology for delivering drugs directly into the skin. In the present study, two different 3D printing processes were utilized to produce coated MNs, namely, digital light processing (DLP) and semisolid extrusion (SSE). Donepezil (DN), a cholinesterase inhibitor administered for the treatment of Alzheimer's disease, was incorporated into the coating material. Physiochemical characterization of the coated MNs confirmed the successful incorporation of donepezil as well as the stability and suitability of the materials for transdermal delivery. Optical microscopy and SEM studies validated the uniform weight distribution and precise dimensions of the MN arrays, while mechanical testing ensured the MNs' robustness, ensuring efficient skin penetration. In vitro studies were conducted to evaluate the produced transdermal patches, indicating their potential use in clinical treatment. Permeation studies revealed a significant increase in DN permeation compared to plain coating material, affirming the effectiveness of the MNs in enhancing transdermal drug delivery. Confocal laser scanning microscopy (CLSM) elucidated the distribution of the API, within skin layers, demonstrating sustained drug release and transcellular transport pathways. Finally, cell studies were also conducted on NIH3T3 fibroblasts to evaluate the biocompatibility and safety of the printed objects for transdermal applications.

Chromenone: An emerging scaffold in anti-Alzheimer drug discovery.

Alzheimer's disease (AD) presents a growing global health concern. In recent decades, natural and synthetic chromenone have emerged as promising drug candidates due to their multi-target potential. Natural chromenone, quercetin, scopoletin, esculetin, coumestrol, umbelliferone, bergapten, and methoxsalen (xanthotoxin), and synthetic chromenone hybrids comprising structures like acridine, 4-aminophenyl, 3-arylcoumarins, quinoline, 1,3,4-oxadiazole, 1,2,3-triazole, and tacrine, have been explored for their potential to combat AD. Key reactions used for synthesis of chromenone hybrids include Perkin and Pechmann condensation. The activity of chromenone hybrids has been reported against several drug targets, including AChE, BuChE, BACE-1, and MAO-A/B. This review comprehensively explores natural, semisynthetic, and synthetic chromenone, elucidating their synthetic routes, possible mode of action/drug targets and structure-activity relationships (SAR). The acquired knowledge provides valuable insights for the development of new chromenone hybrids against AD.

Synthesis, in vitro Α-Glucosidase, and acetylcholinesterase inhibitory activities of novel Indol-Fused Pyrano[2,3-D]Pyrimidine compounds.

In this study, new indol-fused pyrano[2,3-d]pyrimidines were designed and synthesized. These products were obtained in moderate to good yields and their structures were assigned by NMR, MS, and IR analysis. Afterwards, the biological important of the products was highlighted by evaluating in vitro for α-glucosidase inhibitory activity as well as acetylcholinesterase (AChE) inhibitory activity. Eleven products revealed substantial inhibitory activity against α-glucosidase enzyme, among which, two most potent products 11d,e were approximately 93-fold more potent than acarbose as a standard antidiabetic drug. Besides that, product 11k exhibited good AChE inhibition. The substituents on the 5-phenyl ring, attached to the pyran ring, played a critical role in inhibitory activities. The biological potencies have provided an opportunity to further investigations of indol-fused pyrano[2,3-d]pyrimidines as potential anti-diabetic agents.

AChE inhibitory activity of N-substituted natural galanthamine derivatives.

Galanthamine derivatives are known for their AChE inhibitory activity. Among them, galanthamine has been approved for treatment of Alzheimer's disease. N-Acetylnorgalanthamine (narcisine) and N-(2'-methyl)allylnorgalanthamine (the most potent natural AChE inhibitor of galanthamine type) were synthetized using N-norgalanthamine as a precursor. The NMR data described previously for narcisine were revised by two-dimensional 1H-1H and 1H-13C chemical shift correlation experiments. AChE inhibitory assays showed that N-acetylnorgalanthamine and N-formylnorgalanthamine (with previously unknown activity) are 4- and 43-times, respectively, less potent than galanthamine. In vitro (AChE inhibitory) and in silico (docking, ADME) assays and comparison of N-(2'-methyl)allylnorgalanthamine with galanthamine prove that this molecule is a very promising natural AChE inhibitor (33-times more potent than galanthamine) which further in vivo studies would provide better estimation about its applicability as a drug.

Synthesis and biological evaluation of novel aminoquinolines with an n-octyl linker: Impact of halogen substituents on C(7) or a terminal amino group on anticholinesterase and BACE1 activity.

Alzheimer's disease is age-related multifactorial neurodegenerative disease manifested by gradual loss of memory, cognitive decline and changes in personality. Due to rapid and continuous growth of its prevalence, the treatment of Alzheimer's disease calls for development of new and efficacies drugs, especially those that could be able to simultaneously act on more than one of possible targets of action. Aminoquinolines have proven to be a highly promising structural scaffold in the design of such a drug as cholinesterases and ß-secretase 1 inhibitors. In this study, we synthesised twenty-two new 4-aminoquinolines with different halogen atom and its position in the terminal N-benzyl group or with a trifluoromethyl or a chlorine as C(7)-substituents on the quinoline moiety. All compounds were evaluated as multi-target-directedligands by determining their inhibition potency towards human acetylcholinesterase, butyrylcholinesterase and ß-secretase 1. All of the tested derivatives were very potent inhibitors of human acetylcholinesterase and butyrylcholinesterase with inhibition constants (Ki) in the nM to low µM range. Most were estimated to be able to cross the blood-brain barrier by passive transport and were nontoxic toward cells that represented the main models of individual organs.

Synthesis and evaluation of lipoic acid - donepezil hybrids for Alzheimer's disease using a straightforward strategy.

Alzheimer's disease is associated with a progressive loss of neurons and synaptic connections in the cholinergic system. Oxidative stress contributes to neuronal damages and to the development of amyloid plaques and neurofibrillary tangles. Therefore, antioxidants have been widely studied to mitigate the progression of Alzheimer's disease, and among these, lipoic acid has demonstrated a neuroprotective effect. Here, we present the synthesis, the molecular modelling, and the evaluation of lipoic acid-donepezil hybrids based on O-desmethyldonepezil. As compounds 5 and 6 display a high inhibition of acetylcholinesterase (IC50 = 7.6 nM and 9.1 nM, respectively), selective against butyrylcholinesterase, and a notable neuroprotective effect, slightly better than that of lipoic acid, the present study suggests that O-desmethyldonepezil could serve as a platform for the straightforward design of donepezil hybrids.

Metal-free synthesis of functionalized tacrine derivatives and their evaluation for acetyl/butyrylcholinesterase and α-glucosidase inhibition.

A mild and greener protocol was developed for C-C (C(sp3)-H functionalization) and C-N bond formation to synthesize functionalized tacrine derivatives using a biodegradable and reusable deep eutectic solvent [(DES) formed from N,N'-dimethyl urea and L-(+)-tartaric acid in a 3 : 1 ratio at 80 °C]. The condensation of 9-chloro-1,2,3,4-tetrahydroacridines with a variety of aromatic aldehydes gave unsaturated compounds via C(sp3)-H functionalization (at the C-4 position) with good yields. The substituted N-aryl tacrine derivatives were obtained from the condensed products of 9-chloro-1,2,3,4-tetrahydroacridine with substituted anilines via the nucleophilic substitution reaction (SN2 type) in the DES with good yields. This is the first example of C4-functionalized tacrine derivatives, highlighting the dual capacity of the DES to serve as both a catalyst and a solvent for facilitating C-N bond formation on acridine. The generated compounds were evaluated for acetyl/butyrylcholinesterase (AChE/BChE) and α-glucosidase inhibitory activity. It was found that the majority of the compounds reported here were significantly more potent inhibitors than the standard inhibitor tacrine (AChE IC50 = 203.51 nM; BChE IC50 = 204.01 nM). Among the compounds screened, 8m was found to be more potent with IC50 = 125.06 nM and 119.68 nM towards AChE and BChE inhibition respectively. The α-glucosidase inhibitory activity of the compounds was tested using acarbose as a standard drug (IC50 = 23 100 nM) and compound 8j was found to be active with IC50 = 19 400 nM.

A molecular container providing supramolecular protection against acetylcholine hydrolysis.

Alzheimer's disease (AD) is characterized by cognitive decline, often attributed to the deficiency of acetylcholine, which can undergo hydrolysis by acetylcholinesterase (AChE) within the biological milieu. Here, we report a supramolecular strategy that takes advantage of confinement effects to inhibit such a hydrolysis process, shedding some light on AD therapy. A water-soluble and bowl-shaped molecule, hexacarboxylated tribenzotriquinacene (TBTQ-C6), was employed to shield acetylcholine (G1) from enzymatic degradation through host-guest binding interactions. Our study revealed highly efficient host-guest interactions with a binding ratio of 1 : 3, resulting in a significant reduction in acetylcholine hydrolysis from 91.1% to 7.4% in the presence of AChE under otherwise identical conditions. Furthermore, TBTQ-C6 showed potential for attenuating the degradation of butyrylcholine (G2) by butyrylcholinesterase (BChE). The broader implications of this study extend to the potential use of molecular containers in various biochemical and pharmacological applications, opening new avenues for research in the field of neurodegenerative diseases.

Coumarin-azasugar-benzyl conjugates as non-neurotoxic dual inhibitors of butyrylcholinesterase and cancer cell growth.

We have applied the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction to prepare a library of ten coumarin-azasugar-benzyl conjugates and two phthalimide-azasugar-benzyl conjugates with potential anti-Alzheimer and anti-cancer properties. The compounds were evaluated as cholinesterase inhibitors, demonstrating a general preference, of up to 676-fold, for the inhibition of butyrylcholinesterase (BuChE) over acetylcholinesterase (AChE). Nine of the compounds behaved as stronger BuChE inhibitors than galantamine, one of the few drugs in clinical use against Alzheimer's disease. The most potent BuChE inhibitor (IC50 = 74 nM) was found to exhibit dual activities, as it also showed high activity (GI50 = 5.6 ± 1.1 µM) for inhibiting the growth of WiDr (colon cancer cells). In vitro studies on this dual-activity compound on Cerebellar Granule Neurons (CGNs) demonstrated that it displays no neurotoxicity.

Sequential Contrastive and Deep Learning Models to Identify Selective Butyrylcholinesterase Inhibitors.

Butyrylcholinesterase (BChE) is a target of interest in late-stage Alzheimer's Disease (AD) where selective BChE inhibitors (BIs) may offer symptomatic treatment without the harsh side effects of acetylcholinesterase (AChE) inhibitors. In this study, we explore multiple machine learning strategies to identify BIs in silico, optimizing for precision over all other metrics. We compare state-of-the-art supervised contrastive learning (CL) with deep learning (DL) and Random Forest (RF) machine learning, across single and sequential modeling configurations, to identify the best models for BChE selectivity. We used these models to virtually screen a vendor library of 5 million compounds for BIs and tested 20 of these compounds in vitro. Seven of the 20 compounds displayed selectivity for BChE over AChE, reflecting a hit rate of 35% for our model predictions, suggesting a highly efficient strategy for modeling selective inhibition.

Multi-target potential of newly designed tacrine-derived cholinesterase inhibitors: Synthesis, computational and pharmacological study.

Simple and scalable synthetic approach was used for the preparation of thirteen novel tacrine derivatives consisting of tacrine and N-aryl-piperidine-4-carboxamide moiety connected by a five-methylene group linker. An anti-Alzheimer disease (AD) potential of newly designed tacrine derivatives was evaluated against two important AD targets, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE). In vitro pharmacological evaluation showed strong ChE inhibitory activity of all compounds, with IC50 values ranging from 117.5 to 455 nM for AChE and 34 to 324 nM for BuChE. As a representative of the series with the best cytotoxicity / ChE inhibitory activity ratio, expressed as the selectivity index (SI), 2-chlorobenzoyl derivative demonstrated mixed-type inhibition on AChE and BuChE, suggesting binding to both CAS and PAS of the enzymes. It also exhibited antioxidant capacity and neuroprotective potential against amyloid-ß (Aß) toxicity in the culture of neuron-like cells. In-depth computational analysis corroborated well with in vitro ChE inhibition, illuminating that all compounds exhibit significant potential in targeting both enzymes. Molecular dynamics (MD) simulations revealed that 2-chlorobenzoyl derivative, created complexes with AChE and BuChE that demonstrated sufficient stability throughout the observed MD simulation. Computationally predicted ADME properties indicated that these compounds should have good blood-brain barrier (BBB) permeability, an important factor for CNS-targeting drugs. Overall, all tested compounds showed promising pharmacological behavior, highlighting the multi-target potential of 2-chlorobenzoyl derivative which should be further investigated as a new lead in the drug development process.