Unveiling Pyridocarbazole Fused with Carbamate as MAO-B Inhibitors using In silico and In vitro approaches

Ekta Khare^1,2, Zeeshan Fatima¹*, O.P. Tiwari³, Jeevan Patra¹, Nidhi Mishra⁴

¹Amity Institute of Pharmacy, Lucknow, Amity University Uttar Pradesh, Sector 125, Noida, 201313, India.

²GCRG College of Pharmacy, Lucknow, Uttar Pradesh, India.

³Vindhya Gurukul College of Pharmacy, A.K.T.U. Uttar Pradesh Chunar-Mirzapur, India.

⁴Department of Applied Sciences, Indian Institute of Information Technology Allahabad,

Allahabad, Deo ghat, Jhalwa, Allahabad, Uttar Pradesh - 211012, India.

*Corresponding Author E-mail: zfatima@amity.edu

ABSTRACT:

Alzheimer’s disease (AD) a neurodegenerative disorder is one of the most prevalent diseases characterized by multifactorial etiology, including amyloid plaques, tau tangles, as well as neuronal loss. Current therapeutic options are limited and often only provide symptomatic relief. In pursuit of novel therapeutic agents, pyrido-carbazole fused with carbamate (PCC) hybrids were rationally designed and synthesized based on literature and clinically approved candidates. The study involved a comprehensive approach to evaluating these hybrids. Pharmacokinetic and acute toxicity profiles were assessed, followed by molecular modeling to predict a plausible monoamine oxidase B(MAO-B) interactions and in vitro MAO-B inhibition assay. The synthesized hybrids exhibited a range of inhibitory activities against MAO-B, falling at low micromolar concentrations. Notably, compound 4e demonstrated potent and well-balanced activity against MAO-B with an IC₅₀ value of 4.51μM, approaching the efficacy of the standard drug selegiline of IC₅₀ 2.93μM. The percentage inhibition of 4e was higher than the selegiline at a 100 μM concentration. The molecular modeling studies indicated that 4e interacted with the MAO-B catalytic site. These findings highlight the potential of compound 4e as a promising candidate for AD therapy, given its potent MAO-B inhibition, an excellent pharmacokinetic profile, and low toxicity.

KEYWORDS: Alzheimer’s disease, MAO-B, Pyridocarbazole-carbamates, Structure-Activity Relationship, Molecular modeling.

1. INTRODUCTION:

Alzheimer's disease (AD) being a persistent and escalating neurodegenerative condition portraying memory loss, cognitive dysfunction, and behavioral abnormalities¹. Globally, AD has impacted over 50 million individuals, and this number is expected to escalate to 13.8 million by 2060.

Received on 11.09.2024 Revised on 14.01.2025

Accepted on 03.03.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4100-4106.

DOI: 10.52711/0974-360X.2025.00589

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

There are many challenges and severe socioeconomic impacts associated with early diagnosis and the lack of disease-modifying options. The pathogenesis of AD is complex and multifactorial, involving several causative factors which are still uncomprehended. The major crucial factor contributing towards the disease is the deficiency of acetylcholine, a neurotransmitter which is responsible for memory and learning. Another critical factor is oxidative stress, which is exacerbated by increased activity of the enzyme monoamine oxidase (MAO). MAO regulates mood, emotions, and behavior by breaking down monoamines, such as dopamine, serotonin, and noradrenaline. This degradation process generates reactive oxygen species (ROS) and hydrogen peroxide (H₂O₂), leading to oxidative stress and neural cell death through the Fenton reaction mechanism. To ameliorate the MAO-B pathway, many potent MAO-B inhibitors have recently been developed, which reduce the breakdown of neurotransmitters in nerve endings, thereby slowing the progression of neural cell death.^2-3

For several years, researchers have been inspired by the cholinergic pathway hypothesis, focusing on enzymes such as acetylcholinesterase and butyrylcholinesterase to counterbalance cognitive symptoms. A multi-targeted approach has also been envisaged, considering the multifactorial nature of AD, which can control multiple cellular processes. Several potent clinical drugs have already been identified to alleviate cognitive dysfunction at early onset. However, due to their irreversible inhibition, long-term treatment is associated with undesirable, life-threatening adverse effects. Despite all this hype, a paradigm shift is needed to fulfill unmet needs in developing effective disease-modifying drugs. One of the most common approaches is to conjugate multiple potential pharmacophores to form a hybrid, which can amplify bioactivity and offer fewer side effects.^4-6

Carbazole scaffolds have a strong pharmacophoric framework, which attracts researchers for the synthesis of novel drugs and prodrugs. Amino-carbazole is a promising pharmacophore and has gained attention due to its prominent pharmacological actions, including those against Alzheimer’s disease. The rationale for including a carbamate moiety in your Alzheimer's disease molecules is well-supported by existing research and clinical practices. The best notable clinical candidate is the rivastigmine which is known for their efficacy and lower toxicity. Carbamates inhibit cholinesterases by forming a reversible covalent bond with the active serine residue; leading to carbamoylation.⁷ The carbamate derivatives can effectively cross the blood-brain barrier (BBB) and exhibit potent inhibition making them particularly suitable for Alzheimer's therapy.

Considering the above findings and our ongoing efforts to expand the novel chemical diversity inspired by anti-AD clinical drugs, we have previously designed and biologically evaluated pyridocarbazole fused with carbamates (PCC) hybrids against acetylcholinesterase (Figure 1). Our design approach relied on merging the carbazole motif with the carbamate to generate a new chemotype. The designed hybrid compound doesn’t violate any CNS drug filters and has an optimum pharmacokinetic profile as an anti-AD clinical drug. To the best of our knowledge, this designed core has not been previously explored against any anti-AD drug targets. Therefore, exploration of the MAO-B inhibitory activity of this newly designed chemotype will aim to bridge the gap in the structure-activity relationships.^8-9

All the designed compounds were screened against MAO-B using the in vitro fluorometric method to gain full insights into structure-activity relationships. Furthermore, molecular modeling studies of the best-hit molecule and standard control (selegiline) were performed to decipher its structural binding mechanisms.

Figure 1: Rationale for the design and synthesis of pyridocarbazole fused with carbamates (PCC) derivatives

2. EXPERIMENTS:

2.1. Protein and Ligand preparation:

The dimeric crystallographic structure of the MAO-B protein (PDB ID: 2BYB) bound with the native ligand L-deprenyl (selegiline) was obtained from the RCSB PDB bank. The native ligand is well-defined, highlighting crucial insights into the MAO-B inhibition mechanism that are not clearly represented in other MAO-B crystallographic studies. This protein has a distinct active site architecture characterized by a hydrophobic bipartite cavity with an approximate volume of 700 Å³, which can accommodate bulkier ligands. The unique hydrophobic cage-like conformation at the entrance groove of 290 Å³ allows the binding of inhibitors. This site is essential for both its biological role and oxidative deamination catalysis.

The Protein Preparation Wizard, a part of the Schrödinger module, was used for protein preparation.The protein structure was pre-processed to add missing side chains and residues and protonated at 7.0±2.0 pH. Further, hydrogen atoms were added and optimized, and water molecules beyond 3Å were removed. Finally, the optimized protein was restrained for minimization using the OPLS4 force field. The ligand preparation of the designed PCC derivatives was performed using the LigPrep module of Schrödinger. All compounds were ionized at 7.0±2.0 pH using Epik, subjected to produce tautomeric and ionization states, and a maximum of 32 conformers per ligand was generated.

2.2. Receptor Grid Generation and Molecular Docking:

The receptor grid was generated at 10Å around the deprenyl native ligand before the Glide docking process. The docking study was performed using the default parameters of Glide, embedded in the Schrödinger suite, in Xtra-Precision mode.The prepared PCC derivatives were used for virtual screening against the receptor grid of MAO-B under the default parameters of GlideXP. For each ligand, three poses were generated, and the best-scoring states were considered for post-docking analysis.

2.3. Estimation of Binding Free Energy:

Molecular Mechanics Generalized Born Surface Area (MM-GBSA) with the Prime module was used to calculate the binding free energy, employing default parameters within the Schrödinger suite. The local optimization characteristics of Prime were used to obtain the minimized docking poses, and the energies associated with the complexes were calculated using the Generalized Born/Surface Area (GB/SA) continuous solvent model with an OPLS-4 force field. The per-residue decomposition energies of all components were extracted using a Python script provided by Schrödinger.

Molecular Dynamics Simulations:

Molecular dynamics simulations of protein-ligand complexes were performed using Desmond version 2023. Initially, the protein-ligand complex was built using the System Builder with a simple point charge (SPC) solvation model, surrounded by an orthogonal box of 10Å in each direction and neutralized with physiological salts at a 0.15M concentration. Using the steepest descent and the limited memory Broyden-Fletcher-Goldfarb-Shanno (LBFGS) algorithm, the system energy was minimized for a maximum of five thousand steps to reach gradient threshold of 25 kcal/mol/Å. Relaxation of NPT system was done at a temperature of 300K along with pressure of 1.01325 bar, and the simulations were performed for 100 ns under the OPLS4 force field. Trajectory snapshots were recorded every 4.8ps. The principal component analysis (PCA) was performed using MODE-Task after extracting all trajectories frames from the MD simulation .^10-12

2.4. In Vitro inhibition of MAO-B:

The biological evaluation for MAO-B activity was performed based on the reported standardized protocol. The assay involved the preparation of three different solutions. Inhibitor solution having the reference compound and synthesized molecule in 2% DMSO ( 10 ml for each concentration) was prepared. 10ml of enzyme solution comprising of recombinant hMAO-B(0.64U/mL) was prepared in phosphate buffer . Horseradish peroxidase (200U/mL, 100μL), Ampliflu™ Red (20mM, 200μL), and tyramine (100mM, 200μL) dissolved in phosphate buffer and adjusted up to 10ml was used as working solution. The flat black bottom 96-well micro test plate was considered, and the solutions of the inhibitor (20μL/well) and hMAO-B (100μL/well) were added. This plate was incubated at 37°C for thirty min. Further the working solution (100μL/well) was added to the previously incubated plate having inhibitor and enzyme.This mixture was incubated at thirty seven degree for about thirty minutes and the fluorescence (Ex/Em = 535/587nm) was measured at an interval of five min. Two percent DMSO (20μL). Was used as a control. To inspect the effect of inhibitors on horseradish peroxidase, a parallel reading was performed by replacing enzyme solutions with a 3% H₂O₂ solution (20 mM, 100μL/well).The potential of the inhibitors to alter the fluorescence generated because of non-enzymatic inhibition was evaluated by mixing inhibitor and working solutions. The specific fluorescence emission generated to obtain the final results was^13-14calculated after subtraction of the background activity. This was determined from vials containing all components except the hMAO-B, which were replaced by phosphate buffer (100μL/well). All the readings were calculated in triplicate and inhibition percent was calculated using the following equation:

C-T

% Inhibition = ------------- x 100

3. RESULTS AND DISCUSSIONS:

3.1. Screening of PCC hybrids with MAO-B protein:

Initially, the native bound ligand was re-docked to the MAO-B active site and demonstrated an accurate docking pose with the lowest RMSD of 0.186 Å. The re-docked ligand interacts with the entrance pocket tyrosine cage residues, such as Pro104, Leu164, Phe168, Leu171, Cys172, Ile198, Ile199, Ile316, and Tyr398. Amongst the series (4a-4h), compound 4e was revealed to have higher binding free energy (GScore: -12.884kcal/mol). The MMGBSA and non-bonded energies (NBE) confirmed the higher free energies of compound 4e. The non-bonded energies estimate the ability of the ligand to fit perfectly inside the binding pocket. Amongst the series, compound 4e exhibited the highest non-bonded energies. All the PCC hybrids and the standard control selegiline exhibited a similar binding fashion to the co-crystal (Table 1)

Table 1: Binding affinities of best-docked analogs (kcal/mol) and MAO-B activity


C. No.	R	RMSD (Å)	Binding Affinities (kcal/mol)			IC₅₀(µM)
C. No.	R	RMSD (Å)	GScore	MM-GBSA	NBE	IC₅₀(µM)
4a	4-NO₂	0.332	-8.356	-57.68	-50.64	36.83
4b	4-Cl	0.478	-10.159	-63.83	-58.44	29.96
4c	4-F	0.350	-9.889	-62.72	-60.81	39.71
4d	4-CH₃	0.108	-9.675	-71.69	-64.59	7.74
4e	4-OCH₃	0.225	-12.884	-73.85	-92.99	4.51
4f	1,4-Cl	0.197	-9.033	-69.62	-57.01	25.24
4g	3-NO₂	0.435	-8.020	-47.69	-55.40	44.45
4h	3-Cl	0.153	-6.672	-37.72	-27.52	41.91
Selegiline	-	0.224	-11.826	-78.15	-94.70	2.93

3.2. Structure-activity relationships and MAO-B inhibitory activity:

The MAO-B inhibition of designed PCC derivatives was investigated through the fluorometric method, which demonstrated IC₅₀ values ranging from 4.51µM to 44.45µM. Amongst all the derivatives, a few compounds demonstrated optimal to low-micromolar IC₅₀ values, indicating the impact of EWG and EDG substitutions in the phenyl nucleus. Among the series, EDG groups (methoxy and methyl) exhibited better potential activity than the EWG groups (nitro and halogens). In compound 4e, a methoxy-substituted derivative at the para position exhibited the most promising MAO-B inhibitory potential of IC₅₀ 4.5µM, which was similar inhibition to selegiline of IC₅₀ 2.93µM. The compound 4d, a para-methyl derivative, exhibited the second-best MAO-B inhibitory activity of IC₅₀7.74µM, which was two-fold lower inhibition than 4e. Substitution of EWG groups at various positions on the phenyl ring showed modest activity. In most cases, ortho and meta substitutions dramatically reduced activity compared to the para position. In compound 4b, a mono-substituted chloro group at the para position had reasonably low-micromolar inhibition of IC₅₀ 29.96µM compared to the meta position in compound 4h having IC₅₀ 41.91µM. Further, di-substituted chloro at ortho and para positions in compound 4f of IC₅₀ 25.24µM exhibited a similar inhibition pattern to compound 4b. Replacing chloro substituent with fluoro in 4c at the para position weakened the inhibitory concentration showing IC₅₀ 29.96µM compared to the compounds 4b and 4f. Replacement of chloro with nitro at meta position of compound 4g didn’t show any improvement.

3.3. Compound 4e interacts with the orthosteric residues:

The best-identified compound, 4e, determined the efficacy of MAO-B inhibition in correlation with the residual interactions (Figure 2). The binding affinity parameters yielded significantly impressive results (Figure 3). The tyrosine cage framework formed around the pyrido-carbazole scaffold has an aromatic-hydrogen bond, π-π stacking, and hydrophobic contacts. The presence of only one conventional hydrogen bond shows the ketone group in the main pharmacophore interacting with the Met436 residue. The para-substituted methoxy on the phenyl ring projected towards the entrance cavity and showed strong hydrophobic contacts with Phe168, Cys172, Leu171, Ile198, and Ile199. The per-residue energy decomposition for Phe168 (-37.73kcal/mol), Cys172 (-34.22kcal/mol), Leu171 (-36.69kcal/mol), Ile198 (-33.22 kcal/mol), and Ile199 (-29.59kcal/mol) indicated significant interactions. The presence of the EDG methoxy at the distal site of the phenyl ring might be crucial for the formation of hydrophobic interactions and π-π interactions with Cys172 .The p-methoxy substituent on the phenyl ring was projected towards the gating residues Ile199 and Tyr326, forming hydrophobic interactions. These residues at the catalytic site of the MAO-B enzyme govern plasticity and act as a suitable region for inhibitor recognition.^15-17

Figure 2: Molecular interaction of 4e and selegiline with MAO-B protein. The hydrophobic, aromatic-π bonding and hydrogen bonding are represented in grey, yellow, and magenta.

Figure 3: MM-GBSA parameters of compound 4e and selegiline

3.4. Molecular Dynamics:

The best-docked conformer and control selegiline compound were taken for the classical MDS. The molecular dynamics were performed in triplicate for up to 100 ns. The triplicate MDS deviations were small and non-significant. The RMSD trajectories of complexes with selegiline plateaued until equilibration was achieved within the first 30 ns, and they ceased undergoing significant conformational changes (Figure 4). The 4e and selegiline ligands bound with MAO-B stabilized, achieving an average RMSD of 1.86±0.21 Å and 1.50±0.33 Å, respectively.^18-19 The active residues involved in the active binding pocket showed lesser fluctuations. Due to the conformational heterogeneity observed in the RMSD of the MAO-B-4e complex, residues exhibited relatively higher RMSF. As these fluctuations were not occurring in the active binding site, it can be concluded that the protein-ligand complex system remained stable during the simulation period. The average RMSF of 4e and selegiline were 0.67±0.18 Å and 0.59±0.14 Å, respectively. The stability of compound 4e and selegiline bound with MAO-B can be confirmed using multidimensional reduction principal component analysis (PCA) plots (Figure 5). The PCA is plotted using the extracted MDS trajectories frames up to 1,000 frames. The PCA was found to be stabilized during the simulation period, especially with the MAO-B-4e complex. The PCA plot of the complex MAO-B bound with selegiline shows distortions in the initial 200 trajectory frames, which can be corroborated with the RMSD findings. The stability of the complex can also be confirmed based on the molecular binding strengths. Both compounds 4e and selegiline have a maximum number^20-22 of hydrophobic interactions (Figure 6) and few hydrogen bonds^23-24 (Figure 7).

Figure 6: Molecular interaction strength of 4e and selegiline during the simulation course

Figure 7: Interacting residues of compound 4e and selegiline during the simulation. Hydrogen bonding and hydrophobic interactions are represented in green and violet.

4. CONCLUSION:

There is a pressing need for more effective and safer drugs to slow down the advancement of Alzheimer’s disease (AD), as current treatments are primarily symptomatic. This study focuses on the in-silico and in-vitro evaluation of previously synthesized PCC derivatives against AD targets. Among the evaluated series, 4e demonstrated significant micromolar inhibitory activity against MAO-B, outperforming other compounds in the series. The structure-activity relationship analysis showed that para-substituted electron-donating groups enhanced activity. Docking studies revealed that these compounds fit well into the enzyme's active pocket, indicating favorable binding modes and interactions. Molecular dynamics allowed us to confirm the binding stability of the systems based on the trajectories and molecular strengths. Despite the promising potential of 4e against the selected target, further structural modifications are necessary to improve efficacy and safety. Iterative lead optimization of designed pharmacophores and hybrids is required for the improvement of MAO-B potency.

5. ACKNOWLEDGEMENTS:

The authors are grateful to the Amity University Uttar Pradesh (Lucknow Campus) for providing amenities in facilitating our research work.

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Received on 02.02.2024 Revised on 20.08.2024

Accepted on 18.01.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4095-4099.

DOI: 10.52711/0974-360X.2025.00588

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.