Discovery of novel inhibitor of 11 beta-hydroxysteroid dehydrogenase type 1 using in silico structure-based screening approach for the treatment of type 2 diabetes

Abstract

Purpose

The current study is aimed to perform structure-based screening of FDA-approved drugs that can act as novel inhibitor of the 11beta- hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme.

Methods

Structural analogs of carbenoxolone (CBX) were selected from DrugBank database and their Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) parameters were investigated by SwissADME. Molecular docking of CBX analogs against 11β-HSD1 was performed by AutoDock tool, their binding patterns were visualized using PyMOL and the interacting amino acids were determined by ProteinPlus tool. Molecular dynamics simulation was performed on the docked structure of 11β-HSD1 (Protein Data Bank (PDB) code: 2ILT) using GROMACS 2018.1.

Results

The binding energies of hydrocortisone succinate, medroxyprogesterone acetate, testolactone, hydrocortisone cypionate, deoxycorticosterone acetate, and hydrocortisone probutate were lower than that of substrate corticosterone. The molecular dynamics simulation of 11β-HSD1 and hydrocortisone cypionate docked structure showed that it formed a stable complex with the inhibitor. The Root mean square deviation (RMSD) of the protein (0.37 ± 0.05 nm) and ligand (0.41 ± 0.06 nm) shows the stability of the ligand-protein interaction.

Conclusion

The docking study revealed that hydrocortisone cypionate has a higher binding affinity than carbenoxolone and its other analogs. The molecular dynamics simulation indicated the stability of the docked complex of 11β-HSD1 and hydrocortisone cypionate. These findings indicate the potential use of this FDA approved drug in the treatment of type 2 diabetes. However, validation by in vitro inhibitory studies and clinical trials on type 2 diabetes patients is essential to confirm the current findings.

Introduction

The role of glucocorticoid (GC) cortisol in the pathogenesis of diabetes and prediabetic conditions is to antagonize the actions of insulin, leading to insulin resistance (IR) and uncompensated hyperinsulinemia [1, 2]. In people with hypercortisolism and central obesity, IR often persists for years after successful treatment [3]. One of the culprits for hypercortisolism is 11beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1), a key enzyme that catalyzes the intracellular conversion of cortisone to physiologically active cortisol [4, 5] (Fig. 1).

Fig. 1.

Activity of 11β-HSD1 in humans

Structurally, 11β-HSD1 is a short-chain dehydrogenase made of 292 amino acids [6] with seven parallel β sheets and 12 helices. The structure of 11β-HSD1 is represented in Fig. 2.

Fig. 2.

Structure of 11β-HSD1 in Homo sapiens, PDB ID: 2ILT; Green-A chain, Cyan-B chain

(Source: RCSB PDB database)

11β-HSD1 is important for the maintenance of the GC hormonal balance. 11β-HSD1 knock-out mice fed with a high-fat diet are protected against obesity and associated complications [7–9], and develop resistance to diet-induced hyperglycemia due to increased insulin sensitivity and attenuation of hepatic gluconeogenic enzymes. Pharmacological inhibition of 11β-HSD1 in diabetic mice lowers blood glucose, enhances hepatic insulin sensitivity, and causes weight loss [10, 11].

11β-HSD1 inhibition has been recommended for the treatment of type 2 diabetes (T2D) and central obesity [12, 13]. Currently, many selective 11β-HSD1 inhibitors are being tested in clinical trials on T2D mouse models [10]. 11β-HSD1 inhibition by carbenoxolone (CBX), has been shown to induce higher secretion of insulin by beta cells of the pancreas, decrease intrahepatic hydrocortisone concentration, and improve insulin sensitivity in the liver [14]. The details of 11β-HSD1 inhibitors which have completed phase I and phase II clinical trials are mentioned in Table 1.

Table 1.

11β-HSD1 inhibitors in phase I and II clinical trials and their limitations

11β-HSD1 inhibitor	Study participants	Inhibition	Limitation	References
BI187004	Healthy obese males	Hepatic and adipose tissue 11β-HSD1	Lower plasma half-life, inadequate study duration	[15]
BI135585	Healthy and diabetes patients	Hepatic and adipose tissue 11β-HSD1	Unknown molecular mechanism of inhibition, lower bioavailability, rate of inhibition is different in diabetes and healthy participants, inadequate study duration	[16]
RO 5,093,151/RO-150 and RO 5,027,383/RO-838	Diabetes patients	Hepatic 11β-HSD1	Inadequate study duration, increased androgen production in females, no clear correlation between blood glucose and 11β-HSD1 inhibition	[17]
ABT-384	Healthy participants	Hepatic 11β-HSD1	Inadequate study duration, side effects such as nausea, headache, diarrhea, HTN, unknown absolute bioavailability	[18]
MK-0916	Healthy participants	Hepatic 11β-HSD1	Inadequate study duration, clinical effects not explained clearly	[19]
MK-0736	Overweight to obese patients with HTN	Decreased blood pressure, LDL-cholesterol, and body weight	Significant decrease in HDL	[20]
INCB13739	Diabetes patients	Hepatic 11β-HSD1	Increased androstenedione in males, inadequate study duration, patients were on metformin monotherapy	[21]

INCB13739 and RO-151 have been trialed in T2D patients [17, 21], MK-0916 in patients with T2D and metabolic syndrome (metS), MK-0736 has been trialed to treat comorbid obesity and hypertension (HTN) [19, 20]. But the trial has been discontinued. None of these inhibitors showed significant improvement in clinical profile except for INCB13739 which successfully reduced glycated hemoglobin (HbA1c), fasting plasma glucose, and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) levels in people with T2D, but was not further developed or continued for unknown reasons [21]. No human studies of 11β-HSD1 inhibitors have been reported from phase III clinical testing. All the current reports on 11β-HSD1 inhibitors are based on the results of phase 0 to phase II clinical testing [10]. As a result, none of the 11β-HSD1 inhibitors have been licensed for treatment to date [10]. CBX which is an antiulcer drug is prohibited in most countries [10] due to side effects of renal mineralocorticoid excess [22], despite its beneficial effects on glucose reduction and reduced glycogenolysis compared to placebo [23].

CBX is a small molecule drug that resembles the structure of glycyrrhizin found in the root of the licorice plant. It is a triterpenoid with six isoprene units [24]. The chemical interaction of CBX with substrate corticosterone involves hydrogen bonding of the electronegative oxygen atom in CBX with the hydrogen of electronegative nitrogen of I11B (Fig. 3a). Interaction also exists with the T210B, G35B, and I36B of the binding pockets of 11β-HSD1. The chemical interactions between 11β-HSD1 and its substrate corticosterone are hydrogen bonding of electronegative oxygen atom in corticosterone with the hydrogen of electronegative nitrogen of I208A and G206A (Fig. 3b). I111A is the interacting amino acid in the substrate-binding pocket of 11β-HSD1 that binds to corticosterone (Fig. 3b).

Fig. 3a.

Interaction of CBX with 11β-HSD1, Binding energy= -10.6 kcal/mole.

(Source: ProteinPlus)

Fig. 3b.

Interaction of corticosterone with 11β-HSD1, Binding energy= -9.1 kcal/mole.

(Source: ProteinPlus)

It is crucial to design 11β-HSD1 inhibitors that can interact with these amino acids with better binding affinities. Hence, in our study we performed structure-based screening to screen for 11β-HSD1 inhibitors from the Food and Drug Administration (FDA) approved drugs which are structural analogs of CBX that comply with Lipinski’s rule and Absorption, Distribution, Metabolism, and Excretion (ADME).

Materials and methods

In the present study, we have tried to discover a potent, selective analog of CBX by the approach of structure-based screening. The workflow of the screening approach for this purpose has been represented in Fig. 4.

Fig. 4.

Screening of FDA-approved drugs

Protein selection

The protein used for the current study, 11β-HSD1 was selected after a thorough literature survey and based on the presence of highly conserved sequences without mutations against wild-type sequences [25]. The 11β-HSD1 (PDB code: 2ILT) was selected from National Center for Biotechnology Information (NCBI) database [25]. The three-dimensional (3D) crystal structure of 11β-HSD1 in Protein Data Bank (PDB) format was retrieved from the RCSB PDB database [26].

Ligand selection

Structural analogs of CBX (with similarity > 75%) that are also FDA-approved drugs, were collected from the DrugBank database [27] by performing a similarity search. The 3D-structure of these structural analogs was downloaded from the PubChem database [28] in Spatial Data File (SDF) format. The SDF file of the ligand was converted to PDB format using PyMOL [29].

Drug likeliness and ADMET evaluation

We analyzed all the CBX analogs for bioactivity and molecular properties such as lipophilicity parameter- Logarithmic Octanol Water Partition Coefficient (LogP), topological polar surface area (TPSA), the number of atoms (nats), the number of rotatable bonds (nrotb), and the number of violations (n violations). The bioactivity and molecular properties of CBX and its analogs were estimated using Molinspiration [30]. Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) parameters, pharmacokinetic properties, and drug-like nature of CBX and its analogs were predicted using the Lipinski drug filter by SwissADME [31].

Molecular docking

Eleven drugs with suitable drug likeliness were chosen for the docking study. A docking study of FDA-approved CBX analogs was performed by the AutoDock (Version 1.5.6) tool [32] to find binding affinity against the 11β-HSD1 enzyme. AutoDock uses AutoDock4 and AutoDock Vina software to predict the mode of interaction between small molecule drugs and proteins with known 3D structures. The human 11β-HSD1 enzyme (PDB code: 2ILT) was prepared by removing the bound ligands and water molecules, adding polar hydrogens, and assigning Kollman charges. Docking of the ligand (drug) to a set of grids describing the AutoDock 4 was done. AutoDock tools implement the Lamarckian Genetic Algorithm wherein Autogrid precalculates a set of grids describing the target protein to which Autodock performs the docking of the ligand. The center of the docking grid was located in a box 58.957 × 105.209 × 62.493 Å with an exhaustiveness of 8. Then, the lowest binding energy that determines the most stable interactions between each ligand and 2ILT was selected, and ligand-protein interactions were visualized using PyMOL [33]. The 11β-HSD1 was also docked with INCB13739, which is a well-established 11β-HSD1 inhibitor.

PyMOL is used to get the docked file in PDB format for use in the ProteinPlus tool to visualize structures and create two-dimensional (2D) pose depictions. PoseView was used to generate the 2D diagrams of the ligand-protein complex [34].

Redocking using iGEMDOCK

Re-evaluation of the AutoDock docking studies was performed using iGEMDOCK v2.1 software. The docking, virtual screening, and interaction of the compounds were analyzed using iGEMDOCK [35]. The previously prepared protein i.e., human 11β-HSD1 enzyme (PDB code:2ILT) [36] was docked with CBX, and its analogs (Hydrocortisone cypionate, Deoxycorticosterone acetate, Testolactone, Hydrocortisone succinate, Hydrocortisone probutate, Medroxyprogesterone acetate, Clascoterone, Testosterone propionate, Hydrocortisone butyrate, Hydrocortisone valerate, and Gestonorone caproate) separately. Parameters of docking run in iGEMDOCK were set with a population size of n = 200, generations were set to 70 and the number of solutions was set to 2.

Molecular dynamics (MD) simulation

MD simulation was performed using GROMACS 2018.1 on the docked structure of hydrocortisone cypionate with 11β-HSD1 from the previous Sect. [37]. The ligand topology file and parameters were generated using SwissParam [38]. The docked ligand-protein complex was placed in an octahedron periodic box that extended 1.2 nm in all directions. Transferable intermolecular potential with 3 points (TIP3P) was used to model water while the force field used was Charmm36. Na⁺ and Cl^- ions were used to neutralize and salt the system at 0.15 M concentration. Ensemble equilibration was carried out for 1 ns, NVT at 300 K, and NPT at 1 bar. The long-range electrostatic interactions were modeled using the particle mesh Ewald method. Constant pressure simulations were carried out using the Parrinello-Rahman method. The simulations were run for 100 ns with a step size (dt) of 2 fs. The binding energy was calculated using the Molecular mechanics Poisson–Boltzmann surface area (MM-.

PBSA) implemented in the g_mmpbsa tool using snapshots extracted from the trajectory [39]. All the calculations were done for the last 80ns of the simulation to give the system time to equilibrate.

Results

Ligand selection

Structure-based screening of FDA-approved drugs carried out based on the > 75% structural similarity to CBX using the DrugBank database yielded seventeen structurally similar analogs of CBX. The similarity score and implications of these compounds are described in Table 2.

Table 2.

Similarity scores, bioactivity scores, and implications of FDA-approved drugs as 11β-HSD1 inhibitors

Name of FDA-approved drug	Similarity score	Bioactivity score	Implications
Nandrolone decanoate	0.897	0.46	Anemia of renal insufficiency, osteoporosis
Testosterone cypionate	0.897	0.49	Primary hypogonadism, hypogonadotropic hypogonadism
Testosterone enanthate	0.897	0.5	Primary hypogonadism, hypogonadotropic hypogonadism
Testosterone undecanoate	0.897	0.44	Deficiency in endogenous testosterone production
Testosterone propionate	0.879	0.52	Androgen deficiency
Hydroxyprogesterone caproate	0.861	0.48	Prevention of spontaneous preterm births
Gestonorone caproate	0.861	0.49	Not annotated
Drospirenone	0.858	0.22	Oral contraceptive
Medroxyprogesterone acetate	0.833	0.51	Contraceptive, paraphilia in males, endometrial and renal carcinomas, abnormal uterine bleeding, secondary amenorrhea.
Hydrocortisone valerate	0.82	0.59	Dermatoses, inflammation
Clascoterone	0.815	0.55	Acne vulgaris
Hydrocortisone butyrate	0.811	0.6	Dermatoses, inflammation
Hydrocortisone probutate	0.811	0.46	Inflammatory and pruritic corticosteroid-responsive dermatoses
Hydrocortisone succinate	0.811	0.51	Dermatologic and severe allergic diseases, rheumatic, respiratory, renal, ophthalmic, neurological, neoplastic, hematological, gastrointestinal and endocrine disorders.
Hydrocortisone cypionate	0.795	0.46	Inflammatory and pruritic corticosteroid-responsive dermatoses
Testolactone	0.783	0.76	Breast cancer
Deoxycorticosterone acetate	0.77	0.5	Not annotated

Drug likeliness and ADMET evaluation

The molecular properties of the compounds are shown in Table 3 ‘Molecular properties of FDA-approved drugs’. Based on these properties, drug likeliness and ADMET evaluation reports from SwissADME are presented in Table 4.

Table 3.

Molecular properties of FDA approved drugs

Name of FDA approved drug	LogP	TPSA	nats	MW	HBD	HBA	n violations	nrotb	Vol
Carbenoxolone	5.6	117.97	41	570.77	7	2	2	6	553.88
Nandrolone decanoate	7.66	43.38	31	428.66	3	0	1	10	446.23
Testosterone cypionate	5.75	43.38	30	412.61	3	0	1	5	418.29
Testosterone enanthate	6.38	43.38	29	400.6	3	0	1	7	412.06
Testosterone undecanoate	8.34	43.38	33	456.71	3	0	1	11	479.27
Testosterone propionate	4.31	43.38	25	344.5	3	0	0	3	344.85
Hydroxyprogesterone caproate	5.5	60.45	31	428.61	4	0	1	7	430.48
Gestonorone caproate	5.25	60.45	30	414.59	4	0	1	7	414.24
Drospirenone	3.66	43.38	27	366.5	3	0	0	0	345.95
Medroxyprogesterone acetate	4.04	60.45	28	386.53	4	0	0	3	379.86
Hydrocortisone valerate	3.75	100.9	32	446.58	6	2	0	7	429.98
Clascoterone	3.6	80.67	29	402.53	5	1	0	5	388.33
Hydrocortisone butyrate	3.24	100.9	31	432	6	2	0	6	413.18
Hydrocortisone probutate	4.3	106.98	35	488.62	7	1	0	9	466.49
Hydrocortisone succinate	1.59	138.20	33	462.54	8	3	0	7	423.62
Hydrocortisone cypionate	4.13	100.9	35	486.65	6	2	0	7	469.81
Testolactone	3.24	43.38	22	300.4	3	0	0	0	288.48
Deoxycorticosterone acetate	3.5	60.45	27	372.5	4	0	0	4	363.84

LogP- Logarithmic Octanol Water Partition Coefficient; TPSA- topological polar surface area; nats- number of atoms; MW- molecular weight; HBA- number of hydrogen bond acceptors; HBD- number of hydrogen bond donors; nrotb- number of rotatable bonds; Vol- Volume.

Table 4.

Drug likeliness and lead likeliness of CBX and its FDA-approved analog drugs

Name of FDA-approved drug	Violation of Lipinski’s rule of 5 (Drug likeliness)	Lead likeliness property present (Yes/no)
Carbenoxolone (CBX)	Violates; 2 violations are MW > 500, LogP > 4.15	No; 2 violations are MW > 350, LogP3 > 3.5
Nandrolone decanoate	Violates; 1 violation is LogP > 4.15	No; 3 violations are MW > 350, no. of rotatable bonds > 7, LogP3 > 3.5
Testosterone cypionate	Violates; 1 violation is LogP > 4.15	No; 2 violations are MW > 350, LogP3 > 3.5
Testosterone enanthate	Violates; 1 violation is LogP > 4.15	No; 2 violations are MW > 350, LogP3 > 3.5
Testosterone undecanoate	Violates; 1 violation is LogP > 4.15	No; 3 violations are MW > 350, Rotors > 7, LogP3 > 3.5
Testosterone propionate	Does not violate	No; 1 violation is LogP3 > 3.5
Hydroxyprogesterone caproate	Violates; 1 violation is LogP > 4.15	No; 2 violations are MW > 350, LogP3 > 3.5
Gestonorone caproate	Doesnot violate	No; 2 violations are MW > 350, LogP3 > 3.5
Drospirenone	Violates; 1 violation is LogP > 4.15	No; 1 violation is MW > 350
Medroxyprogesterone acetate	Doesnot violate	No; 2 violations are MW > 350, LogP3 > 3.5
Hydrocortisone valerate	Doesnot violate	No; 2 violations are MW > 350, LogP3 > 3.5
Clascoterone	Doesnot violate	No; 2 violations are MW > 350, LogP3 > 3.5
Hydrocortisone butyrate	Doesnot violate	No; 1 violation is MW > 350
Hydrocortisone probutate	Doesnot violate	No; 3 violations are MW > 350, Rotors > 7, LogP3 > 3.5
Hydrocortisone succinate	Doesnot violate	No; 1 violation is MW > 350
Hydrocortisone cypionate	Yes; 0 violation	No; 2 violations are MW > 350, LogP3 > 3.5
Testolactone	Yes; 0 violation	Yes
Desoxycotrticosterone acetate	Yes; 0 violation	No; 1 violation is MW > 350

LogP- Logarithmic Octanol Water Partition Coefficient; MW- molecular weight.

While CBX violated two rules, eleven CBX analogs fulfilled Lipinski’s rule of 5 and were used for molecular docking with 11β-HSD1 (Fig. 5). Testolactone fulfilled both drug likeliness and lead likeliness properties.

Fig. 5.

Chemical structures of CBX analogs: (a) Hydrocortisone butyrate (b) Hydrocortisone succinate (c) Gestonorone caproate (d) Medroxyprogesterone acetate (e) Clascosterone (f) Testolactone (g) Hydrocortisone cypionate (h) Deoxycorticosterone acetate (i) Hydrocortisone probutate (j) Hydrocortisone valerate k. Testosterone propionate.

(Source: Zinc15 database)

The bioactivity scores of CBX and analogs estimated using Molinspiration are given in Table 2. For the CBX analogs that fulfill Lipinski’s rule of 5, the bioactivity scores range from 0.46 to 0.76. Testolactone has the highest bioactivity score of 0.76 and hydrocortisone cypionate has the lowest bioactivity score of 0.46. The bioactivity score of CBX is 0.47.

Molecular docking

The docking of 11β-HSD1 with eleven CBX analogs and FDA-approved drugs with better ADMET properties yielded the binding energies of their interactions. The binding patterns of various ligands with 11β-HSD1 as visualized using PyMOL are represented in Fig. 6. While the binding energy for the substrate and CBX is -9.1 kcal/mole and − 10.6 kcal/mole respectively (Fig. 3a and b), the binding energies of hydrocortisone succinate, medroxyprogesterone acetate, testolactone, hydrocortisone cypionate, deoxycorticosterone acetate, and hydrocortisone probutate were lower than that of the substrate corticosterone (Table 5). Only one drug hydrocortisone cypionate showed binding energy lower than that of CBX. The PoseView results of ProteinPlus showed the interacting amino acids (Fig. 6).

Fig. 6.

Interactions of 11β-HSD1 with CBX analogs that have lower binding energy than that of the substrate corticosterone: (a) Hydrocortisone cypionate (b) Medroxyprogesterone acetate (c) Deoxycorticosterone acetate (d) Hydrocortisone succinate (e) Hydrocortisone probutate. (f) Testolactone

Table 5.

Binding energies of CBX analogs

CBX analogs	Binding energy (ΔG) kcal mol-1
Hydrocortisone cypionate	-10.9
Deoxycorticosterone acetate	-9.9
Testolactone	-9.8
Hydrocortisone succinate	-9.6
Hydrocortisone probutate	-9.5
Medroxyprogesterone acetate	-9.3
Clascoterone	-9.1
Testosterone propionate	-9.1
Hydrocortisone butyrate	-9
Hydrocortisone valerate	-9
Gestonorone caproate	-8.9

Similar to CBX, Hydrocortisone probutate interacts with I111B and Clascosterone interacts with T210B and I36B in the binding pocket of 11β-HSD1. Amino acids interacting with Hydrocortisone valerate and Deoxycorticosterone acetate were similar to those interacting with substrate corticosterone. Hydrocortisone valerate forms a hydrogen bond (HBond) with G206A and interacts with I111A in the binding pocket, while Deoxycorticosterone acetate interacts with I111A in the binding pocket. Hydrocortisone cypionate forms HBond with A162A, S160A, and Y173A and also interacts with L116A, Y167A, A213A, and L207A (Fig. 6).

Figure 7 shows the AutoDock results of INCB13739 as well as the interacting amino acids of 11β-HSD1. The binding energy of INCB13739 with 11β-HSD1 was found to be -11.3 kcal/mol. INCB13739 interacts with I111B, T114B, and Y173B of 11β-HSD1.

Fig. 7.

The interaction of INCB13739 with amino acids in the binding pocket of 11β-HSD1.

(Source: PyMOL and ProteinPlus)

Redocking using iGEMDOCK

The 11β-HSD1 was docked with each of the CBX and its analogs using iGEMDOCK v2.1 software. The interactions of the best-docked CBX and its analogs in complex with 11β-HSD1, as measured by docking score energies are represented in Table 6. Redocking of the protein with CBX showed a total energy of -112.679 kcal mol^− 1. Among all the CBX analogs, hydrocortisone cypionate showed the lowest binding energy of − 115.126 kcal mol^− 1, indicating a high propensity towards binding to the active site of 11β-HSD1 compared to CBX. Table 7 lists the interactions between CBX and its analogs and the 11β-HSD1 binding site residues using iGEMDOCK noted and is compared with AutoDock docking interaction studies. Many interacting amino acid residues were found common for CBX and its analogs, which could be used to determine the ligand binding strategies of the pharmaceutical target.

Table 6.

The energies associated with the interactions between 11β-HSD1 and CBX and its analogs

Compound	Energy kcal mol-1	Van der Waals Interaction (VDW) kcal mol-1	Hydrogen bond (H Bond) kcal mol-1
Hydrocortisone cypionate	-115.126	-88.731	-26.3949
CBX	-112.679	-107.443	-4.31855
Hydrocortisone succinate	-108.028	-91.1242	-16.1632
Hydrocortisone probuatate	-106.797	-100.879	-5.91766
Hydroxycortisone valerate	-102.601	-96.6007	-6
Deoxycorticosterone acetate	-99.4318	-81.9702	-17.4616
Hydrocortisone butyrate	-95.3187	-75.4263	-19.8924
Clascoterone	-94.6273	-81.6983	-12.929
Testolactone	-91.7771	-77.2997	-14.4774
Gestonorone caproate	-89.2675	-80.8628	-8.4047
Medroxyprogesterone acetate	-88.4114	-86.4899	-1.9215
Testosterone propionate	-82.4819	-78.9819	-3.5

Table 7.

Comparing the interactive amino acid residues found by docking studies using AutoDock and iGEMDOCK.

Compounds	Interactions on docking with AutoDock	Interactions on docking with iGEMDOCK
CBX	Thr210, Gly35, Ile36, Ile111	Ser160, Gly31, Lys34, Gly35, Ile36, Asn109 Ile111, Tyr173, Thr210, Thr212
Hydrocortisone cypionate	Ala162, Tyr173, Ser160, Leu207, Ala213, Leu116, Tyr167	Gly35, Ser160, Glu211, Thr212, Lys34, Arg38, Ile111, Leu205, Ile208. Thr210
Hydrocortisone succinate	Ser160, Ala213	Gly35, Ser160, Tyr270, Tyr167, Val170, Tyr173, Thr210, Thr212, Ala213
Hydrocortisone probuatate	Asn109, Ile208, Gly31, Thr212, Ile111, Thr114, Tyr173	Ala213, Tyr167, Val170, Tyr173, Gly206, Leu207
Hydroxycortisone valerate	Gly206, Leu205, Ile111, Tyr173, Ala213, Asn109, Thr212	Leu207, Tyr270, Leu161, Tyr167, Tyr173, Gly206, Ile208, Met223
Deoxycorticosterone acetate	Ser33, Ala213, Ser160, Ile36, Ile111, Thr212	Arg56, Met83, Asn109, Gly31, Lys34, His110, Ile111
Hydrocortisone butyrate	Tyr173, Thr212, Asn109	His120, Asp122, His125, Ala172, Tyr173, Leu118, Phe119, Asp122, His125, Val170
Clascoterone	Ala213, Thr210, Ile36, Asn109	Gly35, Ile36, Ile111, Thr114, Tyr173, Thr212, Ala213
Testolactone	Val217, Tyr173, Tyr270	Thr82, Met83, Glu84, Lys34, Ala55, Arg56 His110
Gestonorone caproate	Asn109, Leu205, Val158, Thr210, Ile36, Ala213	Ser160, Tyr173, Tyr167, Val170, Leu207, Val221
Medroxyprogesterone acetate	Ala213, Thr210, Asn109	Lys34, Asn109, Ile111, Leu205, Thr210, Thr212,
Testosterone propionate	Ser160, Ala213, Ile36, Ile111, Thr212	Ile200, Arg188, Ser192, Arg195, Asn197, Val198, Ser199, Glu244

Molecular Dynamics (MD) simulations

The MD simulation of 11β-HSD1 and hydrocortisone cypionate docked structure obtained from the previous section showed that it formed a stable complex with the inhibitor. It took 26 Na⁺ ions and 27 Cl⁻ ions to neutralize the system. The Root mean square deviation (RMSD) of the protein calculated using the backbone was 0.37 ± 0.05 nm, and the RMSD of the ligand was 0.41 ± 0.06 nm, showing that there was not much structural change occurring that might have compromised the structural integrity of the ligand-protein interaction (Fig. 8a & b). The average radius of gyration for the protein-ligand complex was 1.83 ± 0.01 nm. This property is an indicator of the compactness of the structure, which showed that the protein-ligand complex remained stable (Fig. 8c). The average number of hydrogen bonds between the protein and ligand is 2.01 ± 0.74, which shows that there are favorable interactions that keep the two molecules bound (Fig. 8d). The binding energy calculated from the trajectory using MMPBSA was − 129.450 ± 6.882 kJ/mol.

Fig. 8.

MD simulation results of 11β-HSD1- Hydrocortisone cypionate complex. (a) RMSD of the 11β-HSD1 in the complex. (b) RMSD of Hydrocortisone cypionate in the complex. (c) The radius of gyration of 11β-HSD1- Hydrocortisone cypionate complex (d) Number of HBonds between 11β-HSD1 and Hydrocortisone cypionate during the simulation

Discussion

11β-HSD1 inhibitors could be a therapeutic strategy for the treatment of diabetes and prediabetes conditions [40]. CBX is the potent inhibitor of 11β-HSD1 [41, 42]. However, its use is banned in several countries due to side effects [10]. Various developments in the discovery of 11β-HSD1 inhibitors were based on their ability to form hydrogen bonds and interactions with amino acids that otherwise bind to substrate corticosterone and other potent inhibitors [43]. The results of such studies have been inconsistent. In the current study, analogs of CBX were used as a parent compound for screen through FDA-approved drugs to find small molecules that are structurally similar to CBX. We were able to find eleven CBX analogs that could potentially bind to 11β-HSD1. We carried out docking studies to understand how these eleven drugs interact with 11β-HSD1 and fulfilled Lipinski’s rule of five.

The bioactivity scores of the selected CBX analogs used in the computational analysis ranged from 0.46 to 0.76. We observed that testolactone showed the highest bioactivity score of 0.76 compared to the bioactivity score of CBX, which is 0.47. Unlike CBX, which violated both drug likeliness and lead likeliness properties, testolactone fulfilled both of these properties in this study. Testolactone is an oral small molecule drug used as an antiestrogen agent, and aromatase inhibitor, indicated for the treatment of advanced breast cancer [44–46]. Its use did not report mineralocorticoid excess syndrome as reported in CBX use [22]. Hence, it could be a better small molecule drug, for use in T2D patients as a 11β-HSD1 inhibitor. In vitro studies and clinical studies in T2D patients are essential to confirm 11β-HSD1 inhibitions by these small molecule drugs.

Small molecule FDA-approved drugs such as hydrocortisone succinate, medroxyprogesterone acetate, and deoxycorticosterone acetate also showed better binding to the 11β-HSD1 in the current study. Hydrocortisone succinate is used in the treatment of septic shock, ulcerative colitis, and cancers but the administration is intravenous or intramuscular due to poor gastrointestinal absorptivity [47–49]. Medroxyprogesterone acetate is used as a contraceptive during pregnancy, treat amenorrhea, uterine bleeding, endometrial hyperplasia, and endometrial carcinoma [50–52]. Deoxycorticosterone acetate is used to treat Addison’s disease [53]. The docking results of the current study show that deoxycorticosterone acetate interacts with I111A and hydrocortisone valerate interacts with I111A and forms a hydrogen bond with G206A. The substrate corticosterone binds to these amino acids suggesting their presence in the active substrate-binding site of the enzyme. Hydrocortisone valerate is indicated in the treatment of pruritic dermatoses and inflammation [54].

We observed that hydrocortisone probutate interacts with the amino acids to which CBX binds. Hydrocortisone probutate and CBX form a hydrogen bond with I111B but the binding energy of hydrocortisone probutate is higher (∆G= -9.5 kcal/mole) than that of CBX (∆G= -10.6 kcal/mole) but lower than that of corticosterone (-9.1 kcal/mole). Hydrocortisone probutate is indicated in the treatment of skin related redness and swelling [55].

In the docking results of this study, hydrocortisone cypionate exhibited minimum binding energy (∆G=-10.9 kcal/mole), which was even lower than the binding energies of CBX and corticosterone. The docking of INCB13739 with 11β-HSD1 in the current study shows that INCB13739 binds to 11β-HSD1 with higher affinity compared to hydrocortisone cypionate. Previous studies showed that INCB13739 reduces glycated hemoglobin (HbA1c), fasting plasma glucose, and HOMA-IR levels in T2D patients and improved blood glucose levels in patients who were already on metformin therapy [17, 21], but was not further developed or continued for unknown reasons [21]. No human studies of INCB13739 inhibitors have been reported from phase III clinical testing. Unlike INCB13739, hydrocortisone cypionate is FDA approved drug and has been tested for safe use in humans. This can greatly reduce the time and cost of its development.

iGEMDOCK analysis in the current study showed that hydrocortisone cypionate exhibited the lowest binding energy of − 115.126 kcal mol^-1, which is the lowest among all CBX analogs indicating the high propensity towards binding to the active region of 11β-HSD1 compared to CBX. Validation of docking studies performed by AutoDock and iGEMDOCK in this study showed that hydrocortisone cypionate with the lowest binding energy has a higher tendency than other CBX analogs to bind to the active site of 11β-HSD1. A comparison of interacting amino acid residues found using AutoDock and iGEMDOCK confirmed amino acids that interact with CBX analogs and many interacting amino acid residues were common between CBX and its analogs.

Hydrocortisone cypionate is a small molecule FDA-approved drug for the treatment of inflammation, arthritis, psoriasis, severe asthma, ulcerative colitis, Crohn’s disease, and dermatoses [56]. No studies have reported its use as a 11β-HSD1 inhibitor. Our results are the first of this kind and have opened a new avenue in the direction of its use as an alternative treatment option for the treatment of T2D and prediabetes conditions that are characterized by abnormally high levels of GCs. However, for validation and confirmation, in vitro studies are essential in patients with these conditions.

The evolution of several structural categories of selective inhibitors of 11β-HSD1 indicates that more accurate screening and design of isoform for tissue selectivity may generate prospective therapeutic agents in this area [43]. Oral therapeutic agents that aid in better glycemic control and treat associated disorders of metS are frequently needed in present medical practice. The current study supports ongoing discoveries of 11β-HSD1 inhibitors by pharmaceutical companies such as Incite, Amgen, Merck, etc. to treat type 2 diabetes and other metabolic disorders [40, 57]. Hydrocortisone cypionate that shows highest binding affinity towards 11β-HSD1 in the current study, showed no previous reports of nephrotoxicity, which is the serious side effect of most of the 11β-HSD1 inhibitors. Hydrocortisone cypionate is an FDA-approved drug. Repurposing FDA-approved drugs for 11β-HSD1 inhibition reduces the drug development timeline. As FDA approved drugs have already been demonstrated to be safe in humans, there is no necessity to pass various phases of clinical trials. This reduces the overall cost, time, and risk of the drug development process [58, 59]. Further being a small molecule, hydrocortisone cypionate could show better penetrant effects with better bioavailability [60]. This study is a stepping-stone for unearthing the selective inhibitors of 11β-HSD1 from the repertoire of approved drugs.

Conclusion

The current in silco study suggests that CBX analogs are capable of interacting with crucial amino acids needed for substrate binding of the 11β-HSD1 enzyme. Our docking study shows that hydrocortisone succinate, medroxyprogesterone acetate, testolactone, hydrocortisone cypionate, deoxycorticosterone acetate, and hydrocortisone probutate binds to 11β-HSD1 with higher affinity than substrate corticosterone, with hydrocortisone cypionate having higher binding affinity and lower binding energy than CBX. The MD simulation of the docked complex of 11β-HSD1 and hydrocortisone cypionate shows that it can form a stable complex. This study indicates the potential uses of these FDA-approved inhibitors in the treatment of diabetes and prediabetes conditions. However, confirmation by in vitro inhibitory studies is essential.

Abbreviations

ADMET

Absorption, Distribution, Metabolism, Excretion, and Toxicity

11β-HSD1

11beta-Hydroxysteroid Dehydrogenase Type 1

CBX

Carbenoxolone

2D

Two Dimensional

3D

Three Dimensional

dt

Step Size

Elec

Electrostatic

FDA

Food and Drug Administration

GC

Glucocorticoid

HBA

Number of Hydrogen Bond Acceptors

HbA1c

Glycated Hemoglobin

HBond

Hydrogen Bond

HOMA-IR

Homeostatic Model Assessment for Insulin Resistance

HTN

Hypertension

IR

Insulin Resistance

LogP

Logarithmic Octanol Water Partition Coefficient

MD

Molecular Dynamics

metS

Metabolic Syndrome

MM-PBSA

Molecular Mechanics Poisson–Boltzmann Surface Area.

MW

Molecular Weight

nats

Number of Atoms

nrotb

Number of Rotatable Bonds

ns

Number of Steps

n violations

Number of Violations

NCBI

National Center for Biotechnology Information

NPT

Constant Number of Atoms, Pressure, and Temperature

NVT

Constant Number of Atoms, Volume, and Temperature

PDB

Protein Data Bank

RMSD

Root Mean Square Deviation

SDF

Spatial Data File

T2D

Type 2 Diabetes

TIP3P

Transferable Intermolecular Potential with 3 Points

TPSA

Topological Polar Surface Area

Vol

Volume

VDW

Van Der Waals

Declarations

Competing Interests

Authors declare there is no conflict of interest.