Molecular insight into endosulfan degradation by Ese protein from Arthrobacter: Evidence-based structural bioinformatics and quantum mechanical calculations.

Endosulfan is an organochlorine insecticide widely used for agricultural pest control. Many nations worldwide have restricted or completely banned it due to its extreme toxicity to fish and aquatic invertebrates. Arthrobacter sp. strain KW has the ability to degrade α, ß endosulfan and its intermediate metabolite endosulfate; this degradation is associated with Ese protein, a two-component flavin-dependent monooxygenase (TC-FDM). Employing in silico tools, we obtained the 3D model of Ese protein, and our results suggest that it belongs to the Luciferase Like Monooxygenase family (LLM). Docking studies showed that the residues V59, V315, D316, and T335 interact with α-endosulfan. The residues: V59, T60, V315, D316, and T335 are implicated in the interacting site with ß-endosulfan, and the residues: H17, V315, D316, T335, N364, and Q363 participate in the interaction with endosulfate. Topological analysis of the electron density by means of the Quantum Theory of Atoms in Molecules (QTAIM) and the Non-Covalent Interaction (NCI) index reveals that the Ese-ligands complexes are formed mainly by dispersive forces, where Cl atoms have a predominant role. As Ese is a monooxygenase member, we predict the homodimer formation. However, enzymatic studies must be developed to investigate the Ese protein's enzymatic and catalytic activity.

Design and simulation of a caprylic acid enzymatically modified phosphatidylcholine micelle using a coarse-grained molecular dynamics simulations approach.

Computationally simulated micelle models provide useful structural information on the molecular and biological sciences. One strategy to study the self-aggregation process of surfactant molecules that make up a micelle is through molecular dynamics (MD) simulations. In this study, a theoretical approach with a coarse-grained MD simulation (CG-MD) was employed to evaluate the critical micellar concentration (CMC), the micellization process, building a tridimensional (3D) model system of a micelle using data from the experimentally enzymatically modified phospholipids (PL) by phospholipase A1 (PA1). This required enzymatic interesterification of soybean phosphatidylcholine (PC) with caprylic acid, along with purification and characterization by chromatographic techniques to measure the esterified fatty acids and the corresponding PL composition. The number of molecules used in the CG-MD simulation system was determined from the experimental CMC data which was 0.025%. The molecular composition of the system is: 1 C 18:2, 2 C 8:0/8:0, 3 C 8:0/18:3n-9, 4 C 8:0/18:0, 5 C8:0/18:2n-6, 6 C8:0/18:1n-9, and 7 C 8:0/16:0. According to our theoretical results, the micelle model is structurally stable with an average Rg of 3.64 ± 0.10 Å, and might have an elliptical form with a radius of 24.6 Å. Regarding CMC value there was a relationship between the experimental data of the modified PLs and the theoretical analysis by GC-MD, which suggest that the enzymatic modification of PLs does not affect their self-aggregation properties. Finally, the micellar system obtained in the current research can be used as a simple and useful model to design optimal biocompatible nanoemulsions as possible vehicles for bioactive small molecules.Communicated by Ramaswamy H. Sarma.

Cytotoxic evaluation of YSL-109 in a triple negative breast cancer cell line and toxicological evaluations.

Breast cancer (BC) is the leading cause of cancer-related death in women worldwide. Triple negative breast cancer (TNBC) is the most aggressive form of BC being with the worst prognosis and the worst survival rates. There is no specific pharmacological target for the treatment of TNBC; conventional therapy includes the use of non-specific chemotherapy that generally has a poor prognosis. Therefore, the search of effective therapies against to TNBC continues at both preclinical and clinical level. In this sense, the exploration of different pharmacological targets is a continue task that pave the way to epigenetic modulation using novel small molecules. Lately, the inhibition of histone deacetylases (HDACs) has been explored to treat different BC, including TNBC. HDACs remove the acetyl groups from the ɛ-amino lysine resides on histone and non-histone proteins. In particular, the inhibition of HDAC6 has been suggested to be useful for the treatment of TNBC due to it is overexpressed in TNBC. Therefore, in this work, an HDAC6 selective inhibitor, the (S)-4-butyl-N-(1-(hydroxyamino)-3-(naphthalen-1-yl)-1-oxopropan-2-yl) benzamide (YSL-109), was assayed on TNBC cell line (MDA-MB231) showing an antiproliferative activity (IC50 = 50.34 ± 1.11 µM), whereas on fibroblast, it was lesser toxic. After corroborating the in vitro antiproliferative activity of YSL-109 in TNBC, the toxicological profile was explored using combined approach with in silico tools and experimental assays. YSL-109 shows moderate mutagenic activity on TA-98 strain at 30 and 100 µM in the Ames test, whereas YSL-109 did not show in vivo genotoxicity and its oral acute toxicity (LD50) in CD-1 female mice was higher than 2000 mg/kg, which is in agreement with our in silico predictions. According to these results, YSL-109 represents an interesting compound to be explored for the treatment of TNBC under preclinical in vivo models.

Dihydropyrazole-Carbohydrazide Derivatives with Dual Activity as Antioxidant and Anti-Proliferative Drugs on Breast Cancer Targeting the HDAC6.

Breast cancer (BC) is the most frequently diagnosed cancer and is the second-most common cause of death in women worldwide. Because of this, the search for new drugs and targeted therapy to treat BC is an urgent and global need. Histone deacetylase 6 (HDAC6) is a promising anti-BC drug target associated with its development and progression. In the present work, the design and synthesis of a new family of dihydropyrazole-carbohydrazide derivatives (DPCH) derivatives focused on HDAC6 inhibitory activity is presented. Computational chemistry approaches were employed to rationalize the design and evaluate their physicochemical and toxic-biological properties. The new family of nine DPCH was synthesized and characterized. Compounds exhibited optimal physicochemical and toxicobiological properties for potential application as drugs to be used in humans. The in silico studies showed that compounds with -Br, -Cl, and -OH substituents had good affinity with the catalytic domain 2 of HDAC6 like the reference compounds. Nine DPCH derivatives were assayed on MCF-7 and MDA-MB-231 BC cell lines, showing antiproliferative activity with IC50 at µM range. Compound 2b showed, in vitro, an IC50 value of 12 ± 3 µM on human HDAC6. The antioxidant activity of DPCH derivatives showed that all the compounds exhibit antioxidant activity similar to that of ascorbic acid. In conclusion, the DPCH derivatives are promising drugs with therapeutic potential for the epigenetic treatment of BC, with low cytotoxicity towards healthy cells and important antioxidant activity.

HDAC Inhibition as Neuroprotection in COVID-19 Infection.

The SARS-CoV-2 virus is responsible for COVID-19 affecting millions of humans around the world. COVID-19 shows various clinical symptoms (fever, cough, fatigue, diarrhea, body aches, headaches, anosmia, and hyposmia). Approximately 30% of patients with COVID-19 showed neurological symptoms, from mild to severe manifestations including headache, dizziness, impaired consciousness, encephalopathy, anosmia, hypogeusia, and hyposmia, among others. The neurotropism of the SARS-CoV-2 virus explains its neuroinvasion provoking neurological damage such as acute demyelination, neuroinflammation, etc. At the molecular level, the COVID-19 patients had higher levels of cytokines and chemokines known as cytokines storms which disrupt the blood-brain barrier allowing the entrance of monocytes and lymphocytes, causing neuroinflammation, neurodegeneration, and demyelination. In addition, the proinflammatory cytokines have been observed in ischemic, hemorrhagic strokes, seizures, and encephalopathy. In this sense, early neuroprotective management should be adopted to avoid or decrease neurological damage due to SARS-CoV-2 infection. Several approaches can be used; one of them includes using HDAC inhibitors (HDACi) due to their neuroprotective effects. Also, the HDACi down-regulates the proinflammatory cytokines (IL-6 and TNF-α) decreasing the neurotoxicity. HDACi can also avoid and prevent the entrance of the virus into the central nervous System (CNS) and decrease the virus replication by downregulating the virus receptors. Here we review the mechanisms that could explain how the SARS-CoV-2 virus could reach the CNS, induce neurological damage and symptoms, and the possibility to use HDACi as neuroprotective therapy.

In silico design of HDAC6 inhibitors with neuroprotective effects.

HDAC6 has emerged as a molecular target to treat neurodegenerative disorders, due to its participation in protein aggregate degradation, oxidative stress process, mitochondrial transport, and axonal transport. Thus, in this work we have designed a set of 485 compounds with hydroxamic and bulky-hydrophobic moieties that may function as HDAC6 inhibitors with a neuroprotective effect. These compounds were filtered by their predicted ADMET properties and their affinity to HDAC6 demonstrated by molecular docking and molecular dynamics simulations. The combination of in silico with in vitro neuroprotective results allowed the identification of a lead compound (FH-27) which shows neuroprotective effect that could be due to HDAC6 inhibition. Further, FH-27 chemical moiety was used to design a second series of compounds improving the neuroprotective effect from 2- to 10-fold higher (YSL-99, YSL-109, YSL-112, YSL-116 and YSL-121; 1.25 ± 0.67, 1.82 ± 1.06, 7.52 ± 1.78, 5.59 and 5.62 ± 0.31 µM, respectively). In addition, the R enantiomer of FH-27 (YSL-106) was synthesized, showing a better neuroprotective effect (1.27 ± 0.60 µM). In conclusion, we accomplish the in silico design, synthesis, and biological evaluation of hydroxamic acid derivatives with neuroprotective effect as suggested by an in vitro model. Communicated by Ramaswamy H. Sarma.

Computational study of DMPC liposomes loaded with the N-(2-Hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) and determination of its antiproliferative activity in vitro in NIH-3T3 cells.

N-(2-Hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a valproic acid (VPA) derivative that has shown promising antiproliferative effects in different cancer cell lines, such as A204, HeLa, and MDA-MB-231. However, its low water solubility could reduce its therapeutic effectiveness. To solve this problem, in this work, we incorporated HO-AAVPA into dimyristoyl-phosphatidylcholine (DMPC) liposomes in the presence or absence of cholesterol (CHOL). Using differential scanning calorimetry (DSC), we found that the transition enthalpy (ΔHtr) of DMPC liposomes is reduced in the presence of CHOL and/or HO-AAVPA, indicating the favorable interactions between CHOL and/or HO-AAVPA and DMPC. Further, by molecular dynamics simulations it was possible to observed that HO-AAVPA migrates from the center of the bilayer toward the water and lipid interface of the DPMC bilayer systems exposing the amine group to water and the aliphatic chain toward the interior of the bilayer. As a consequence, we observed an ordering of the lipid bilayer. Moreover, CHOL harbors into the inner bilayer membrane, increasing the order parameter of the system. The liposomal solutions loaded with HO-AAVPA were tested in the NIH3T3 cell line, showing a reduction in cell proliferation compared to those cells presented without liposomes.Communicated by Ramaswamy H. Sarma.

Targeting Several Biologically Reported Targets of Glioblastoma Multiforme by Assaying 2D and 3D Cultured Cells.

Glioblastoma multiforme (GBM) is account for 70% of all primary malignancies of the central nervous system. The median survival of human patients after treatment is around 15 months. There are several biological targets which have been reported that can be pursued using ligands with varied structures to treat this disease. In our group, we have developed several ligands that target a wide range of proteins involved in anticancer effects, such as histone deacetylase (HDACs), G protein-coupled estrogen receptor 1 (GPER), estrogen receptor-beta (ERß) and NADPH oxidase (NOX), that were screened on bidimensional (2D) and tridimensional (3D) GBM stem cells like (GSC). Our results show that some HDAC inhibitors show antiproliferative properties at 21-32 µM. These results suggest that in this 3D culture, HDACs could be the most relevant targets that are modulated to induce the antiproliferative effects that require in the future further experimental studies.

Drug repositioning to target NSP15 protein on SARS-CoV-2 as possible COVID-19 treatment.

SARS-CoV and SARS-CoV-2 belong to the subfamily Coronaviridae and infect humans, they are constituted by four structural proteins: Spike glycoprotein (S), membrane (M), envelope (E) and nucleocapsid (N), and nonstructural proteins, such as Nsp15 protein which is exclusively present on nidoviruses and is absent in other RNA viruses, making it an ideal target in the field of drug design. A virtual screening strategy to search for potential drugs was proposed, using molecular docking to explore a library of approved drugs available in the DrugBank database in order to identify possible NSP15 inhibitors to treat Covid19 disease. We found from the docking analysis that the antiviral drugs: Paritaprevir and Elbasvir, currently both approved for hepatitis C treatment which showed some of the lowest free binding energy values were considered as repositioning drugs to combat SARS-CoV-2. Furthermore, molecular dynamics simulations of the Apo and Holo-Nsp15 systems were performed in order to get insights about the stability of these protein-ligand complexes.

Structural insights into SARS-CoV-2 spike protein and its natural mutants found in Mexican population.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus responsible for coronavirus disease 2019 (COVID-19); it become a pandemic since March 2020. To date, there have been described three lineages of SARS-CoV-2 circulating worldwide, two of them are found among Mexican population, within these, we observed three mutations of spike (S) protein located at amino acids H49Y, D614G, and T573I. To understand if these mutations could affect the structural behavior of S protein of SARS-CoV-2, as well as the binding with S protein inhibitors (cepharanthine, nelfinavir, and hydroxychloroquine), molecular dynamic simulations and molecular docking were employed. It was found that these punctual mutations affect considerably the structural behavior of the S protein compared to wild type, which also affect the binding of its inhibitors into their respective binding site. Thus, further experimental studies are needed to explore if these affectations have an impact on drug-S protein binding and its possible clinical effect.

N-(2'-Hydroxyphenyl)-2-Propylpentanamide (HO-AAVPA) Inhibits HDAC1 and Increases the Translocation of HMGB1 Levels in Human Cervical Cancer Cells.

N-(2'-hydroxyphenyl)-2-propylpentanamide (HO-AAVPA) is a VPA derivative designed to be a histone deacetylase (HDAC) inhibitor. HO-AAVPA has better antiproliferative effect than VPA in cancer cell lines. Therefore, in this work, the inhibitory effect of HO-AAVPA on HDAC1, HDAC6, and HDAC8 was determined by in silico and in vitro enzymatic assay. Furthermore, its antiproliferative effect on the cervical cancer cell line (SiHa) and the translocation of HMGB1 and ROS production were evaluated. The results showed that HO-AAVPA inhibits HDAC1, which could be related with HMGB1 translocation from the nucleus to the cytoplasm due to HDAC1 being involved in the deacetylation of HMGB1. Furthermore, an increase in ROS production was observed after the treatment with HO-AAVPA, which also could contribute to HMGB1 translocation. Therefore, the results suggest that one of the possible antiproliferative mechanisms of HO-AAVPA is by HDAC1 inhibition which entails HMGB1 translocation and ROS increased levels that could trigger the cell apoptosis.

Hydroxamic acid derivatives as HDAC1, HDAC6 and HDAC8 inhibitors with antiproliferative activity in cancer cell lines.

Histone deacetylases (HDACs) belong to a family of enzymes that remove acetyl groups from the ɛ-amino of histone and nonhistone proteins. Additionally, HDACs participate in the genesis and development of cancer diseases as promising therapeutic targets to treat cancer. Therefore, in this work, we designed and evaluated a set of hydroxamic acid derivatives that contain a hydrophobic moiety as antiproliferative HDAC inhibitors. For the chemical structure design, in silico tools (molecular docking, molecular dynamic (MD) simulations, ADME/Tox properties were used to target Zn2+ atoms and HDAC hydrophobic cavities. The most promising compounds were assayed in different cancer cell lines, including hepatocellular carcinoma (HepG2), pancreatic cancer (MIA PaCa-2), breast cancer (MCF-7 and HCC1954), renal cancer (RCC4-VHL and RCC4-VA) and neuroblastoma (SH-SY5Y). Molecular docking and MD simulations coupled to the MMGBSA approach showed that the target compounds have affinity for HDAC1, HDAC6 and HDAC8. Of all the compounds evaluated, YSL-109 showed the best activity against hepatocellular carcinoma (HepG2 cell line, IC50 = 3.39 µM), breast cancer (MCF-7 cell line, IC50 = 3.41 µM; HCC1954 cell line, IC50 = 3.41 µM) and neuroblastoma (SH-SY5Y cell line, IC50 = 6.42 µM). In vitro inhibition assays of compound YSL-109 against the HDACs showed IC50 values of 259.439 µM for HDAC1, 0.537 nM for HDAC6 and 2.24 µM for HDAC8.

O-Alkyl Hydroxamates Display Potent and Selective Antileishmanial Activity.

Leishmania (L.) infantum causes visceral, cutaneous, and mucosal leishmaniasis in humans and canine leishmaniasis in dogs. Herein, we describe that O-alkyl hydroxamate derivatives displayed potent and selective in vitro activity against the amastigote stage of L. infantum while no activity was observed against promastigotes. Compound 5 showed potent in vivo activity against L. infantum. Moreover, the combination of compound 5 supported on gold nanoparticles and meglumine antimoniate was also effective in vivo and improved the activity of these compounds compared to that of the individual treatment. Docking studies showed that compound 5 did not reach highly conserved pocket C and established interactions with the semiconserved residues V44, A45, R242, and E243 in pocket A of LiSIR2rp1. The surface space determined by these four amino acids is not conserved in human sirtuins. Compound 5 represents a new class of selective ligands with antileishmanial activity.

DNA methylation of the RE-1 silencing transcription factor in peripheral blood mononuclear cells and gene expression of antioxidant enzyme in patients with late-onset Alzheimer disease.

Late-onset Alzheimer disease (LOAD) is the most frequent cause of dementia in elderly adults. However, the factors determining disease onset remain unclear. In the elderly, the activation and expression of the gene encoding RE-1 silencing transcription factor (REST) may be a determinant of neuroprotective mechanisms and good amyloidogenic pathway management. In the present study, the minimal promoter region of REST1 was genetically and epigenetically analyzed in blood samples from 21 subjects with LOAD and 20 cognitively healthy elderly subjects. Genomic DNA was isolated, treated with bisulfite and pyrosequenced, and gene expression was determined using real-time PCR. Notably, subjects with LOAD exhibited hypermethylation and significantly diminished expression of REST1 compared with healthy subjects (p = 0.001). In the LOAD group, the gene expression of CAT, SOD2 and GPX also showed a significant decrease and an increase in malondialdehyde. A docking analysis revealed that the first zinc finger protein Sp1 recognized and bound the methylated sequence in subjects with LOAD differently than the binding observed in control subjects. These results reveal that in patients with LOAD the methylation of specific sites in the promoter sequence of REST suppresses its expression and this could be regulating the decreased expression of CAT, SOD and GPX, besides interfering with the action of transcription factors as Sp1.

Exploring the inhibitory activity of valproic acid against the HDAC family using an MMGBSA approach.

Valproic acid (VPA) is a compound currently used in clinical practice for the treatment of epilepsy as well as bipolar and mood disorders. VPA targets histone deacetylases (HDACs), which participate in the removal of acetyl groups from lysine in several proteins, regulating a wide variety of functions within the organism. An imbalance or malfunction of these enzymes is associated with the development and progression of several diseases, such as cancer and neurodegenerative diseases. HDACs are divided into four classes, but VPA only targets Class I (HDAC1-3 and 8) and Class IIa (HDAC4-5, 7 and 9) HDACs; however, structural and energetic information regarding the manner by which VPA inhibits these HDACs is lacking. Here, the structural and energetic features that determine this recognition were studied using molecular docking and molecular dynamics (MD) simulation. It was found that VPA reaches the catalytic site in HDAC1-3 and 7, whereas in HDAC6, VPA only reaches the catalytic tunnel. In HDAC4, VPA was bound adjacent to L1 and L2, a zone that participates in corepressor binding, and in HDAC8, VPA was bound to the hydrophobic active site channel (HASC), in line with previous reports.

Identification of the NADP+ Structural Binding Site and Coenzyme Effect on the Fused G6PD::6PGL Protein from Giardia lamblia.

Giardia lambia is a flagellated protozoan parasite that lives in the small intestine and is the causal agent of giardiasis. It has been reported that G. lamblia exhibits glucose-6-phosphate dehydrogenase (G6PD), the first enzyme in the pentose phosphate pathway (PPP). Our group work demonstrated that the g6pd and 6pgl genes are present in the open frame that gives rise to the fused G6PD::6PGL protein; where the G6PD region is similar to the 3D structure of G6PD in Homo sapiens. The objective of the present work was to show the presence of the structural NADP+ binding site on the fused G6PD::6PGL protein and evaluate the effect of the NADP+ molecule on protein stability using biochemical and computational analysis. A protective effect was observed on the thermal inactivation, thermal stability, and trypsin digestions assays when the protein was incubated with NADP+. By molecular docking, we determined the possible structural-NADP+ binding site, which is located between the Rossmann fold of G6PD and 6PGL. Finally, molecular dynamic (MD) simulation was used to test the stability of this complex; it was determined that the presence of both NADP+ structural and cofactor increased the stability of the enzyme, which is in agreement with our experimental results.

Structural and energetic basis for the inhibitory selectivity of both catalytic domains of dimeric HDAC6.

HDAC6 is a protein involved in cancer, neurodegenerative disease and inflammatory disorders. To date, the full three-dimensional (3D) structure of human HDAC6 has not been elucidated; however, there are some experimental 3D structural homologs to HDAC6 that can be used as templates. In this work, we utilized molecular modeling procedures to model both of the catalytic domains of HDAC6 connected by the linker region where DMB region is placed. Once the 3D structure of human HDAC6 was obtained, it was structurally evaluated and submitted to docking and molecular dynamic (MD) simulations along with Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) method to explore the stability and the binding free energy properties of the HDAC6-ligand complexes. In addition, its structural and energetic behavior was explored with each one of the catalytic domains in the molecular recognition of six selective HDAC6 inhibitors, HPOB, CAY10603, Nexturastat, Rocilinostat, Tubacin and Tubastatin A for DD2, and with the so-called 9-peptide which is DD1-HDAC6 selective substrate. The use of the whole system (DD1-DMB-DD2) showed a tendency toward the ligand affinity of DD2, CAY10603> Tubacin > Rocilinostat > Nexturastat > HPOB > Tubastatin > 9-peptide, which is in line with experimental reports. However, 9-peptide showed a higher affinity for DD1, which agrees with experimental reports elsewhere. Principal component analysis provided important information about the structural changes linked to the molecular recognition process, whereas per-residue decomposition analysis revealed the energetic contribution of the key residues in the molecular binding and structural characteristics that could assist in drug design.

Insights into structural features of HDAC1 and its selectivity inhibition elucidated by Molecular dynamic simulation and Molecular Docking.

Histone deacetylases (HDACs) are a family of proteins whose main function is the removal of acetyl groups from lysine residues located on histone and non-histone substrates, which regulates gene transcription and other activities in cells. HDAC1 dysfunction has been implicated in cancer development and progression; thus, its inhibition has emerged as a new therapeutic strategy. Two additional metal binding sites (Site 1 and Site 2) in HDACs have been described that are primarily occupied by potassium ions, suggesting a possible structural role that affects HDAC activity. In this work, we explored the structural role of potassium ions in Site 1 and Site 2 and how they affect the interactions of compounds with high affinities for HDAC1 (AC1OCG0B, Chlamydocin, Dacinostat and Quisinostat) and SAHA (a pan-inhibitor) using molecular docking and molecular dynamics (MD) simulations in concert with a Molecular-Mechanics-Generalized-Born-Surface-Area (MMGBSA) approach. Four models were generated: one with a potassium ion (K+) in both sites (HDAC1k), a second with K+ only at site 1 (HDAC1ks1), a third with K+ only at site 2 (HDAC1ks2) and a fourth with no K+ (HDAC1wk). We found that the presence or absence of K+ not only impacted the structural flexibility of HDAC1, but also its molecular recognition, consistent with experimental findings. These results could therefore be useful for further structure-based drug design studies addressing new HDAC1 inhibitors.

Design, Synthesis and Biological Evaluation of a Phenyl Butyric Acid Derivative, N-(4-chlorophenyl)-4-phenylbutanamide: A HDAC6 Inhibitor with Anti-proliferative Activity on Cervix Cancer and Leukemia Cells.

BACKGROUND: The epigenetic regulation of genes in cancer could be targeted by inhibiting Histone deacetylase 6 (HDAC6), an enzyme involved in several types of cancer such as lymphoma, leukemia, ovarian cancer, etc. OBJECTIVE: Through in silico methods, a set of Phenyl butyric acid derivatives with possible HDAC6 inhibitory activity were designed, rendering monophenylamides and biphenylamides using tubacin (HDAC6 selective inhibitor) as reference. METHOD: The target compounds were submitted to theoretical ADMET analyses and their binding properties on different HDAC6 conformers were evaluated through docking calculations. RESULTS: These in silico studies allowed us to identify a compound named B-R2B. In order to have more information about the B-R2B binding recognition properties on HDAC6, the B-R2B-HDAC6 complex was submitted through 100 ns-long Molecular Dynamics (MD) simulation coupled to MMGBSA approach, revealing that B-R2B is located at the entrance of HDAC6 active pocket, blocking the passage of the substrate without reaching the HDAC6 binding site. Based on these results, B-R2B was synthesized, characterized and biologically tested. The HDAC6 fluorometric drug discovery kit Fluor-de-Lys (ENZO Life Sciences Inc.) was used to determine the HDAC6 human inhibitory activity (IC50 value) of B-R2B compound. In addition, B-R2B show IC50 values on cancer cell lines (HeLa; IC50 = 72.6 µM), acute myeloid leukemia (THP-1; IC50 = 16.5 µM), human mast leukemia (HMC; IC50 = 79.29 µM) and chronic myelogenous leukemia (Kasumi; IC50 = 101 µM). CONCLUSION: These results show that B-R2B is a HDAC6 inhibitor, specifically a non-competitive type in a similar way that tubacin does, according to MD simulations.

Searching the conformational complexity and binding properties of HDAC6 through docking and molecular dynamic simulations.

Histone deacetylases (HDACs) are a family of proteins involved in the deacetylation of histones and other non-histones substrates. HDAC6 belongs to class II and shares similar biological functions with others of its class. Nevertheless, its three-dimensional structure that involves the catalytic site remains unknown for exploring the ligand recognition properties. Therefore, in this contribution, homology modeling, 100-ns-long Molecular Dynamics (MD) simulation and docking calculations were combined to explore the conformational complexity and binding properties of the catalytic domain 2 from HDAC6 (DD2-HDAC6), for which activity and affinity toward five different ligands have been reported. Clustering analysis allowed identifying the most populated conformers present during the MD simulation, which were used as starting models to perform docking calculations with five DD2-HDAC6 inhibitors: Cay10603 (CAY), Rocilinostat (RCT), Tubastatin A (TBA), Tubacin (TBC), and Nexturastat (NXT), and then were also submitted to 100-ns-long MD simulations. Docking calculations revealed that the five inhibitors bind at the DD2-HDAC6 binding site with the lowest binding free energy, the same binding mode is maintained along the 100-ns-long MD simulations. Overall, our results provide structural information about the molecular flexibility of apo and holo DD2-HDAC6 states as well as insight of the map of interactions between DD2-HDAC6 and five well-known DD2-HDAC6 inhibitors allowing structural details to guide the drug design. Finally, we highlight the importance of combining different theoretical approaches to provide suitable structural models for structure-based drug design.