Development of Computational Approaches with a Fragment-Based Drug Design Strategy: In Silico Hsp90 Inhibitors Discovery

A semi-exhaustive approach and a heuristic search algorithm use a fragment-based drug design (FBDD) strategy for designing new inhibitors in an in silico process. A deconstruction reconstruction process uses a set of known Hsp90 ligands for generating new ones. The deconstruction process consists of cutting off a known ligand in fragments. The reconstruction process consists of coupling fragments to develop a new set of ligands. For evaluating the approaches, we compare the binding energy of the new ligands with the known ligands.     

Inhibition of Hepatitis C Replication by Targeting the Molecular Chaperone Hsp90: Synthesis and Biological Evaluation of 4,5,6,7-Tetrahydrobenzo[1,2-d]thiazole Derivatives

Cellular chaperones that belong to the heat-shock protein 90 (Hsp90) family are a prerequisite for successful viral propagation for most viruses. The hepatitis C virus (HCV) uses Hsp90 for maturation, folding, and modification of viral proteins. Based on our previous discovery that marine alkaloid analogues with a 4,5,6,7-tetrahydrobenzo[1,2-d]thiazole-2-amine structure show inhibition of HCV replication and binding to Hsp90, a series of twelve novel compounds based on this scaffold was designed and synthesized. The aim was improved Hsp90 affinity and anti-HCV activity. Through structural optimization, improved binding to Hsp90 and specific HCV inhibition in genotype 1b and 2a replicon models was achieved for three compounds belonging to the newly synthesized series. Furthermore, these compounds efficiently inhibited replication of full-length HCV genotype 2a in a reporter virus RNA assay with IC₅₀ values ranging from 0.03 to 0.6 μm .     

Structure‐Based and in silico Design of Hsp90 Inhibitors

The molecular chaperone Hsp90 is responsible for activation and stabilization of several oncoproteins in cancer cells, and has emerged as an important target in cancer treatment because of this pivotal role. In recent years, interests have arisen around structure‐based design of small molecules aimed at inhibiting the chaperone activity of Hsp90. In this review, we illustrate the recent advances in structure‐based and in silico strategies aimed at discovering and optimizing Hsp90 inhibitors.     

New,Non‐quinone Fluorogeldanamycin Derivatives Strongly Inhibit Hsp90

Streptomyces hygroscopicus is a natural producer of geldanamycin. Mutasynthetic supplementation of an AHBA‐blocked mutant with all possible monofluoro 3‐aminobenzoic acids provided new fluorogeldanamycins. These showed strong antiproliferative activity and inhibitory effects on human heat shock protein Hsp90. Binding to Hsp90 in the low nanomolar range was determined from molecular modelling, AFM analysis and by calorimetric studies.     

The Chaperone System in Breast Cancer: Roles and Therapeutic Prospects of the Molecular Chaperones Hsp27, Hsp60, Hsp70, and Hsp90

Breast cancer (BC) is a major public health problem, with key pieces of information needed for developing preventive and curative measures still missing. For example, the participation of the chaperone system (CS) in carcinogenesis and anti-cancer responses is poorly understood, although it can be predicted to be a crucial factor in these mechanisms. The chief components of the CS are the molecular chaperones, and here we discuss four of them, Hsp27, Hsp60, Hsp70, and Hsp90, focusing on their pro-carcinogenic roles in BC and potential for developing anti-BC therapies. These chaperones can be targets of negative chaperonotherapy, namely the elimination/blocking/inhibition of the chaperone(s) functioning in favor of BC, using, for instance, Hsp inhibitors. The chaperones can also be employed in immunotherapy against BC as adjuvants, together with BC antigens. Extracellular vesicles (EVs) in BC diagnosis and management are also briefly discussed, considering their potential as easily accessible carriers of biomarkers and as shippers of anti-cancer agents amenable to manipulation and controlled delivery. The data surveyed from many laboratories reveal that, to enhance the understanding of the role of the CS in BS pathogenesis, one must consider the CS as a physiological system, encompassing diverse members throughout the body and interacting with the ubiquitin–proteasome system, the chaperone-mediated autophagy machinery, and the immune system (IS). An integrated view of the CS, including its functional partners and considering its highly dynamic nature with EVs transporting CS components to reach all the cell compartments in which they are needed, opens as yet unexplored pathways leading to carcinogenesis that are amenable to interference by anti-cancer treatments centered on CS components, such as the molecular chaperones.     

Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay �C Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90αN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90αN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding.     

Hsp90 Activity Is Necessary for the Maturation of Rabies Virus Polymerase

Mononegavirales is an order of viruses with a genome in the form of a non-segmented negative-strand RNA that encodes several proteins. The functional polymerase complex of these viruses is composed of two proteins: a large protein (L) and a phosphoprotein (P). The replication of viruses from this order depends on Hsp90 chaperone activity. Previous studies have demonstrated that Hsp90 inhibition results in the degradation of mononegaviruses L protein, with exception of the rabies virus, for which the degradation of P protein was observed. Here, we demonstrated that Hsp90 inhibition does not affect the expression of rabies L and P proteins, but it inhibits binding of the P protein and L protein into functional viral polymerase. Rabies and the vesicular stomatitis virus, but not the measles virus, L proteins can be expressed independently of the presence of a P protein and in the presence of an Hsp90 inhibitor. Our results suggest that the interaction of L proteins with P proteins and Hsp90 in the process of polymerase maturation may be a process specific to particular viruses.     

Structural Determination of Three Different Series of Compounds as Hsp90 Inhibitors Using 3D-QSAR Modeling, Molecular Docking and Molecular Dynamics Methods

Hsp90 is involved in correcting, folding, maturation and activation of a diverse array of client proteins; it has also been implicated in the treatment of cancer in recent years. In this work, comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), molecular docking and molecular dynamics were performed on three different series of Hsp90 inhibitors to build 3D-QSAR models, which were based on the ligand-based or receptor-based methods. The optimum 3D-QSAR models exhibited reasonable statistical characteristics with averaging internal q(2) > 0.60 and external r(2) (pred) > 0.66 for Benzamide tetrahydro-4H-carbazol-4-one analogs (BT), AT13387 derivatives (AT) and Dihydroxylphenyl amides (DA). The results revealed that steric effects contributed the most to the BT model, whereas H-bonding was more important to AT, and electrostatic, hydrophobic, H-bond donor almost contributed equally to the DA model. The docking analysis showed that Asp93, Tyr139 and Thr184 in Hsp90 are important for the three series of inhibitors. Molecular dynamics simulation (MD) further indicated that the conformation derived from docking is basically consistent with the average structure extracted from MD simulation. These results not only lead to a better understanding of interactions between these inhibitors and Hsp90 receptor but also provide useful information for the design of new inhibitors with a specific activity.     

Discovery and biological activity of 6BrCaQ as an inhibitor of the Hsp90 protein folding machinery

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy, due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and viability. Here, a novel series of Hsp90 inhibitors containing a quinolein‐2‐one scaffold was synthesized and evaluated in cell proliferation assays. Results from these structure–activity relationships studies enabled identification of the simplified 3‐aminoquinolein‐2‐one analogue 2 b (6BrCaQ), which manifests micromolar activity against a panel of cancer cell lines. The molecular signature of Hsp90 inhibition was assessed by depletion of standard known Hsp90 client proteins. Finally, processing and activation of caspases 7, 8, and 9, and the subsequent cleavage of PARP by 6BrCaQ, suggest stimulation of apoptosis through both extrinsic and intrinsic pathways.     

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9b_S53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b–TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.     

Design,Synthesis, and Characterization of Macrocyclic Inhibitors of the Proprotein Convertase Furin

The activation of viral glycoproteins by the host protease furin is an essential step in the replication of numerous pathogenic viruses. Thus, effective inhibitors of furin could serve as broad-spectrum antiviral drugs. A crystal structure of an inhibitory hexapeptide derivative in complex with furin served as template for the rational design of various types of new cyclic inhibitors. Most of the prepared derivatives are relatively potent furin inhibitors with inhibition constants in the low nanomolar or even sub-nanomolar range. For seven derivatives the crystal structures in complex with furin could be determined. In three complexes, electron density was found for the entire inhibitor. In the other cases the structures could be determined only for the P6/P5-P1 segments, which directly interact with furin. The cyclic derivatives together with two non-cyclic reference compounds were tested as inhibitors of the proteolytic activation and replication of respiratory syncytial virus in cells. Significant antiviral activity was found for both linear reference inhibitors, whereas a negligible efficacy was determined for the cyclic derivatives.     

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys¹⁸ in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg¹⁷ was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys¹⁸ does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.     

Comparative Binding Energy (COMBINE) Analysis for Understanding the Binding Determinants of Type II Dehydroquinase Inhibitors

Herein we report comparative binding energy (COMBINE) analyses to derive quantitative structure–activity relationship (QSAR) models that help rationalize the determinants of binding affinity for inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway. Independent COMBINE models were derived for Helicobacter pylori and Mycobacterium tuberculosis DHQ2, which is an essential enzyme in both these pathogenic bacteria that has no counterpart in human cells. These studies quantify the importance of the hydrogen bonding interactions between the ligands and the water molecule involved in the DHQ2 reaction mechanism. They also highlight important differences in the ligand interactions with the interface pocket close to the active site that could provide guides for future inhibitor design.     

Insights to the Structural Basis for the Stereospecificity of the Escherichia coli Phytase,AppA

AppA, the Escherichia coli periplasmic phytase of clade 2 of the histidine phosphatase (HP2) family, has been well-characterized and successfully engineered for use as an animal feed supplement. AppA is a 1D-6-phytase and highly stereospecific but transiently accumulates 1D-myo-Ins(2,3,4,5)P₄ and other lower phosphorylated intermediates. If this bottleneck in liberation of orthophosphate is to be obviated through protein engineering, an explanation of its rather rigid preference for the initial site and subsequent cleavage of phytic acid is required. To help explain this behaviour, the role of the catalytic proton donor residue in determining AppA stereospecificity was investigated. Four variants were generated by site-directed mutagenesis of the active site HDT amino acid sequence motif containing the catalytic proton donor, D304. The identity and position of the prospective proton donor residue was found to strongly influence stereospecificity. While the wild-type enzyme has a strong preference for 1D-6-phytase activity, a marked reduction in stereospecificity was observed for a D304E variant, while a proton donor-less mutant (D304A) displayed exclusive 1D-1/3-phytase activity. High-resolution X-ray crystal structures of complexes of the mutants with a non-hydrolysable substrate analogue inhibitor point to a crucial role played by D304 in stereospecificity by influencing the size and polarity of specificity pockets A and B. Taken together, these results provide the first evidence for the involvement of the proton donor residue in determining the stereospecificity of HP2 phytases and prepares the ground for structure-informed engineering studies targeting the production of animal feed enzymes capable of the efficient and complete dephosphorylation of dietary phytic acid.     

TAS-116, a Well-Tolerated Hsp90 Inhibitor,Prevents the Activation of the NLRP3 Inflammasome in Human Retinal Pigment Epithelial Cells

Chronic inflammation has been associated with several chronic diseases, such as age-related macular degeneration (AMD). The NLRP3 inflammasome is a central proinflammatory signaling complex that triggers caspase-1 activation leading to the maturation of IL-1β. We have previously shown that the inhibition of the chaperone protein, Hsp90, prevents NLRP3 activation in human retinal pigment epithelial (RPE) cells; these are cells which play a central role in the pathogenesis of AMD. In that study, we used a well-known Hsp90 inhibitor geldanamycin, but it cannot be used as a therapy due to its adverse effects, including ocular toxicity. Here, we have tested the effects of a novel Hsp90 inhibitor, TAS-116, on NLRP3 activation using geldanamycin as a reference compound. Using our existing protocol, inflammasome activation was induced in IL-1α-primed ARPE-19 cells with the proteasome and autophagy inhibitors MG-132 and bafilomycin A1, respectively. Intracellular caspase-1 activity was determined using a commercial caspase-1 activity kit and the FLICA assay. The levels of IL-1β were measured from cell culture medium samples by ELISA. Cell viability was monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test and lactate dehydrogenase (LDH) measurements. Our findings show that TAS-116 could prevent the activation of caspase-1, subsequently reducing the release of mature IL-1β. TAS-116 has a better in vitro therapeutic index than geldanamycin. In summary, TAS-116 appears to be a well-tolerated Hsp90 inhibitor, with the capability to prevent the activation of the NLRP3 inflammasome in human RPE cells.     

Beyond Heparinization: Design of Highly Potent Thrombin Inhibitors Suitable for Surface Coupling

During extracorporeal circulation, when blood comes in contact with artificial surfaces, patients receive a standard treatment with anticoagulants to avoid blood coagulation. Dialysis patients in particular are systemically treated with heparin up to four times a week, causing a high burden for the body. For potential anticoagulant modification of external materials, such as dialysis equipment, a series of highly potent thrombin inhibitors was developed. All inhibitors share the general formula arylsulfonyl‐P3‐Pro‐4‐amidinobenzylamide, where P3 is glycyl or a trifunctional amino acid residue in L ‐configuration. Among this series, several derivatives inhibit thrombin with K_i values of less than 1 nM . Specificity measurements revealed that this inhibitor type is highly specific for thrombin with negligible activity against related trypsin‐like serine proteases. X‐ray analysis of the most potent analogue in complex with thrombin demonstrated that the N‐terminal arylsulfonyl group occupies the aryl binding site, whereas the P3 side chain is directed into the solvent and therefore is well suited for further coupling. Based on their in vitro profile, these inhibitors are suitable candidates for the development of hemocompatible materials with anticoagulant properties.     

Structural Analysis of Small‐Molecule Binding to the BAZ2A and BAZ2B Bromodomains

The bromodomain‐containing protein BAZ2A is a validated target in prostate cancer research, whereas the function of its paralogue BAZ2B is still undefined. The bromodomains of BAZ2A and BAZ2B have a similar binding site for their natural ligand, the acetylated lysine side chain. Here, we present an analysis of the binding modes of eight compounds belonging to three distinct chemical classes. For all compounds, the moiety mimicking the natural ligand engages in essentially identical interactions in the BAZ2A and BAZ2B bromodomains. In contrast, the rest of the molecule is partially solvent‐exposed and adopts different orientations with different interactions in the two bromodomains. Some of these differences could be exploited for designing inhibitors with selectivity within the BAZ2 bromodomain subfamily.     

Further Insight into Crystal Structures of Escherichia coli IspH/LytB in Complex with Two Potent Inhibitors of the MEP Pathway: A Starting Point for Rational Design of New Antimicrobials

IspH, also called LytB, a protein involved in the biosynthesis of isoprenoids through the methylerythritol phosphate pathway, is an attractive target for the development of new antimicrobial drugs. Here, we report crystal structures of Escherichia coli IspH in complex with the two most potent inhibitors: (E)-4-mercapto-3-methylbut-2-en-1-yl diphosphate (TMBPP) and (E)-4-amino-3-methylbut-2-en-1-yl diphosphate (AMBPP) at 1.95 and 1.7 Å resolution, respectively. The structure of the E. coli IspH:TMBPP complex exhibited two conformers of the inhibitor. This unexpected feature was exploited to design and evolve new antimicrobial candidates in silico.     

Probing the S1′ Site for the Identification of Non‐Zinc‐Binding MMP‐2 Inhibitors

Matrix metalloproteinases (MMPs) are zinc‐dependent enzymes involved in several pathological states. Among them, MMP‐2 is a relevant therapeutic target because of its role in cancer development and progression. Many MMP inhibitors (MMPIs) have been discovered over the last 30 years, and the majority of them contain a functional group that binds the zinc ion (zinc‐binding group; ZBG). Unfortunately, no MMPIs have reached the market yet, owing to toxic effects due to unselective interactions of the ZBG. The new generation of MMPIs that do not bind the zinc ion could overcome problems of selectivity and toxicity, but have so far been developed only for MMP‐8, ‐12, and ‐13. In this work, a virtual screening protocol was established by combining ligand‐ and structure‐based methods to identify non‐zinc‐binding MMP‐2 inhibitors using a new‐generation MMP‐8 inhibitor as a probe to find unexplored interactions in the MMP‐2 S1′ site. The screening allowed the identification of micromolar MMP‐2 inhibitors that putatively avoid binding the zinc ion, as demonstrated by docking calculations. The LIA model, built to correlate predicted and experimental binding energies of the identified non‐zinc‐binding MMP‐2 hits, underpins the reliability of the predicted docking poses.