Linkers as Game-changers in PROTAC Technology: Emphasizing General Trends in PROTAC Pharmacokinetics for their Rational Design

Proteolysis Targeting Chimeras (PROTACs) are heterobifunctional molecules that act as degraders. They selectively remove disease-associated proteins by hijacking the Ubiquitin-Proteasome System (UPS). Chemically, they consist of three parts: an E3 ligase ligand, a target of interest (TOI) ligand, and a linker, which connects the two moieties. The rapid expansion of PROTAC Technology as an innovative therapeutic modality in cancer fostered the drug discovery effort to optimize their physicochemical properties. Due to their large size, their features are far from the traditional ‘drug-like’ properties. This short review highlights some of the structural modifications in the linker component to optimize the PROTAC Drug Metabolism and Pharmacokinetics (DMPK) profile. In particular, we discussed aspects related to solubility, cell permeability, active transporters efflux and, metabolic stability

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer's disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positiv

Breaking the Aggregation of the Monoclonal Antibody Bevacizumab (Avastin®) by Dexamethasone Phosphate: Insights from Molecular Modelling and Asymmetrical Flow Field-Flow Fractionation

ABSTRACT: Purpose: To investigate the mechanism behind the aggregation breaking properties of dexamethasone phosphate and related corticosteroids on the IgG1 antibody bevacizumab (Avastin®). Methods: An in silico 3D dimer model is developed to identify the bevacizumab-bevacizumab interface, and different corticosteroids are docked onto the model to distinguish preferred binding sites. In silico predictions are validated by in vitro stability studies, where the antibody is stressed in presence or absence of each corticosteroid and formed aggregates are quantified by asymmetrical flow field-flow fractionation. Results: The dimer model features one close crystal contact area: Lys445 on the Fc region interacts with one Fab arm of the second bevacizumab. Docking reveals an interaction between the phosphate group of dexamethasone phosphate and Lys445, while the rest of the molecule is hindering dimer formation. Predictions are confirmed in vitro, demonstrating that dexamethasone phosphate and betamethasone phosphate partly prevent antibody aggregation, whereas triamcinolone acetonide phosphate does not. Conclusions: Results suggest that bevacizumab monomers follow a specific mechanism to form dimers in which a protein-protein interaction hotspot can be distinguished. The dimer formation can be hindered by corticosteroids in a specific way. This approach allows a simple way to stabilize IgG1 antibodie

The rational of catalytic activity of herpes simplex virus thymidine kinase. a combined biochemical and quantum chemical study.

Most antiherpes therapies exploit the large substrate acceptance of herpes simplex virus type 1 thymidine kinase (TK(HSV1)) relative to the human isoenzyme. The enzyme selectively phosphorylates nucleoside analogs that can either inhibit viral DNA polymerase or cause toxic effects when incorporated into viral DNA. To relate structural properties of TK(HSV1) ligands to their chemical reactivity we have carried out ab initio quantum chemistry calculations within the density functional theory framework in combination with biochemical studies. Calculations have focused on a set of ligands carrying a representative set of the large spectrum of sugar-mimicking moieties and for which structural information of the TK(HSV1)-ligand complex is available. The k(cat) values of these ligands have been measured under the same experimental conditions using an UV spectrophotometric assay. The calculations point to the crucial role of electric dipole moment of ligands and its interaction with the negatively charged residue Glu(225). A striking correlation is found between the energetics associated with this interaction and the k(cat) values measured under homogeneous conditions. This finding uncovers a fundamental aspect of the mechanism governing substrate diversity and catalytic turnover and thus represents a significant step toward the rational design of novel and powerful prodrugs for antiviral and TK(HSV1)-linked suicide gene therapies

Substrate diversity of herpes simplex virus thymidine kinase. Impact Of the kinematics of the enzyme.

Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches

Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation

In the resolution of inflammatory responses, neutrophils rapidly undergo apoptosis. We describe a new proapoptotic pathway in which cathepsin D directly activates caspase-8. Cathepsin D is released from azurophilic granules in neutrophils in a caspase-independent but reactive oxygen species–dependent manner. Under inflammatory conditions, the translocation of cathepsin D in the cytosol is blocked. Pharmacological or genetic inhibition of cathepsin D resulted in delayed caspase activation and reduced neutrophil apoptosis. Cathepsin D deficiency or lack of its translocation in the cytosol prolongs innate immune responses in experimental bacterial infection and in septic shock. Thus, we identified a new function of azurophilic granules that is in addition to their role in bacterial defense mechanisms: to regulate the life span of neutrophils and, therefore, the duration of innate immune responses through the release of cathepsin D

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer's disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positiv

Establishment and Validation of Whole-Cell Based Fluorescence Assays to Identify Anti-Mycobacterial Compounds Using the Acanthamoeba castellanii - Mycobacterium marinum Host-Pathogen System

Tuberculosis is considered to be one of the world's deadliest disease with 2 million deaths each year. The need for new antitubercular drugs is further exacerbated by the emergence of drug-resistance strains. Despite multiple recent efforts, the majority of the hits discovered by traditional target-based screening showed low efficiency in vivo. Therefore, there is heightened demand for whole-cell based approaches directly using host-pathogen systems. The phenotypic host-pathogen assay described here is based on the monitoring of GFP-expressing Mycobacterium marinum during infection of the amoeba Acanthamoeba castellanii. The assay showed straight-forward medium-throughput scalability, robustness and ease of manipulation, demonstrating its qualities as an efficient compound screening system. Validation with a series of known antitubercular compounds highlighted the advantages of the assay in comparison to previously published macrophage-Mycobacterium tuberculosis-based screening systems. Combination with secondary growth assays based on either GFP-expressing D. discoideum or M. marinum allowed us to further fine-tune compound characterization by distinguishing and quantifying growth inhibition, cytotoxic properties and antibiotic activities of the compounds. The simple and relatively low cost system described here is most suitable to detect anti-infective compounds, whether they present antibiotic activities or not, in which case they might exert anti-virulence or host defense boosting activities, both of which are largely overlooked by classical screening approaches

CEMIP ( HYBID , KIAA1199 ): structure, function and expression in health and disease

CEMIP (cell migration-inducing protein), also known as KIAA1199 or HYBID, is a protein involved in the depolymerisation of hyaluronic acid (HA), a major glycosaminoglycan component of the extracellular matrix. CEMIP was originally described in patients affected by nonsyndromic hearing loss and has subsequently been shown to play a key role in tumour initiation and progression, as well as arthritis, atherosclerosis and idiopathic pulmonary fibrosis. Despite the vast literature associating CEMIP with these diseases, its biology remains elusive. The present review article summarises all the major scientific evidence regarding its structure, function, role and expression, and attempts to cast light on a protein that modulates EMT, fibrosis and tissue inflammation, an unmet key aspect in several inflammatory disease conditions

5-ethyl-2'-deoxyuridine fragilizes Klebsiella pneumoniae outer wall and facilitates intracellular killing by phagocytic cells

Klebsiella pneumoniae is the causative agent of a variety of severe infections. Many K. pneumoniae strains are resistant to multiple antibiotics, and this situation creates a need for new antibacterial molecules. K. pneumoniae pathogenicity relies largely on its ability to escape phagocytosis and intracellular killing by phagocytic cells. Interfering with these escape mechanisms may allow to decrease bacterial virulence and to combat infections. In this study, we used Dictyostelium discoideum as a model phagocyte to screen a collection of 1,099 chemical compounds. Phg1A KO D. discoideum cells cannot feed upon K. pneumoniae bacteria, unless bacteria bear mutations decreasing their virulence. We identified 3 non-antibiotic compounds that restored growth of phg1A KO cells on K. pneumoniae, and we characterized the mode of action of one of them, 5-ethyl-2'-deoxyuridine (K2). K2-treated bacteria were more rapidly killed in D. discoideum phagosomes than non-treated bacteria. They were more sensitive to polymyxin and their outer membrane was more accessible to a hydrophobic fluorescent probe. These results suggest that K2 acts by rendering the membrane of K. pneumoniae accessible to antibacterial effectors. K2 was effective on three different K. pneumoniae strains, and acted at concentrations as low as 3 μM. K2 has previously been used to treat viral infections but its precise molecular mechanism of action in K. pneumoniae remains to be determined