ABSTRACT
The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) posed a major threat to global health. Although the World Health Organization ended the public health emergency status, antiviral drugs are needed to address new variants of SARS-CoV-2 and future pandemics. To identify novel broad-spectrum coronavirus drugs, we developed a high-content imaging platform compatible with high-throughput screening. The platform is broadly applicable as it can be adapted to include various cell types, viruses, antibodies, and dyes. We demonstrated that the antiviral activity of compounds against SARS-CoV-2 variants (Omicron BA.5 and Omicron XBB.1.5), SARS-CoV, and human coronavirus 229E could easily be assessed. The inclusion of cellular dyes and immunostaining in combination with in-depth image analysis enabled us to identify compounds that induced undesirable phenotypes in host cells, such as changes in cell morphology or in lysosomal activity. With the platform, we screened â¼900K compounds and triaged hits, thereby identifying potential candidate compounds carrying broad-spectrum activity with limited off-target effects. The flexibility and early-stage identification of compounds with limited host cell effects provided by this high-content imaging platform can facilitate coronavirus drug discovery. We anticipate that its rapid deployability and fast turnaround can also be applied to combat future pandemics.
Subject(s)
Coronavirus Infections , Coronavirus , Humans , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , Coronavirus Infections/drug therapy , High-Throughput Screening Assays/methods , Coloring Agents/pharmacology , Coloring Agents/therapeutic use , PandemicsABSTRACT
The ongoing COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is partly under control by vaccination. However, highly potent and safe antiviral drugs for SARS-CoV-2 are still needed to avoid development of severe COVID-19. We report the discovery of a small molecule, Z-Tyr-Ala-CHN2, which was identified in a cell-based antiviral screen. The molecule exerts sub-micromolar antiviral activity against SARS-CoV-2, SARS-CoV-1, and human coronavirus 229E. Time-of-addition studies reveal that Z-Tyr-Ala-CHN2 acts at the early phase of the infection cycle, which is in line with the observation that the molecule inhibits cathepsin L. This results in antiviral activity against SARS-CoV-2 in VeroE6, A549-hACE2, and HeLa-hACE2 cells, but not in Caco-2 cells or primary human nasal epithelial cells since the latter two cell types also permit entry via transmembrane protease serine subtype 2 (TMPRSS2). Given their cell-specific activity, cathepsin L inhibitors still need to prove their value in the clinic; nevertheless, the activity profile of Z-Tyr-Ala-CHN2 makes it an interesting tool compound for studying the biology of coronavirus entry and replication.
ABSTRACT
We recently disclosed a set of heteroaryl-fused piperazine inhibitors of BACE1 that combined nanomolar potency with good intrinsic permeability and low Pgp-mediated efflux. Herein we describe further work on two prototypes of this family of inhibitors aimed at modulating their basicity and reducing binding to the human ether-a-go-go-related gene (hERG) channel. This effort has led to the identification of compound 36, a highly potent (hAß42 cell IC50 = 1.3 nM), cardiovascularly safe, and orally bioavailable compound that elicited sustained Aß42 reduction in mouse and dog animal models.
ABSTRACT
Despite several years of research, only a handful of ß-secretase (BACE) 1 inhibitors have entered clinical trials as potential therapeutics against Alzheimer's disease. The intrinsic basic nature of low molecular weight, amidine-containing BACE 1 inhibitors makes them far from optimal as central nervous system drugs. Herein we present a set of novel heteroaryl-fused piperazine amidine inhibitors designed to lower the basicity of the key, enzyme binding, amidine functionality. This study resulted in the identification of highly potent (IC50 ≤ 10 nM), permeable lead compounds with a reduced propensity to suffer from P-glycoprotein-mediated efflux.
ABSTRACT
In previous studies, the introduction of electron withdrawing groups to 1,4-oxazine BACE1 inhibitors reduced the p Ka of the amidine group, resulting in compound 2 that showed excellent in vivo efficacy, lowering Aß levels in brain and CSF. However, a suboptimal cardiovascular safety margin, based on QTc prolongation, prevented further progression. Further optimization resulted in the replacement of the 2-fluoro substituent by a CF3-group, which reduced hERG inhibition. This has led to compound 3, with an improved cardiovascular safety margin and sufficiently safe in GLP toxicity studies to progress into clinical trials.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Protease Inhibitors/chemistry , Protease Inhibitors/pharmacology , Administration, Intravenous , Administration, Oral , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/cerebrospinal fluid , Animals , Aspartic Acid Endopeptidases/metabolism , Biological Availability , Cardiovascular Diseases/chemically induced , Chemical and Drug Induced Liver Injury/etiology , Dogs , Drug Evaluation, Preclinical/methods , Drug Stability , ERG1 Potassium Channel/metabolism , Guinea Pigs , Humans , Male , Mice, Inbred Strains , Oxazines/chemistry , Peptide Fragments/cerebrospinal fluid , Protease Inhibitors/administration & dosage , Protease Inhibitors/adverse effects , Rats, Sprague-Dawley , Structure-Activity RelationshipABSTRACT
The metabotropic glutamate subtypeâ 2 (mGlu2 ) receptor is a presynaptic membrane receptor distributed widely in brain that provides feedback inhibitory control of glutamate release. Inhibition of the mGlu2 receptor function with a negative allosteric modulator (NAM) enhances activity-dependent glutamate release, which may be of therapeutic benefit for the treatment of neurological and psychiatric disorders. An attractive pyrazole hit was identified after a high-throughput screening (HTS) campaign. The evolution of this hit is described by structure-activity relationship (SAR) studies on specific parts of the molecule. From near micromolar potency we could obtain compounds with single-digit nanomolar activity in the mGlu2 NAM GTPγS assay. In addition to SAR on inâ vitro potency, a more detailed overview is given with a specific set of compounds on the excellent agreement between inâ vitro potency, free brain concentration, and exâ vivo mGlu2 receptor occupancy. Finally, to obtain improved drug-like compounds, plans for future research are suggested toward increasing free brain concentration while maintaining high inâ vitro potency.
Subject(s)
Pyrazoles/pharmacology , Receptors, Metabotropic Glutamate/chemistry , Receptors, Metabotropic Glutamate/metabolism , Allosteric Regulation/drug effects , Animals , Cell Line , Dose-Response Relationship, Drug , HEK293 Cells , Humans , Microsomes, Liver/chemistry , Microsomes, Liver/metabolism , Molecular Structure , Permeability/drug effects , Pyrazoles/chemistry , Rats , Structure-Activity RelationshipABSTRACT
1,4-Oxazines are presented, which show good in vitro inhibition in enzymatic and cellular BACE1 assays. We describe lead optimization focused on reducing the amidine pKa while optimizing interactions in the BACE1 active site. Our strategy permitted modulation of properties such as permeation and especially P-glycoprotein efflux. This led to compounds which were orally bioavailable, centrally active, and which demonstrated robust lowering of brain and CSF Aß levels, respectively, in mouse and dog models. The amyloid lowering potential of these molecules makes them valuable leads in the search for new BACE1 inhibitors for the treatment of Alzheimer's disease.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Brain/metabolism , Oxazines/chemical synthesis , Oxazines/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/cerebrospinal fluid , Animals , Biological Availability , Blood Proteins/metabolism , Blood-Brain Barrier , Cell Line, Tumor , Cytochrome P-450 Enzyme Inhibitors/chemical synthesis , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Dogs , Drug Design , Female , Humans , Male , Mice , Models, Molecular , Oxazines/pharmacokinetics , Protein BindingABSTRACT
The synthesis of new fluorinated pyrrolidones starting from unprotected amino esters and amino nitriles through a Michael addition-lactamization sequence is described. The resulting CF3 -containing building blocks, bearing a quaternary stereogenic center adjacent to the fluorinated group, have been converted into amino pyrrolidines that display potent ß-secretase 1 (BACE1) inhibitory activity. This work constitutes an example of selective fluorination as a valid strategy for the modulation of physicochemical and biological properties of lead compounds in drug discovery.
Subject(s)
Amidines/chemical synthesis , Amidines/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/chemistry , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/chemistry , Hydrocarbons, Fluorinated/chemistry , Hydrocarbons, Fluorinated/pharmacology , Pyrrolidinones/chemistry , Pyrrolidinones/pharmacology , Cyclization , Drug Discovery , Molecular Structure , StereoisomerismABSTRACT
We report the continuation of a focused medicinal chemistry program aimed to further optimize a series of imidazo[1,2-a]pyrazines as a novel class of potent and selective phosphodiesterase 10A (PDE10A) inhibitors. In vitro and in vivo pharmacokinetic and pharmacodynamic evaluation allowed the selection of compound 25a for its assessment in preclinical models of psychosis. The evolution of our medicinal chemistry program, structure-activity relationship (SAR) analysis, as well as a detailed pharmacological profile for optimized lead 25a are described.
Subject(s)
Phosphodiesterase Inhibitors/chemical synthesis , Phosphoric Diester Hydrolases , Administration, Oral , Animals , Biological Availability , Disease Models, Animal , Phosphodiesterase Inhibitors/pharmacology , Phosphodiesterase Inhibitors/therapeutic use , Rats , Rats, Wistar , Schizophrenia/drug therapy , Structure-Activity RelationshipABSTRACT
The synthesis of enantiomerically pure cis- and trans-2-phenyl-3-(trifluoromethyl)piperazines is described. It involved, as the key step, a diastereoselective nucleophilic addition of the Ruppert-Prakash reagent (TMSCF3) to α-amino sulfinylimines bearing Ellman's auxiliary. This methodology allows an entry into hitherto unknown trifluoromethylated and stereochemically defined piperazines, key scaffold components in medicinal chemistry.
Subject(s)
Hydrocarbons, Fluorinated/chemistry , Indicators and Reagents/chemistry , Piperazines/chemistry , Piperazines/chemical synthesis , Silanes/chemistry , Catalysis , Drug Discovery , Molecular Structure , StereoisomerismABSTRACT
The 1,2-addition of alkyl Grignard reagents to readily available N-tert-butanesulfinyl ketimines, bearing an α-silyloxy substituent, proceeds in high yields and excellent diastereocontrol. The utility of the present method was demonstrated by the synthesis, in enantiomerically pure form, of one recently disclosed ß-secretase (BACE1) inhibitor.
Subject(s)
Amino Alcohols/chemical synthesis , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Alzheimer Disease/drug therapy , Amino Alcohols/chemistry , Enzyme Inhibitors/chemistry , Humans , Imines/chemical synthesis , Imines/chemistry , Nitriles/chemical synthesis , Nitriles/chemistryABSTRACT
Polycationic oligo(chiral bicyclic guanidines) constitute useful non-peptidic penetrating agents for cell uptake and protein surface recognition. We report herein improved and selective procedures for the preparation of oligoguanidinium scaffolds linked through thioether bonds, with similar or different groups and functions at both ends of the chain. Two synthetic strategies were developed to obtain these compounds in relatively good yields from a common thioacetate precursor: generation of a disulfide intermediate or thiolate formation. Thus, tetraguanidinium intermediates 8 and 22 are best synthesized by the disulfide route, whereas hexamer 29, octamer 31, and trimer 37 arise from a combination of both the disulfide and the thioacetate routes. Finally, tetramer 28 can be readily obtained from either strategy.
Subject(s)
Bridged Bicyclo Compounds/chemistry , Bridged Bicyclo Compounds/metabolism , Guanidine/chemistry , Guanidine/metabolism , Bridged Bicyclo Compounds/chemical synthesis , Cell Membrane Permeability , Chemistry Techniques, Synthetic , Disulfides/chemical synthesis , Disulfides/chemistry , Guanidine/chemical synthesis , HeLa Cells , Humans , Polyamines/chemical synthesis , Polyamines/chemistry , Polyamines/metabolism , Polyelectrolytes , Sulfhydryl Compounds/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
Aminopiperazinone inhibitors of BACE were identified by rational design. Structure based design guided idea prioritization and initial racemic hit 18a showed good activity. Modification in decoration and chiral separation resulted in the 40 nM inhibitor, (-)-37, which showed in vivo reduction of amyloid beta peptides. The crystal structure of 18a showed a binding mode driven by interaction with the catalytic aspartate dyad and distribution of the biaryl amide decoration towards S1 and S3 pockets.
Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemical synthesis , Piperazines/chemistry , Amyloid Precursor Protein Secretases/metabolism , Binding Sites , Computer Simulation , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Piperazines/chemical synthesis , Piperazines/pharmacology , Protein Structure, Tertiary , StereoisomerismABSTRACT
Oligoguanidinium-based cell delivery systems have gained broad interest in the drug delivery field since one decade ago. Thus, arginine-containing peptides as Tat or Antp, oligoarginine peptides, and derived peptoids have been described as shuttles for delivering nonpermeant drugs inside cancer cells. Herein we report a new family of tetraguanidinium cell penetrating vectors efficiently internalized in human tumor cells. Their high internalization, studied by confocal microscopy and flow cytometry, as well as their specific accumulation in mitochondria makes these new vectors likely vehicles for the targeted delivery of anticancer drugs to mitochondria.
Subject(s)
Guanidine/pharmacokinetics , Mitochondria/metabolism , Nylons/pharmacokinetics , Amino Acid Sequence , Antennapedia Homeodomain Protein , Drug Delivery Systems , Flow Cytometry , Gene Products, tat/pharmacokinetics , Guanidine/pharmacology , HeLa Cells , Homeodomain Proteins/pharmacokinetics , Homeodomain Proteins/pharmacology , Humans , Microscopy, Confocal , Molecular Sequence Data , Nuclear Proteins/pharmacokinetics , Nuclear Proteins/pharmacology , Nylons/chemical synthesis , Nylons/pharmacology , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Peptide Fragments/pharmacokinetics , Peptide Fragments/pharmacology , Transcription Factors/pharmacokinetics , Transcription Factors/pharmacologyABSTRACT
Original inhibitors of HIV-1 protease based on a chiral bicyclic guanidinium scaffold linked to short peptidic mimics of the terminal protease sequences and to a lipophilic group were designed. These inhibitors prevent dimerization of the native protease by an interfacial structure at the highly conserved antiparallel beta-strand involving both the N and C termini that substantially account for dimerization. The preorganized guanidinium spacer introduces additional electrostatic hydrogen-bonding interactions with the C-terminal Phe-99 carboxylate. Lipophilic residues linked to side chains and the guanidinium scaffold are essential for dimerization inhibition as ascertained by Zhang kinetics (4, K(id) = 290 nM; 6 or 6', K(id) = 150 nM; 8, K(id) = 400 nM) combined with a circular dichroism study on the enzyme thermal stability. Remarkably, less hydrophobic compounds result in mixed dimerization (1a and 3) or active site inhibitors (5). Removal of the guanidinium hydrophobic groups leads to less active or inactive ligands.
Subject(s)
Bridged Bicyclo Compounds, Heterocyclic/chemistry , Guanidines/chemistry , HIV Protease Inhibitors/chemistry , HIV Protease/metabolism , Binding Sites , Circular Dichroism , Dimerization , Hydrophobic and Hydrophilic Interactions , Kinetics , Models, Molecular , Protein Structure, Secondary , Serine/chemistry , Stereoisomerism , Tyrosine/chemistryABSTRACT
A number of artificial carriers for the transport of zwitterionic aromatic amino acids across bulk model membranes (U-tube type) have been prepared and evaluated. 1,2-Dichloroethane and dichloromethane were employed in the organic phase. All compounds are based on a bicyclic chiral guanidinium scaffold that ideally complements the carboxylate function. The guanidinium central moiety was attached to crown ethers or lasalocid A as specific subunits for ammonium recognition as well as to aromatic or hydrophobic residues to evaluate their potential interaction with the side chains of the guest amino acids. The subunits were linked to the guanidinium through ester or amide connectors. Amides were found to be better carriers than esters, though less enantioselective. On the other hand, crown ethers were superior to lasalocid derivatives. As expected, transport rates were dependent on the carrier concentration in the liquid membrane. Reciprocally, enantioselectivities were much higher at lower carrier concentrations. The results show that our previously proposed three-point binding model (J. Am. Chem. Soc. 1992, 114, 1511-1512), involving the participation of the aromatic or hydrophobic residue to interact with the side chains of the amino acid guest, is unnecessary to explain the high enantioselectivities observed. Molecular dynamics fully support a two-point model involving only the guanidinium and crown ether moieties. These molecules constitute the first examples of chiral selectors for underivatized amino acids acting as carriers under neutral conditions.