ABSTRACT
Structural optimization of a previously reported agonist of ĀµOR, PZM21 is described resulting in the discovery of a novel series of amides with at least 4-folds enhanced CNS penetration in rat. Furthermore, these efforts yielded compounds with varying levels of efficacy on the receptor ranging from high efficacy agonists such as compound 20 to antagonists, such as 24. The correlation between in vitro activation of ĀµOR and relative activity in models of analgesia for these compounds is discussed. The compelling results obtained in these studies demonstrate the potential utility of these newly discovered compounds in the treatment of pain and opioid use disorder.
Subject(s)
Opioid-Related Disorders , Pain , Rats , Animals , Pain/drug therapy , Amides , Brain/metabolism , Receptors, Opioid, mu/agonists , Analgesics, Opioid/pharmacology , Analgesics, Opioid/therapeutic useABSTRACT
The bromodomain and extra-terminal (BET) family of proteins, consisting of the bromodomains containing protein 2 (BRD2), BRD3, BRD4, and the testis-specific BRDT, are key epigenetic regulators of gene transcription and has emerged as an attractive target for anticancer therapy. Herein, we describe the discovery of a novel potent BET bromodomain inhibitor, using a systematic structure-based approach focused on improving potency, metabolic stability, and permeability. The optimized dimethylisoxazole aryl-benzimidazole inhibitor exhibited high potency towards BRD4 and related BET proteins in biochemical and cell-based assays and inhibited tumor growth in two proof-of-concept preclinical animal models.
Subject(s)
Benzimidazoles/pharmacology , Drug Discovery , Isoxazoles/pharmacology , Multiple Myeloma/drug therapy , Transcription Factors/antagonists & inhibitors , Administration, Oral , Animals , Benzimidazoles/chemistry , Benzimidazoles/metabolism , Biological Availability , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Humans , Isoxazoles/administration & dosage , Isoxazoles/chemistry , Isoxazoles/metabolism , Mice , Molecular Structure , Multiple Myeloma/metabolism , Multiple Myeloma/pathology , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Protein Domains/drug effects , Structure-Activity Relationship , Transcription Factors/metabolismABSTRACT
Bromodomain and extraterminal domain protein inhibitors (BETi) hold great promise as a novel class of cancer therapeutics. Because acquired resistance typically limits durable responses to targeted therapies, it is important to understand mechanisms by which tumor cells adapt to BETi. Here, through pooled shRNA screening of colorectal cancer cells, we identified tripartite motif-containing protein 33 (TRIM33) as a factor promoting sensitivity to BETi. We demonstrate that loss of TRIM33 reprograms cancer cells to a more resistant state through at least two mechanisms. TRIM33 silencing attenuates down-regulation of MYC in response to BETi. Moreover, loss of TRIM33 enhances TGF-Ć receptor expression and signaling, and blocking TGF-Ć receptor activity potentiates the antiproliferative effect of BETi. These results describe a mechanism for BETi resistance and suggest that combining inhibition of TGF-Ć signaling with BET bromodomain inhibition may offer new therapeutic benefits.
Subject(s)
Azepines/pharmacology , Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-myc/metabolism , Transcription Factors/metabolism , Transforming Growth Factor beta/metabolism , Triazoles/pharmacology , Azepines/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Proliferation/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Drug Resistance/genetics , Gene Expression Regulation, Neoplastic/drug effects , HCT116 Cells , HEK293 Cells , Humans , Molecular Structure , Proteins/metabolism , Proto-Oncogene Proteins c-myc/genetics , RNA Interference , Receptors, Transforming Growth Factor beta/genetics , Receptors, Transforming Growth Factor beta/metabolism , Signal Transduction/drug effects , Signal Transduction/genetics , Transcription Factors/genetics , Transforming Growth Factor beta/genetics , Triazoles/chemistryABSTRACT
GS-5806 is a small-molecule inhibitor of human respiratory syncytial virus fusion protein-mediated viral entry. During viral entry, the fusion protein undergoes major conformational changes, resulting in fusion of the viral envelope with the host cell membrane. This process is reproduced in vitro using a purified, truncated respiratory syncytial virus (RSV) fusion protein. GS-5806 blocked these conformational changes, suggesting a possible mechanism for antiviral activity.
Subject(s)
Antiviral Agents/pharmacology , Pyrazoles/pharmacology , Respiratory Syncytial Virus, Human/drug effects , Respiratory Syncytial Virus, Human/metabolism , Sulfonamides/pharmacology , Viral Proteins/chemistry , Viral Proteins/metabolism , Indazoles , Protein Conformation , Respiratory Syncytial Virus InfectionsABSTRACT
Dilated cardiomyopathy (DCM) is characterized by reduced cardiac output, as well as thinning and enlargement of left ventricular chambers. These characteristics eventually lead to heart failure. Current standards of care do not target the underlying molecular mechanisms associated with genetic forms of heart failure, driving a need to develop novel therapeutics for DCM. To identify candidate therapeutics, we developed an in vitro DCM model using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) deficient in B-cell lymphoma 2 (BCL2)-associated athanogene 3 (BAG3). With these BAG3-deficient iPSC-CMs, we identified cardioprotective drugs using a phenotypic screen and deep learning. From a library of 5500 bioactive compounds and siRNA validation, we found that inhibiting histone deacetylase 6 (HDAC6) was cardioprotective at the sarcomere level. We translated this finding to a BAG3 cardiomyocyte-knockout (BAG3cKO) mouse model of DCM, showing that inhibiting HDAC6 with two isoform-selective inhibitors (tubastatin A and a novel inhibitor TYA-018) protected heart function. In BAG3cKO and BAG3E455K mice, HDAC6 inhibitors improved left ventricular ejection fraction and reduced left ventricular diameter at diastole and systole. In BAG3cKO mice, TYA-018 protected against sarcomere damage and reduced Nppb expression. Based on integrated transcriptomics and proteomics and mitochondrial function analysis, TYA-018 also enhanced energetics in these mice by increasing expression of targets associated with fatty acid metabolism, protein metabolism, and oxidative phosphorylation. Our results demonstrate the power of combining iPSC-CMs with phenotypic screening and deep learning to accelerate drug discovery, and they support developing novel therapies that address underlying mechanisms associated with heart disease.
Subject(s)
Cardiomyopathy, Dilated , Deep Learning , Heart Failure , Adaptor Proteins, Signal Transducing/metabolism , Animals , Apoptosis Regulatory Proteins/metabolism , Cardiomyopathy, Dilated/diagnosis , Cardiomyopathy, Dilated/drug therapy , Cardiomyopathy, Dilated/genetics , Disease Models, Animal , Heart Failure/metabolism , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Mice , Myocytes, Cardiac/metabolism , Stroke Volume , Ventricular Function, LeftABSTRACT
A diphosphate of a novel cyclopentyl based nucleoside phosphonate with potent inhibition of HIV reverse transcriptase (RT) (20, IC(50)=0.13 microM) has been discovered. In cell culture the parent phosphonate diacid 9 demonstrated antiviral activity EC(50)=16 microM, within two-fold of GS-9148, a prodrug of which is currently under clinical investigation, and within 5-fold of tenofovir (PMPA). In vitro cellular metabolism studies using 9 confirmed that the active diphosphate metabolite is produced albeit at a lower efficiency relative to GS-9148.
Subject(s)
Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Nucleosides/chemical synthesis , Organophosphonates/chemical synthesis , Reverse Transcriptase Inhibitors/chemical synthesis , Cell Line, Tumor , Drug Design , HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , Humans , Models, Molecular , Molecular Structure , Nucleosides/chemistry , Nucleosides/pharmacology , Organophosphonates/chemistry , Organophosphonates/pharmacology , Prodrugs/chemical synthesis , Prodrugs/pharmacokinetics , Prodrugs/pharmacology , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/therapeutic use , Structure-Activity RelationshipABSTRACT
Cyclophilins are a family of peptidyl-prolyl isomerases that are implicated in a wide range of diseases including hepatitis C. Our aim was to discover through total synthesis an orally bioavailable, non-immunosuppressive cyclophilin (Cyp) inhibitor with potent anti-hepatitis C virus (HCV) activity that could serve as part of an all oral antiviral combination therapy. An initial lead 2 derived from the sanglifehrin A macrocycle was optimized using structure based design to produce a potent and orally bioavailable inhibitor 3. The macrocycle ring size was reduced by one atom, and an internal hydrogen bond drove improved permeability and drug-like properties. 3 demonstrates potent Cyp inhibition ( Kd = 5 nM), potent anti-HCV 2a activity (EC50 = 98 nM), and high oral bioavailability in rat (100%) and dog (55%). The synthetic accessibility and properties of 3 support its potential as an anti-HCV agent and for interrogating the role of Cyp inhibition in a variety of diseases.
Subject(s)
Cyclophilins/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/pharmacokinetics , Administration, Oral , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Antiviral Agents/pharmacokinetics , Antiviral Agents/pharmacology , Biological Availability , Cell Line , Cyclophilins/chemistry , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Hepacivirus/drug effects , Lactones/administration & dosage , Lactones/chemistry , Lactones/pharmacokinetics , Lactones/pharmacology , Models, Molecular , Protein Conformation , Spiro Compounds/administration & dosage , Spiro Compounds/chemistry , Spiro Compounds/pharmacokinetics , Spiro Compounds/pharmacologyABSTRACT
Hepsin is an integral membrane protein that may participate in cell growth and in maintaining proper cell morphology and is overexpressed in a number of primary tumors. We have determined the 1.75 A resolution structure of the extracellular component of human hepsin. This structure includes a 255-residue trypsin-like serine protease domain and a 109-residue region that forms a novel, poorly conserved, scavenger receptor cysteine-rich (SRCR) domain. The two domains are associated with each other through a single disulfide bond and an extensive network of noncovalent interactions. The structure suggests how the extracellular region of hepsin may be positioned with respect to the plasma membrane.
Subject(s)
Extracellular Space/chemistry , Receptors, Immunologic/chemistry , Serine Endopeptidases/chemistry , Amino Acid Sequence , Cell Membrane/chemistry , Humans , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , Protein Structure, Tertiary , Receptors, Scavenger , Sequence AlignmentABSTRACT
GS-5806 is a novel, orally bioavailable RSV fusion inhibitor discovered following a lead optimization campaign on a screening hit. The oral absorption properties were optimized by converting to the pyrazolo[1,5-a]-pyrimidine heterocycle, while potency, metabolic, and physicochemical properties were optimized by introducing the para-chloro and aminopyrrolidine groups. A mean EC50 = 0.43 nM was found toward a panel of 75 RSV A and B clinical isolates and dose-dependent antiviral efficacy in the cotton rat model of RSV infection. Oral bioavailability in preclinical species ranged from 46 to 100%, with evidence of efficient penetration into lung tissue. In healthy human volunteers experimentally infected with RSV, a potent antiviral effect was observed with a mean 4.2 log10 reduction in peak viral load and a significant reduction in disease severity compared to placebo. In conclusion, a potent, once daily, oral RSV fusion inhibitor with the potential to treat RSV infection in infants and adults is reported.
Subject(s)
Antiviral Agents/pharmacology , Drug Discovery , Pyrazoles/pharmacology , Respiratory Syncytial Virus Infections/prevention & control , Respiratory Syncytial Virus Infections/virology , Respiratory Syncytial Viruses/drug effects , Sulfonamides/pharmacology , Virus Internalization/drug effects , Administration, Oral , Animals , Antiviral Agents/administration & dosage , Antiviral Agents/chemistry , Dogs , Dose-Response Relationship, Drug , Humans , Indazoles , Macaca fascicularis , Microbial Sensitivity Tests , Molecular Structure , Pyrazoles/administration & dosage , Pyrazoles/chemistry , Rats , Respiratory Syncytial Viruses/physiology , Structure-Activity Relationship , Sulfonamides/administration & dosage , Sulfonamides/chemistryABSTRACT
We report the discovery of 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(4-methylphenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (2a) as an inducer of apoptosis using our proprietary cell- and caspase-based HTS assay. Through structure activity relationship (SAR) studies, 5-(4-hydroxy-6-methyl-2-oxo-2H-pyran-3-yl)-7-(2-methoxy-4-(methylthio)phenyl)-(E)-2,3,6,7-tetrahydro-1,4-thiazepine (5d) was identified as a potent apoptosis inducer with an EC(50) value of 0.08 microM in T47D cells, which was >15-fold more potent than screening hit 2a. Compound 5d also was found to be highly active in a growth inhibition assay with a GI(50) value of 0.05 microM in T47D cells and to function as an inhibitor of tubulin polymerization.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis , Caspases/metabolism , Chemistry, Pharmaceutical/methods , Neoplasms/drug therapy , Thiazepines/chemistry , Cell Line, Tumor , Cell Proliferation , Drug Design , Drug Screening Assays, Antitumor , Humans , Models, Chemical , Structure-Activity Relationship , Thiazepines/pharmacology , Tubulin/chemistryABSTRACT
Using a scaleable, directed library approach based on orthogonally protected advanced intermediates, we have prepared a series of potent keto-1,2,4-oxadiazoles designed to explore the P(2) binding pocket of human mast cell tryptase, while building in a high degree of selectivity over human trypsin and other serine proteases.
Subject(s)
Enzyme Inhibitors/chemical synthesis , Mast Cells/drug effects , Oxadiazoles/chemical synthesis , Serine Endopeptidases/drug effects , Animals , Binding Sites/drug effects , Drug Design , Drug Evaluation, Preclinical , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Haplorhini , Humans , Mast Cells/enzymology , Mice , Molecular Structure , Oxadiazoles/chemistry , Oxadiazoles/pharmacology , Stereoisomerism , Structure-Activity Relationship , TryptasesABSTRACT
A series of novel alpha-keto-[1,2,4]-oxadiazoles has been synthesized as human tryptase inhibitors for evaluation as a new class of anti-asthmatic agent. The inhibitor design is focused on using a prime-side hydrophobic pocket and the S2 pocket of beta-tryptase to achieve inhibition potency and selectivity over other serine proteases.
Subject(s)
Oxazoles/pharmacology , Serine Endopeptidases/drug effects , Crystallography, X-Ray , Humans , Kinetics , Oxazoles/chemistry , TryptasesABSTRACT
The metabolites of the tryptase inhibitor CRA-9249 were identified after exposure to liver microsomes. CRA-9249 was found to be degraded rapidly in liver microsomes from rabbit, dog, cynomolgus monkey, and human, and less rapidly in microsomes from rat. The key metabolites included cleavage of an aryl ether, in addition to an unexpected hydroxylation of the amide side chain adjacent to the amide nitrogen. The chemical structures of both metabolites were confirmed by synthesis and comparison to material isolated from the liver microsomes. Several suspected hydroxylated metabolites were also synthesized and analyzed as part of the structure identification process.
Subject(s)
Benzimidazoles/metabolism , Enzyme Inhibitors/metabolism , Serine Endopeptidases/drug effects , Animals , Chromatography, High Pressure Liquid , Dogs , Hydroxylation , Macaca fascicularis , Magnetic Resonance Spectroscopy , Rats , TryptasesABSTRACT
Improved peptide-based inhibitors of human beta tryptase were discovered using information gleaned from tripeptide library screening and structure-guided design methods, including fragment screening. Our efforts sought to improve this class of inhibitors by replacing the traditional Lys or Arg P1 element. The optimized compounds display low nanomolar potency against the mast cell target and several hundred-fold selectivity with respect to serine protease off targets. Thus, replacement of Lys/Arg at P1 in a peptide-like scaffold does not need to be accompanied by a loss in target affinity.
Subject(s)
Serine Endopeptidases/drug effects , Serine Proteinase Inhibitors/chemistry , Crystallography, X-Ray , Models, Molecular , Protein Conformation , Serine Proteinase Inhibitors/pharmacology , TryptasesABSTRACT
The synthesis of novel [1,2,4]oxadiazoles and their structure-activity relationship (SAR) for the inhibition of tryptase and related serine proteases is presented. Elaboration of the P'-side afforded potent, selective, and orally bioavailable tryptase inhibitors.
Subject(s)
Enzyme Inhibitors/pharmacology , Serine Endopeptidases/drug effects , Administration, Oral , Biological Availability , Enzyme Inhibitors/administration & dosage , Enzyme Inhibitors/chemistry , Models, Molecular , Structure-Activity Relationship , TryptasesABSTRACT
As a continuation of our efforts to discover novel apoptosis inducers as anticancer agents using a cell-based caspase HTS assay, 2-phenyl-oxazole-4-carboxamide derivatives were identified. The structure-activity relationships of this class of molecules were explored. Compound 1k, with EC(50) of 270 nM and GI(50) of 229 nM in human colorectal DLD-1 cells, was selected and demonstrated the ability to cleave PARP and displayed DNA laddering, the hallmarks of apoptosis. Compound 1k showed 63% tumor growth inhibition in human colorectal DLD-1 xenograft mouse model at 50 mpk, bid.
Subject(s)
Amides/chemistry , Amides/pharmacology , Apoptosis/drug effects , Oxazoles/chemistry , Oxazoles/pharmacology , Amides/chemical synthesis , Animals , Cell Line, Tumor , Female , Humans , Mice , Molecular Structure , Oxazoles/chemical synthesis , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Screening of a diverse set of bisbenzimidazoles for inhibition of the hepatitis C virus (HCV) serine protease NS3/NS4A led to the identification of a potent Zn(2+)-dependent inhibitor (1). Optimization of this screening hit afforded a 10-fold more potent inhibitor (46) under Zn(2+) conditions (K(i)=27nM). This compound (46) binds also to NS3/NS4A in a Zn(2+) independent fashion (K(i)=1microM). The SAR of this class of compounds under Zn(2+) conditions is highly divergent compared to the SAR in the absence of Zn(2+), suggesting two distinct binding modes.