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1.
J Med Chem ; 65(2): 1206-1224, 2022 01 27.
Article in English | MEDLINE | ID: mdl-34734694

ABSTRACT

Multiple Sclerosis is a chronic autoimmune neurodegenerative disorder of the central nervous system (CNS) that is characterized by inflammation, demyelination, and axonal injury leading to permeant disability. In the early stage of MS, inflammation is the primary driver of the disease progression. There remains an unmet need to develop high efficacy therapies with superior safety profiles to prevent the inflammation processes leading to disability. Herein, we describe the discovery of BIIB091, a structurally distinct orthosteric ATP competitive, reversible inhibitor that binds the BTK protein in a DFG-in confirmation designed to sequester Tyr-551, an important phosphorylation site on BTK, into an inactive conformation with excellent affinity. Preclinical studies demonstrated BIB091 to be a high potency molecule with good drug-like properties and a safety/tolerability profile suitable for clinical development as a highly selective, reversible BTKi for treating autoimmune diseases such as MS.


Subject(s)
Agammaglobulinaemia Tyrosine Kinase , Drug Discovery , Multiple Sclerosis , Protein Kinase Inhibitors , Animals , Male , Rats , Agammaglobulinaemia Tyrosine Kinase/antagonists & inhibitors , Macaca fascicularis , Multiple Sclerosis/drug therapy , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Rats, Sprague-Dawley , Tissue Distribution
2.
J Org Chem ; 86(4): 3377-3421, 2021 02 19.
Article in English | MEDLINE | ID: mdl-33544599

ABSTRACT

Molecular design, synthesis, and biological evaluation of tubulysin analogues, linker-drugs, and antibody-drug conjugates are described. Among the new discoveries reported is the identification of new potent analogues within the tubulysin family that carry a C11 alkyl ether substituent, rather than the usual ester structural motif at that position, a fact that endows the former with higher plasma stability than that of the latter. Also described herein are X-ray crystallographic analysis studies of two tubulin-tubulysin complexes formed within the α/ß interface between two tubulin heterodimers and two highly potent tubulysin analogues, one of which exhibited a different binding mode to the one previously reported for tubulysin M. The X-ray crystallographic analysis-derived new insights into the binding modes of these tubulysin analogues explain their potencies and provide inspiration for further design, synthesis, and biological investigations within this class of antitumor agents. A number of these analogues were conjugated as payloads with appropriate linkers at different sites allowing their attachment onto targeting antibodies for cancer therapies. A number of such antibody-drug conjugates were constructed and tested, both in vivo and in vitro, leading to the identification of at least one promising ADC (Herceptin-LD3), warranting further investigations.


Subject(s)
Immunoconjugates , Pharmaceutical Preparations , Immunoconjugates/pharmacology , Structure-Activity Relationship , Tubulin , X-Rays
3.
J Org Chem ; 86(3): 2499-2521, 2021 02 05.
Article in English | MEDLINE | ID: mdl-33417458

ABSTRACT

Thailanstatin A and spliceostatin D, two naturally occurring molecules endowed with potent antitumor activities by virtue of their ability to bind and inhibit the function of the spliceosome, and their natural siblings and designed analogues, constitute an appealing family of compounds for further evaluation and optimization as potential drug candidates for cancer therapies. In this article, the design, synthesis, and biological investigation of a number of novel thailanstatin A analogues, including some accommodating 1,1-difluorocyclopropyl and tetrahydrooxazine structural motifs within their structures, are described. Important findings from these studies paving the way for further investigations include the identification of several highly potent compounds for advancement as payloads for antibody-drug conjugates (ADCs) as potential targeted cancer therapies and/or small molecule drugs, either alone or in combination with other anticancer agents.


Subject(s)
Antineoplastic Agents , Immunoconjugates , Antineoplastic Agents/pharmacology , Pyrans/pharmacology
4.
Bioorg Med Chem Lett ; 30(24): 127640, 2020 12 15.
Article in English | MEDLINE | ID: mdl-33127540

ABSTRACT

PNU-159682 is a highly potent secondary metabolite of nemorubicin belonging to the anthracycline class of natural products. Due to its extremely high potency and only partially understood mechanism of action, it was deemed an interesting starting point for the development of a new suite of linker drugs for antibody drug conjugates (ADCs). Structure activity relationships were explored on the small molecule which led to six linker drugs being developed for conjugation to antibodies. Herein we describe the synthesis of novel PNU-159682 derivatives and the subsequent linker drugs as well as the corresponding biological evaluations of the small molecules and ADCs.


Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Doxorubicin/analogs & derivatives , Immunoconjugates/chemistry , Immunoconjugates/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Cell Line, Tumor , Doxorubicin/chemical synthesis , Doxorubicin/chemistry , Doxorubicin/pharmacology , Humans , Mice, Inbred NOD , Mice, SCID , Neoplasms/drug therapy
5.
J Am Chem Soc ; 142(5): 2549-2561, 2020 02 05.
Article in English | MEDLINE | ID: mdl-31976660

ABSTRACT

The family of anthraquinone-fused enediyne antitumor antibiotics was established by the discovery of dynemicin A and deoxy-dynemicin A. It was then expanded, first by the isolation of uncialamycin, and then by the addition to the family of tiancimycins A-F and yangpumicin A. This family of natural products provides opportunities in total synthesis, biology, and medicine due to their novel and challenging molecular structures, intriguing biological properties and mechanism of action, and potential in targeted cancer therapies. Herein, the total syntheses of tiancimycins A and B, yangpumicin A, and a number of related anthraquinone-fused enediynes are described. Biological evaluation of the synthesized compounds revealed extremely potent cytotoxicities against a number of cell lines, thus enriching the structure-activity relationships within this class of compounds. The findings of these studies may facilitate future investigations directed toward antibody-drug conjugates for targeted cancer therapies and provide inspiration for further advances in total synthesis and chemical biology.


Subject(s)
Anthraquinones/chemistry , Antibiotics, Antineoplastic/pharmacology , Enediynes/chemical synthesis , Enediynes/pharmacology , Antibiotics, Antineoplastic/chemistry , Humans , Structure-Activity Relationship
6.
Bioorg Med Chem Lett ; 29(12): 1522-1531, 2019 06 15.
Article in English | MEDLINE | ID: mdl-30981576

ABSTRACT

Disruption of interleukin-13 (IL-13) signaling with large molecule antibody therapies has shown promise in diseases of allergic inflammation. Given that IL-13 recruits several members of the Janus Kinase family (JAK1, JAK2, and TYK2) to its receptor complex, JAK inhibition may offer an alternate small molecule approach to disrupting IL-13 signaling. Herein we demonstrate that JAK1 is likely the isoform most important to IL-13 signaling. Structure-based design was then used to improve the JAK1 potency of a series of previously reported JAK2 inhibitors. The ability to impede IL-13 signaling was thereby significantly improved, with the best compounds exhibiting single digit nM IC50's in cell-based assays dependent upon IL-13 signaling. Appropriate substitution was further found to influence inhibition of a key off-target, LRRK2. Finally, the most potent compounds were found to be metabolically labile, which makes them ideal scaffolds for further development as topical agents for IL-13 mediated diseases of the lungs and skin (for example asthma and atopic dermatitis, respectively).


Subject(s)
Dermatitis, Atopic/genetics , Interleukin-13/metabolism , Janus Kinase 1/antagonists & inhibitors , Janus Kinase 2/antagonists & inhibitors , Humans , Signal Transduction
7.
J Med Chem ; 62(4): 2140-2153, 2019 02 28.
Article in English | MEDLINE | ID: mdl-30715878

ABSTRACT

Pim kinases have been targets of interest for a number of therapeutic areas. Evidence of durable single-agent efficacy in human clinical trials validated Pim kinase inhibition as a promising therapeutic approach for multiple myeloma patients. Here, we report the compound optimization leading to GDC-0339 (16), a potent, orally bioavailable, and well tolerated pan-Pim kinase inhibitor that proved efficacious in RPMI8226 and MM.1S human multiple myeloma xenograft mouse models and has been evaluated as an early development candidate.


Subject(s)
Antineoplastic Agents/therapeutic use , Multiple Myeloma/drug therapy , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrazoles/therapeutic use , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/metabolism , Cell Line, Tumor , Dogs , Female , Humans , Macaca fascicularis , Madin Darby Canine Kidney Cells , Male , Mice, SCID , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/metabolism , Proto-Oncogene Proteins c-pim-1/metabolism , Pyrazoles/chemical synthesis , Pyrazoles/metabolism , Rats, Sprague-Dawley , Structure-Activity Relationship , Xenograft Model Antitumor Assays
8.
J Med Chem ; 61(8): 3641-3659, 2018 04 26.
Article in English | MEDLINE | ID: mdl-29590749

ABSTRACT

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel expressed in sensory neurons where it functions as an irritant sensor for a plethora of electrophilic compounds and is implicated in pain, itch, and respiratory disease. To study its function in various disease contexts, we sought to identify novel, potent, and selective small-molecule TRPA1 antagonists. Herein we describe the evolution of an N-isopropylglycine sulfonamide lead (1) to a novel and potent (4 R,5 S)-4-fluoro-5-methylproline sulfonamide series of inhibitors. Molecular modeling was utilized to derive low-energy three-dimensional conformations to guide ligand design. This effort led to compound 20, which possessed a balanced combination of potency and metabolic stability but poor solubility that ultimately limited in vivo exposure. To improve solubility and in vivo exposure, we developed methylene phosphate prodrug 22, which demonstrated superior oral exposure and robust in vivo target engagement in a rat model of AITC-induced pain.


Subject(s)
Prodrugs/pharmacology , Proline/analogs & derivatives , Proline/pharmacology , Sulfonamides/pharmacology , TRPA1 Cation Channel/antagonists & inhibitors , Animals , Dogs , Drug Discovery , Drug Stability , Humans , Ligands , Madin Darby Canine Kidney Cells , Microsomes, Liver/metabolism , Models, Molecular , Molecular Conformation , Prodrugs/chemical synthesis , Prodrugs/chemistry , Prodrugs/pharmacokinetics , Proline/chemical synthesis , Proline/pharmacokinetics , Rats , Solubility , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistry , Sulfonamides/pharmacokinetics , TRPA1 Cation Channel/chemistry
9.
J Med Chem ; 60(19): 8083-8102, 2017 10 12.
Article in English | MEDLINE | ID: mdl-28929759

ABSTRACT

Significant data exists to suggest that dual leucine zipper kinase (DLK, MAP3K12) is a conserved regulator of neuronal degeneration following neuronal injury and in chronic neurodegenerative disease. Consequently, there is considerable interest in the identification of DLK inhibitors with a profile compatible with development for these indications. Herein, we use structure-based drug design combined with a focus on CNS drug-like properties to generate compounds with superior kinase selectivity and metabolic stability as compared to previously disclosed DLK inhibitors. These compounds, exemplified by inhibitor 14, retain excellent CNS penetration and are well tolerated following multiple days of dosing at concentrations that exceed those required for DLK inhibition in the brain.


Subject(s)
Alzheimer Disease/drug therapy , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , MAP Kinase Kinase Kinases/antagonists & inhibitors , Amyloid beta-Protein Precursor/biosynthesis , Amyloid beta-Protein Precursor/genetics , Animals , Brain/drug effects , Brain/metabolism , Drug Design , Humans , Macaca fascicularis , Mice , Mice, Inbred C57BL , Models, Molecular , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship
10.
Protein Sci ; 26(2): 152-162, 2017 02.
Article in English | MEDLINE | ID: mdl-27727493

ABSTRACT

Germinal-center kinase-like kinase (GLK, Map4k3), a GCK-I family kinase, plays multiple roles in regulating apoptosis, amino acid sensing, and immune signaling. We describe here the crystal structure of an activation loop mutant of GLK kinase domain bound to an inhibitor. The structure reveals a weakly associated, activation-loop swapped dimer with more than 20 amino acids of ordered density at the carboxy-terminus. This C-terminal PEST region binds intermolecularly to the hydrophobic groove of the N-terminal domain of a neighboring molecule. Although the GLK activation loop mutant crystallized demonstrates reduced kinase activity, its structure demonstrates all the hallmarks of an "active" kinase, including the salt bridge between the C-helix glutamate and the catalytic lysine. Our compound displacement data suggests that the effect of the Ser170Ala mutation in reducing kinase activity is likely due to its effect in reducing substrate peptide binding affinity rather than reducing ATP binding or ATP turnover. This report details the first structure of GLK; comparison of its activation loop sequence and P-loop structure to that of Map4k4 suggests ideas for designing inhibitors that can distinguish between these family members to achieve selective pharmacological inhibitors.


Subject(s)
Mutation, Missense , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/chemistry , Amino Acid Substitution , Crystallography, X-Ray , Humans , Protein Domains , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Protein Structure, Secondary
11.
Bioorg Med Chem Lett ; 26(2): 495-498, 2016 Jan 15.
Article in English | MEDLINE | ID: mdl-26706172

ABSTRACT

A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.


Subject(s)
Nerve Tissue Proteins/antagonists & inhibitors , Pyrrolidines/pharmacology , Sulfonamides/pharmacology , Transient Receptor Potential Channels/antagonists & inhibitors , Animals , Calcium Channels , Humans , Microsomes, Liver/metabolism , Pyrrolidines/chemical synthesis , Rats , Stereoisomerism , Structure-Activity Relationship , Sulfonamides/chemical synthesis , TRPA1 Cation Channel
12.
ACS Med Chem Lett ; 6(12): 1241-6, 2015 Dec 10.
Article in English | MEDLINE | ID: mdl-26713112

ABSTRACT

Signaling pathways intersecting with the p21-activated kinases (PAKs) play important roles in tumorigenesis and cancer progression. By recognizing that the limitations of FRAX1036 (1) were chiefly associated with the highly basic amine it contained, we devised a mitigation strategy to address several issues such as hERG activity. The 5-amino-1,3-dioxanyl moiety was identified as an effective means of reducing pK a and logP simultaneously. When positioned properly within the scaffold, this group conferred several benefits including potency, pharmacokinetics, and selectivity. Mouse xenograft PK/PD studies were carried out using an advanced compound, G-5555 (12), derived from this approach. These studies concluded that dose-dependent pathway modulation was achievable and paves the way for further in vivo investigations of PAK1 function in cancer and other diseases.

13.
Nature ; 527(7578): 323-8, 2015 Nov 19.
Article in English | MEDLINE | ID: mdl-26536114

ABSTRACT

Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteremia , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Intracellular Space/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Vancomycin/pharmacology , Animals , Anti-Bacterial Agents/therapeutic use , Bacteremia/drug therapy , Bacteremia/microbiology , Carrier State/drug therapy , Carrier State/microbiology , Drug Design , Female , Immunoconjugates/chemistry , Intracellular Space/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Microbial Sensitivity Tests , Phagosomes/drug effects , Phagosomes/metabolism , Phagosomes/microbiology , Staphylococcal Infections/drug therapy , Staphylococcal Infections/pathology , Staphylococcus aureus/pathogenicity , Vancomycin/therapeutic use
14.
Bioorg Med Chem Lett ; 25(22): 5258-64, 2015 Nov 15.
Article in English | MEDLINE | ID: mdl-26459208

ABSTRACT

Pim kinase inhibitors are promising cancer therapeutics. Pim-2, among the three Pim isoforms, plays a critical role in multiple myeloma yet inhibition of Pim-2 is challenging due to its high affinity for ATP. A co-crystal structure of a screening hit 1 bound to Pim-1 kinase revealed the key binding interactions of its indazole core within the ATP binding site. Screening of analogous core fragments afforded 1H-pyrazolo[3,4-c]pyridine (6-azaindazole) as a core for the development of pan-Pim inhibitors. Fragment and structure based drug design led to identification of the series with picomolar biochemical potency against all three Pim isoforms. Desirable cellular potency was also achieved.


Subject(s)
Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins c-pim-1/antagonists & inhibitors , Pyrazoles/pharmacology , Pyridines/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Crystallography, X-Ray , Humans , Indazoles/chemical synthesis , Indazoles/chemistry , Indazoles/pharmacology , Mice , Models, Molecular , Proto-Oncogene Proteins c-pim-1/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Pyridines/chemical synthesis , Pyridines/chemistry , Structure-Activity Relationship
15.
Toxicol Mech Methods ; 25(6): 478-86, 2015.
Article in English | MEDLINE | ID: mdl-26275124

ABSTRACT

Application of safety lead optimization screening strategies during the early stage of drug discovery led to the identification of a series of CNS-active small molecule inhibitors with opioid off-target effects, as evidenced by potent agonistic activity in functional cell-based assays for mu (MOP), kappa (KOP) and delta (DOP) opioid receptors. The translation of these effects was confirmed in vivo with the following observations: hypoactivity and decreased fecal production in rats (characteristic of MOP agonism); increased urine production in rats (characteristic of KOP agonism); and decreased intestinal transit time in mice, which was partially blocked by the MOP antagonist naloxone, demonstrating that the in vivo effects were specific for MOP. Based on the confirmation of in vitro-in vivo translatability, an in vitro screening strategy was implemented that resulted in the identification of an optimized backup molecule, devoid of in vivo off-target opioid effects. In addition, in silico modeling by docking of the various molecules to the opioid receptors allowed the identification of the structural drivers of these off-target effects, which can be applied to future chemical-design criteria. Thus, implementation of the safety lead optimization strategy described in this article demonstrates the utility and impact of such approaches on risk mitigation and identification of lead small molecules with improved safety profiles.


Subject(s)
Amyloid Precursor Protein Secretases/antagonists & inhibitors , Aspartic Acid Endopeptidases/antagonists & inhibitors , Drug Design , Protease Inhibitors/pharmacology , Receptors, Opioid/agonists , Amyloid Precursor Protein Secretases/metabolism , Animals , Aspartic Acid Endopeptidases/metabolism , Binding Sites , CHO Cells , Cricetulus , Defecation/drug effects , Dose-Response Relationship, Drug , Female , Gastrointestinal Transit/drug effects , Guinea Pigs , Ileum/drug effects , Ileum/metabolism , In Vitro Techniques , Mice , Molecular Docking Simulation , Narcotic Antagonists/pharmacology , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protease Inhibitors/toxicity , Protein Binding , Protein Conformation , Rats, Sprague-Dawley , Receptors, Opioid/genetics , Receptors, Opioid/metabolism , Structure-Activity Relationship , Time Factors , Transfection , Urination/drug effects
16.
ACS Med Chem Lett ; 6(8): 925-9, 2015 Aug 13.
Article in English | MEDLINE | ID: mdl-26288695

ABSTRACT

Time-dependent inhibition (TDI) of cytochrome P450 (CYP) enzymes may incur serious undesirable drug-drug interactions and in rare cases drug-induced idiosyncratic toxicity. The reactive metabolites are often generated through multiple sequential biotransformations and form adducts with CYP enzymes to inactivate their function. The complexity of these processes makes addressing TDI liability very challenging. Strategies to mitigate TDI are therefore highly valuable in discovering safe therapies to benefit patients. In this Letter, we disclose our simplified approach toward addressing CYP3A TDI liabilities, guided by metabolic mechanism hypotheses. By adding a methyl group onto the α carbon of a basic amine, TDI activities of both the truncated and full molecules (7a and 11) were completely eliminated. We propose that truncated molecules, albeit with caveats, may be used as surrogates for full molecules to investigate TDI.

17.
J Med Chem ; 58(12): 5053-74, 2015 Jun 25.
Article in English | MEDLINE | ID: mdl-25988399

ABSTRACT

Checkpoint kinase 1 (ChK1) plays a key role in the DNA damage response, facilitating cell-cycle arrest to provide sufficient time for lesion repair. This leads to the hypothesis that inhibition of ChK1 might enhance the effectiveness of DNA-damaging therapies in the treatment of cancer. Lead compound 1 (GNE-783), the prototype of the 1,7-diazacarbazole class of ChK1 inhibitors, was found to be a highly potent inhibitor of acetylcholine esterase (AChE) and unsuitable for development. A campaign of analogue synthesis established SAR delineating ChK1 and AChE activities and allowing identification of new leads with improved profiles. In silico docking using a model of AChE permitted rationalization of the observed SAR. Compounds 19 (GNE-900) and 30 (GNE-145) were identified as selective, orally bioavailable ChK1 inhibitors offering excellent in vitro potency with significantly reduced AChE activity. In combination with gemcitabine, these compounds demonstrate an in vivo pharmacodynamic effect and are efficacious in a mouse p53 mutant xenograft model.


Subject(s)
Acetylcholinesterase/metabolism , Carbazoles/chemistry , Carbazoles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Protein Kinases/metabolism , Acetylcholinesterase/chemistry , Acetylcholinesterase/pharmacokinetics , Acetylcholinesterase/therapeutic use , Animals , Aza Compounds/chemistry , Aza Compounds/pharmacokinetics , Aza Compounds/pharmacology , Aza Compounds/therapeutic use , Cell Line, Tumor , Checkpoint Kinase 1 , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacokinetics , Cholinesterase Inhibitors/pharmacology , Cholinesterase Inhibitors/therapeutic use , Crystallography, X-Ray , Dogs , Humans , Mice , Mice, Nude , Models, Molecular , Neoplasms/drug therapy , Neoplasms/enzymology , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/therapeutic use , Protein Kinases/chemistry , Rats
18.
Sci Transl Med ; 7(273): 273ra15, 2015 Feb 04.
Article in English | MEDLINE | ID: mdl-25653221

ABSTRACT

Inhibition of the kinase activity of leucine-rich repeat kinase 2 (LRRK2) is under investigation as a possible treatment for Parkinson's disease. However, there is no clinical validation as yet, and the safety implications of targeting LRRK2 kinase activity are not well understood. We evaluated the potential safety risks by comparing human and mouse LRRK2 mRNA tissue expression, by analyzing a Lrrk2 knockout mouse model, and by testing selective brain-penetrating LRRK2 kinase inhibitors in multiple species. LRRK2 mRNA tissue expression was comparable between species. Phenotypic analysis of Lrrk2 knockout mice revealed morphologic changes in lungs and kidneys, similar to those reported previously. However, in preclinical toxicity assessments in rodents, no pulmonary or renal changes were induced by two distinct LRRK2 kinase inhibitors. Both of these kinase inhibitors induced abnormal cytoplasmic accumulation of secretory lysosome-related organelles known as lamellar bodies in type II pneumocytes of the lung in nonhuman primates, but no lysosomal abnormality was observed in the kidney. The pulmonary change resembled the phenotype of Lrrk2 knockout mice, suggesting that this was LRRK2-mediated rather than a nonspecific or off-target effect. A biomarker of lysosomal dysregulation, di-docosahexaenoyl (22:6) bis(monoacylglycerol) phosphate (di-22:6-BMP), was also decreased in the urine of Lrrk2 knockout mice and nonhuman primates treated with LRRK2 kinase inhibitors. Our results suggest a role for LRRK2 in regulating lysosome-related lamellar bodies and that pulmonary toxicity may be a critical safety liability for LRRK2 kinase inhibitors in patients.


Subject(s)
Lung/enzymology , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Alveolar Epithelial Cells/drug effects , Alveolar Epithelial Cells/pathology , Animals , Biomarkers/blood , Biomarkers/urine , Dose-Response Relationship, Drug , Female , HEK293 Cells , Humans , Kidney/abnormalities , Kidney/drug effects , Kidney/pathology , Kidney/ultrastructure , Leucine-Rich Repeat Serine-Threonine Protein Kinase-2 , Lung/abnormalities , Lung/pathology , Lung/ultrastructure , Macaca fascicularis , Male , Mice, Inbred C57BL , Mice, Knockout , Morpholines/chemistry , Morpholines/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Pyrazoles/chemistry , Pyrazoles/pharmacology , Pyrimidines/chemistry , Pyrimidines/pharmacology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley
19.
J Med Chem ; 58(1): 401-18, 2015 Jan 08.
Article in English | MEDLINE | ID: mdl-25341110

ABSTRACT

Dual leucine zipper kinase (DLK, MAP3K12) was recently identified as an essential regulator of neuronal degeneration in multiple contexts. Here we describe the generation of potent and selective DLK inhibitors starting from a high-throughput screening hit. Using proposed hinge-binding interactions to infer a binding mode and specific design parameters to optimize for CNS druglike molecules, we came to focus on the di(pyridin-2-yl)amines because of their combination of desirable potency and good brain penetration following oral dosing. Our lead inhibitor GNE-3511 (26) displayed concentration-dependent protection of neurons from degeneration in vitro and demonstrated dose-dependent activity in two different animal models of disease. These results suggest that specific pharmacological inhibition of DLK may have therapeutic potential in multiple indications.


Subject(s)
MAP Kinase Kinase Kinases/antagonists & inhibitors , Nerve Degeneration/prevention & control , Neurodegenerative Diseases/prevention & control , Protein Kinase Inhibitors/pharmacology , Animals , Disease Models, Animal , Dogs , Dose-Response Relationship, Drug , Drug Discovery , HEK293 Cells , Humans , Madin Darby Canine Kidney Cells , Mice, Inbred C57BL , Models, Chemical , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Rats
20.
Eur J Drug Metab Pharmacokinet ; 40(2): 171-85, 2015 Jun.
Article in English | MEDLINE | ID: mdl-24696325

ABSTRACT

(R)-2-Amino-1,3',3'-trimethyl-7'-(pyrimidin-5-yl)-3',4'-dihydro-2'H-spiro[imidazole-4,1'-naphthalen]-5(1H)-one (GNE-892) is an orally administered inhibitor of ß-secretase 1 (ß-site amyloid precursor protein cleaving enzyme 1, BACE1) that was developed as an intervention therapy against Alzheimer's disease. A clinical microdosing strategy was being considered for de-risking the potential pharmacokinetic liabilities of GNE-892. We tested whether dose-proportionality was observed in cynomolgus monkey as proof-of-concept for a human microdosing study. With cryopreserved monkey hepatocytes, concentration-dependency for substrate turnover and the relative contribution of P450- versus AO-mediated metabolism were observed. Characterization of the kinetics of these metabolic pathways demonstrated differences in the affinities of P450 and AO for GNE-892, which supported the metabolic profiles that had been obtained. To test if this metabolic shift occurred in vivo, mass balance studies in monkeys were conducted at doses of 0.085 and 15 mg/kg. Plasma exposure of GNE-892 following oral administration was more than 20-fold greater than dose proportional at the high-dose. P-gp-mediated efflux was unable to explain the discrepancy. The profiles of metabolites in circulation and excreta were indicative that oxidative metabolism limited the exposure to unchanged GNE-892 at the low dose. Further, the in vivo data supported the concentration-dependent metabolic shift between P450 and AO. In conclusion, microdosing of GNE-892 was not predictive of pharmacokinetics at a more pharmacologically relevant dose due to saturable absorption and metabolism. Therefore, it is important to consider ADME liabilities and their potential concentration-dependency when deciding upon a clinical microdosing strategy.


Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/physiology , Aldehyde Oxidase/physiology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Cytochrome P-450 Enzyme System/physiology , Enzyme Inhibitors/metabolism , Imidazoles/metabolism , Spiro Compounds/metabolism , Animals , Macaca fascicularis , Male
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