ABSTRACT
Glutamate plays a key role in cognition and mood, and it has been shown that inhibiting ionotropic glutamate receptors disrupts cognition, while enhancing ionotropic receptor activity is pro-cognitive. One approach to elevating glutamatergic tone has been to antagonize presynaptic metabotropic glutamate receptor 2 (mGluR2). A desire for selectivity over the largely homologous mGluR3 motivated a strategy to achieve selectivity through the identification of mGluR2 negative allosteric modulators (NAMs). Extensive screening and optimization efforts led to the identification of a novel series of 4-arylquinoline-2-carboxamides. This series was optimized for mGluR2 NAM potency, clean off-target activity, and desirable physical properties, which resulted in the identification of improved C4 and C7 substituents. The initial lead compound from this series was Ames-positive in a single strain with metabolic activation, indicating that a reactive metabolite was likely responsible for the genetic toxicity. Metabolic profiling and Ames assessment across multiple analogs identified key structure-activity relationships associated with Ames positivity. Further optimization led to the Ames-negative mGluR2 negative allosteric modulator MK-8768.
ABSTRACT
Lens epithelial-derived growth factor (LEDGF) increases the efficiency of proviral DNA integration into the host genome by interacting with HIV integrase (IN) and directing it to a chromatin environment that favors viral transcription. Allosteric integrase inhibitors (ALLINIs), such as known 2-(tert-butoxy)acetic acid (1), bind to the LEDGF pocket on the catalytic core domain (CCD) of IN, but exert more potent antiviral activities by inhibition of late-stage HIV-1 replication events than through disruption of proviral integration at an earlier phase. A high-throughput screen (HTS) for compounds that disrupt IN-LEDGF interaction led to the identification of a novel arylsulfonamide series, as exemplified by 2, possessing ALLINI-like properties. Further SAR studies led to more potent compound 21 and provided key chemical biology probes revealing that arylsulfonamides are a novel class of ALLINIs with a distinct binding mode than that of 2-(tert-butoxy)acetic acids.
Subject(s)
Anti-HIV Agents , HIV Integrase Inhibitors , HIV Integrase , HIV Integrase Inhibitors/pharmacology , HIV Integrase Inhibitors/chemistry , Allosteric Regulation , Catalytic Domain , HIV Integrase/metabolismABSTRACT
Antiretroviral therapy inhibits HIV-1 replication but is not curative due to establishment of a persistent reservoir after virus integration into the host genome. Reservoir reduction is therefore an important HIV-1 cure strategy. Some HIV-1 nonnucleoside reverse transcriptase inhibitors induce HIV-1 selective cytotoxicity in vitro but require concentrations far exceeding approved dosages. Focusing on this secondary activity, we found bifunctional compounds with HIV-1-infected cell kill potency at clinically achievable concentrations. These targeted activator of cell kill (TACK) molecules bind the reverse transcriptase-p66 domain of monomeric Gag-Pol and act as allosteric modulators to accelerate dimerization, resulting in HIV-1+ cell death through premature intracellular viral protease activation. TACK molecules retain potent antiviral activity and selectively eliminate infected CD4+ T cells isolated from people living with HIV-1, supporting an immune-independent clearance strategy.
Subject(s)
HIV Infections , HIV-1 , Humans , HIV Infections/drug therapy , Antiviral Agents/therapeutic use , Apoptosis , Cell Death , CD4-Positive T-Lymphocytes , Virus ReplicationABSTRACT
Although current antiretroviral therapy can control HIV-1 replication and prevent disease progression, it is not curative. Identifying mechanisms that can lead to eradication of persistent viral reservoirs in people living with HIV-1 (PLWH) remains an outstanding challenge to achieving cure. Utilizing a phenotypic screen, we identified a novel chemical class capable of killing HIV-1 infected peripheral blood mononuclear cells. Tool compounds ICeD-1 and ICeD-2 ("inducer of cell death-1 and 2"), optimized for potency and selectivity from screening hits, were used to deconvolute the mechanism of action using a combination of chemoproteomic, biochemical, pharmacological, and genetic approaches. We determined that these compounds function by modulating dipeptidyl peptidase 9 (DPP9) and activating the caspase recruitment domain family member 8 (CARD8) inflammasome. Efficacy of ICeD-1 and ICeD-2 was dependent on HIV-1 protease activity and synergistic with efavirenz, which promotes premature activation of HIV-1 protease at high concentrations in infected cells. This in vitro synergy lowers the efficacious cell kill concentration of efavirenz to a clinically relevant dose at concentrations of ICeD-1 or ICeD-2 that do not result in complete DPP9 inhibition. These results suggest engagement of the pyroptotic pathway as a potential approach to eliminate HIV-1 infected cells.
Subject(s)
HIV Infections , HIV-1 , Alkynes , Benzoxazines , CARD Signaling Adaptor Proteins/metabolism , Cyclopropanes , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/metabolism , HIV Infections/drug therapy , HIV-1/metabolism , Humans , Inflammasomes/metabolism , Leukocytes, Mononuclear , Neoplasm Proteins/metabolismABSTRACT
Small molecule developability challenges have been well documented over the last two decades. One of these critical developability parameters is aqueous solubility. In general, more soluble compounds have improved oral absorption. While enabling formulation technologies exist to improve bioperformance for low solubility compounds, these are often more complex, expensive, and challenging to scale up. Therefore, to avoid these development issues, medicinal chemists need tools to rapidly profile and improve the physicochemical properties of molecules during discovery. Dose number (Do) is a simple metric to predict whether a compound will be reasonably absorbed based on solubility at an expected clinical dose and represents a valuable parameter to the medicinal chemist defining a clinical candidate. The goal of this mini-Perspective is to present the background of the Do equation and how it can be effectively used to rapidly predict oral absorption potential for molecules in the discovery space.
Subject(s)
Chemistry, Pharmaceutical/methods , Drug Discovery/methods , Pharmaceutical Preparations/chemistry , Administration, Oral , Animals , Dose-Response Relationship, Drug , Humans , Oral Mucosal Absorption , Pharmaceutical Preparations/administration & dosage , SolubilityABSTRACT
Two orthogonal approaches for hit identification in drug discovery are large-scale in vitro and in silico screening. In recent years, due to the emergence of new targets and a rapid increase in the size of the readily synthesizable chemical space, there is a growing emphasis on the integration of the two techniques to improve the hit finding efficiency. Here, we highlight three examples of drug discovery projects at Merck & Co., Inc., Kenilworth, NJ, USA in which different virtual screening (VS) techniques, each specifically tailored to leverage knowledge available for the target, were utilized to augment the selection of high-quality chemical matter for in vitro assays and to enhance the diversity and tractability of hits. Central to success is a fully integrated workflow combining in silico and experimental expertise at every stage of the hit identification process. We advocate that workflows encompassing VS as part of an integrated hit finding plan should be widely adopted to accelerate hit identification and foster cross-functional collaborations in modern drug discovery.
Subject(s)
Drug Discovery , High-Throughput Screening Assays , Computer Simulation , Small Molecule LibrariesABSTRACT
Antagonism of the mGluR2 receptor has the potential to provide therapeutic benefit to cognitive disorders by elevating synaptic glutamate, the primary excitatory neurotransmitter in the brain. Selective antagonism of the mGluR2 receptor, however, has so far been elusive, given the very high homology of this receptor with mGluR3, particularly at the orthosteric binding site. Given that inhibition of mGluR3 has been implicated in undesired effects, we sought to identify selective mGluR2 negative allosteric modulators. Herein we describe the discovery of the highly potent and selective class of mGluR2 negative allosteric modulators, 4-arylquinoline-2-carboxamides, following a successful HTS campaign and medicinal chemistry optimization, showing potent in vivo efficacy in rodent.
Subject(s)
Drug Discovery , Receptors, Metabotropic Glutamate/antagonists & inhibitors , Adjuvants, Anesthesia/toxicity , Amino Acids/pharmacology , Amphetamines/pharmacology , Animals , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Glutamic Acid/metabolism , High-Throughput Screening Assays , Mice , Molecular Structure , Scopolamine/toxicity , Structure-Activity RelationshipABSTRACT
Allosteric integrase inhibitors (ALLINIs) bind to the lens epithelial-derived growth factor (LEDGF) pocket on HIV-1 integrase (IN) and possess potent antiviral effects. Rather than blocking proviral integration, ALLINIs trigger IN conformational changes that have catastrophic effects on viral maturation, rendering the virions assembled in the presence of ALLINIs noninfectious. A high-throughput screen for compounds that disrupt the IN·LEDGF interaction was executed, and extensive triage led to the identification of a t-butylsulfonamide series, as exemplified by 1. The chemical, biochemical, and virological characterization of this series revealed that 1 and its analogs produce an ALLINI-like phenotype through engagement of IN sites distinct from the LEDGF pocket. Key to demonstrating target engagement and differentiating this new series from the existing ALLINIs was the development of a fluorescence polarization probe of IN (FLIPPIN) based on the t-butylsulfonamide series. These findings further solidify the late antiviral mechanism of ALLINIs and point toward opportunities to develop structurally and mechanistically novel antiretroviral agents with unique resistance patterns.
Subject(s)
Allosteric Regulation/drug effects , HIV Infections/drug therapy , HIV Integrase Inhibitors/chemistry , HIV Integrase Inhibitors/pharmacology , HIV Integrase/metabolism , HIV-1/drug effects , Cell Line , Drug Discovery , HIV Infections/metabolism , HIV Infections/virology , HIV-1/metabolism , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Protein Interaction Maps/drug effects , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
The development of a C(sp2)-C(sp3) cross-coupling reaction for rapid, parallel synthesis of analogues of two HIV NNRTI clinical candidates is described. This method allowed easy access to the C-ring space using a practical alkylation with commercially available tributyl(iodomethyl)stannane followed by a palladium-catalyzed coupling with a variety of aryl halides (I, Br) in the presence of copper chloride. Optimization and scope of this method are reported.
ABSTRACT
The NaV1.7 voltage-gated sodium channel is a highly valued target for the treatment of neuropathic pain due to its expression in pain-sensing neurons and human genetic mutations in the gene encoding NaV1.7, resulting in either loss-of-function (e.g., congenital analgesia) or gain-of-function (e.g., paroxysmal extreme pain disorder) pain phenotypes. We exploited existing technologies in a novel manner to identify selective antagonists of NaV1.7. A full-deck high-throughput screen was developed for both NaV1.7 and cardiac NaV1.5 channels using a cell-based membrane potential dye FLIPR assay. In assay development, known local anesthetic site inhibitors produced a decrease in maximal response; however, a subset of compounds exhibited a concentration-dependent delay in the onset of the response with little change in the peak of the response at any concentration. Therefore, two methods of analysis were employed for the screen: one to measure peak response and another to measure area under the curve, which would capture the delay-to-onset phenotype. Although a number of compounds were identified by a selective reduction in peak response in NaV1.7 relative to 1.5, the AUC measurement and a subsequent refinement of this measurement were able to differentiate compounds with NaV1.7 pharmacological selectivity over NaV1.5 as confirmed in electrophysiology.
Subject(s)
High-Throughput Screening Assays/methods , NAV1.5 Voltage-Gated Sodium Channel/metabolism , NAV1.7 Voltage-Gated Sodium Channel/metabolism , Neuralgia/drug therapy , Humans , Kinetics , Membrane Potentials/drug effects , Molecular Targeted Therapy , NAV1.5 Voltage-Gated Sodium Channel/drug effects , NAV1.7 Voltage-Gated Sodium Channel/drug effects , Neurons/drug effects , Neurons/pathology , Pain/drug therapy , Rectum/abnormalitiesABSTRACT
Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer's disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.
Subject(s)
Adenosine/analogs & derivatives , Drug Design , Hydrolases/antagonists & inhibitors , S-Adenosylhomocysteine , Adenosine/chemistry , Adenosine/pharmacology , Animals , Brain Chemistry , Enzyme Activation/drug effects , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Homocysteine/blood , Hydrogen Bonding , Inhibitory Concentration 50 , Models, Molecular , Rats , S-Adenosylhomocysteine/chemistry , Substrate SpecificityABSTRACT
Numerous changes occur during aging and Alzheimer's disease (AD) progression, including a decline in cholinergic functioning and cognition, as well as alterations in gene expression and activity in the nitric oxide/cyclic guanosine monophosphate (NO/cGMP) pathway. Donepezil, the current standard of care for Alzheimer's disease, improves cholinergic functioning and has demonstrated effects on multiple domains of cognition, including memory and attention in both preclinical species and patients. We previously found that increasing activation of the NO/cGMP pathway via phosphodiesterase 9 (PDE9) inhibition also improves memory in rodents and suggested that PDE9 might be a promising target for novel treatments for AD. Here we investigated whether PDE9 inhibition also enhances attention using a novel attention task in rats. We validated this task using several pharmacological manipulations and showed that the selective PDE9 inhibitor PF-04447943 produced effects similar to those of donepezil. These data confirm and extend the hypothesis that PDE9 inhibition might serve as a novel treatment for AD and age-related cognitive decline.
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/antagonists & inhibitors , Cognition Disorders/drug therapy , Cognition Disorders/enzymology , Nootropic Agents/pharmacology , Phosphodiesterase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidinones/pharmacology , 3',5'-Cyclic-AMP Phosphodiesterases/physiology , Animals , Cognition Disorders/chemically induced , Disease Models, Animal , Male , Nootropic Agents/therapeutic use , Phosphodiesterase Inhibitors/therapeutic use , Pyrazoles/therapeutic use , Pyrimidinones/therapeutic use , Rats , Rats, Wistar , Scopolamine/adverse effectsABSTRACT
A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.
Subject(s)
Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Protein Kinases/drug effects , Quinazolines/chemical synthesis , Quinazolines/pharmacology , Binding Sites , Checkpoint Kinase 1 , Combinatorial Chemistry Techniques , Crystallography, X-Ray , Humans , Molecular Conformation , Molecular Structure , Protein Kinase Inhibitors/chemistry , Protein Kinases/chemistry , Protein Kinases/metabolism , Quinazolines/chemistryABSTRACT
2,6-Dichloro-9-thiabicyclo[3.3.1]nonane, easily available by an improved condensation of sulfur dichloride, sulfuryl chloride, and 1,5-cyclooctadiene, is a well- behaved scaffold for the nucleophilic substitution of azides and cyanides via neighboring- group participation by the sulfur atom. The products are isolated in high yields with purity >95% by simple extraction and washing protocols.
Subject(s)
Alkanes/chemistry , Azides/chemistry , Bridged Bicyclo Compounds/chemistry , Cyanides/chemistry , Magnetic Resonance Spectroscopy , Sulfur/chemistrySubject(s)
Bridged-Ring Compounds/chemical synthesis , Limonins/chemical synthesis , Bridged-Ring Compounds/chemistry , Insecticides/chemical synthesis , Insecticides/chemistry , Limonins/chemistry , Models, Molecular , Molecular Conformation , Molecular Structure , Naphthalenes/chemical synthesis , Naphthalenes/chemistryABSTRACT
With proper activation of the leaving group, sulfur mustards react with Grignard reagents with neighboring group participation of the sulfur atom. 2,6-Dichloro-9-thiabicyclo[3.3.1]nonane is especially useful in this regard, providing clean reactivity with organomagnesium nucleophiles on a topologically constrained scaffold.