Inhibitors of FAP-fluorogen interaction as a multiplex assay tool compound for receptor internalization assays

<p>A novel assay using fluorogen activating peptide (FAP) technology for G protein-coupled receptor (GPCR) activation and internalization was applied to the human β2AR. This technology avoids microscopy and antibody-based detection methods. A major goal for the project was to identify G-protein independent/β2AR ligands or β2AR biased ligands that induce β2AR internalization. Analysis of the most potent hits in the primary project revealed that they interfered with fluorogen activation by the FAP rather than interacting with the receptor itself. These molecules were pursued further because they had the potential to enable improved assay protocols to monitor receptor trafficking and receptor location in real time. A highly potent compound (ML342, CID 2953239) was declared as a Molecular Libraries Probe Center Network (MLPCN) probe molecule.</p

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)‑1<i>H</i>‑indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ‑Thrombin

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based <b>3</b>. Of these, <b>9j</b> (PAR4 IC₅₀ = 445 nM, PAR1 response IC₅₀ > 30 μM) and <b>10h</b> (PAR4 IC₅₀ = 179 nM, PAR1 response IC₅₀ > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin

Development of a Series of (1-Benzyl-3-(6-methoxypyrimidin-3-yl)-5-(trifluoromethoxy)‑1<i>H</i>‑indol-2-yl)methanols as Selective Protease Activated Receptor 4 (PAR4) Antagonists with in Vivo Utility and Activity Against γ‑Thrombin

Here, we describe the development of a series of highly selective PAR4 antagonists with nanomolar potency and selectivity versus PAR1, derived from the indole-based <b>3</b>. Of these, <b>9j</b> (PAR4 IC₅₀ = 445 nM, PAR1 response IC₅₀ > 30 μM) and <b>10h</b> (PAR4 IC₅₀ = 179 nM, PAR1 response IC₅₀ > 30 μM) maintained an overall favorable in vitro DMPK profile, encouraging rat/mouse in vivo pharmacokinetics (PK) and activity against γ-thrombin

High-Affinity Small-Molecule Inhibitors of the Menin-Mixed Lineage Leukemia (MLL) Interaction Closely Mimic a Natural Protein–Protein Interaction

The protein–protein interaction (PPI) between menin and mixed lineage leukemia (MLL) plays a critical role in acute leukemias, and inhibition of this interaction represents a new potential therapeutic strategy for MLL leukemias. We report development of a novel class of small-molecule inhibitors of the menin–MLL interaction, the hydroxy- and aminomethylpiperidine compounds, which originated from HTS of ∼288000 small molecules. We determined menin–inhibitor co-crystal structures and found that these compounds closely mimic all key interactions of MLL with menin. Extensive crystallography studies combined with structure-based design were applied for optimization of these compounds, resulting in <b>MIV</b>-<b>6<i>R</i></b>, which inhibits the menin–MLL interaction with IC₅₀ = 56 nM. Treatment with <b>MIV</b>-<b>6</b> demonstrated strong and selective effects in MLL leukemia cells, validating specific mechanism of action. Our studies provide novel and attractive scaffold as a new potential therapeutic approach for MLL leukemias and demonstrate an example of PPI amenable to inhibition by small molecules

Identification of Specific Ligand–Receptor Interactions That Govern Binding and Cooperativity of Diverse Modulators to a Common Metabotropic Glutamate Receptor 5 Allosteric Site

A common metabotropic glutamate receptor 5 (mGlu₅) allosteric site is known to accommodate diverse chemotypes. However, the structural relationship between compounds from different scaffolds and mGlu₅ is not well understood. In an effort to better understand the molecular determinants that govern allosteric modulator interactions with mGlu₅, we employed a combination of site-directed mutagenesis and computational modeling. With few exceptions, six residues (P654, Y658, T780, W784, S808, and A809) were identified as key affinity determinants across all seven allosteric modulator scaffolds. To improve our interpretation of how diverse allosteric modulators occupy the common allosteric site, we sampled the wealth of mGlu₅ structure–activity relationship (SAR) data available by docking 60 ligands (actives and inactives) representing seven chemical scaffolds into our mGlu₅ comparative model. To spatially and chemically compare binding modes of ligands from diverse scaffolds, the ChargeRMSD measure was developed. We found a common binding mode for the modulators that placed the long axes of the ligands parallel to the transmembrane helices 3 and 7. W784 in TM6 not only was identified as a key NAM cooperativity determinant across multiple scaffolds, but also caused a NAM to PAM switch for two different scaffolds. Moreover, a single point mutation in TM5, G747V, altered the architecture of the common allosteric site such that 4-nitro-<i>N</i>-(1,3-diphenyl-1<i>H</i>-pyrazol-5-yl)­benzamide (VU29) was noncompetitive with the common allosteric site. Our findings highlight the subtleties of allosteric modulator binding to mGlu₅ and demonstrate the utility in incorporating SAR information to strengthen the interpretation and analyses of docking and mutational data

Discovery, Synthesis, And Structure-Based Optimization of a Series of <i>N</i>‑(<i>tert</i>-Butyl)-2‑(<i>N</i>‑arylamido)-2-(pyridin-3-yl) Acetamides (ML188) as Potent Noncovalent Small Molecule Inhibitors of the Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) 3CL Protease

A high-throughput screen of the NIH molecular libraries sample collection and subsequent optimization of a lead dipeptide-like series of severe acute respiratory syndrome (SARS) main protease (3CLpro) inhibitors led to the identification of probe compound ML188 (<b>16-(<i>R</i>)</b>, (<i>R</i>)-<i>N</i>-(4-(<i>tert</i>-butyl)­phenyl)-<i>N</i>-(2-(<i>tert</i>-butylamino)-2-oxo-1-(pyridin-3-yl)­ethyl)­furan-2-carboxamide, Pubchem CID: 46897844). Unlike the majority of reported coronavirus 3CLpro inhibitors that act via covalent modification of the enzyme, <b>16-(<i>R</i>)</b> is a noncovalent SARS-CoV 3CLpro inhibitor with moderate MW and good enzyme and antiviral inhibitory activity. A multicomponent Ugi reaction was utilized to rapidly explore structure–activity relationships within S_1′, S₁, and S₂ enzyme binding pockets. The X-ray structure of SARS-CoV 3CLpro bound with <b>16-(<i>R</i>)</b> was instrumental in guiding subsequent rounds of chemistry optimization. <b>16-(<i>R</i>)</b> provides an excellent starting point for the further design and refinement of 3CLpro inhibitors that act by a noncovalent mechanism of action

Discovery of Potent 2‑Aryl-6,7-dihydro‑5<i>H</i>‑pyrrolo[1,2‑<i>a</i>]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5<i>H</i>-pyrrolo­[1,2-<i>a</i>]­imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer

Discovery of Potent 2‑Aryl-6,7-dihydro‑5<i>H</i>‑pyrrolo[1,2‑<i>a</i>]imidazoles as WDR5-WIN-Site Inhibitors Using Fragment-Based Methods and Structure-Based Design

WDR5 is a chromatin-regulatory scaffold protein overexpressed in various cancers and a potential epigenetic drug target for the treatment of mixed-lineage leukemia. Here, we describe the discovery of potent and selective WDR5-WIN-site inhibitors using fragment-based methods and structure-based design. NMR-based screening of a large fragment library identified several chemically distinct hit series that bind to the WIN site within WDR5. Members of a 6,7-dihydro-5<i>H</i>-pyrrolo­[1,2-<i>a</i>]­imidazole fragment class were expanded using a structure-based design approach to arrive at lead compounds with dissociation constants <10 nM and micromolar cellular activity against an AML-leukemia cell line. These compounds represent starting points for the discovery of clinically useful WDR5 inhibitors for the treatment of cancer

A Novel M₁ PAM VU0486846 Exerts Efficacy in Cognition Models without Displaying Agonist Activity or Cholinergic Toxicity

Selective activation of the M₁ subtype of muscarinic acetylcholine receptor, via positive allosteric modulation (PAM), is an exciting strategy to improve cognition in schizophrenia and Alzheimer’s disease patients. However, highly potent M₁ ago-PAMs, such as MK-7622, PF-06764427, and PF-06827443, can engender excessive activation of M₁, leading to agonist actions in the prefrontal cortex (PFC) that impair cognitive function, induce behavioral convulsions, and result in other classic cholinergic adverse events (AEs). Here, we report a fundamentally new and highly selective M₁ PAM, VU0486846. VU0486846 possesses only weak agonist activity in M₁-expressing cell lines with high receptor reserve and is devoid of agonist actions in the PFC, unlike previously reported ago-PAMs MK-7622, PF-06764427, and PF-06827443. Moreover, VU0486846 shows no interaction with antagonist binding at the orthosteric acetylcholine (ACh) site (e.g., neither bitopic nor displaying negative cooperativity with [³H]-NMS binding at the orthosteric site), no seizure liability at high brain exposures, and no cholinergic AEs. However, as opposed to ago-PAMs, VU0486846 produces robust efficacy in the novel object recognition model of cognitive function. Importantly, we show for the first time that an M₁ PAM can reverse the cognitive deficits induced by atypical antipsychotics, such as risperidone. These findings further strengthen the argument that compounds with modest in vitro M₁ PAM activity (EC₅₀ > 100 nM) and pure-PAM activity in native tissues display robust procognitive efficacy without AEs mediated by excessive activation of M₁. Overall, the combination of compound assessment with recombinant in vitro assays (mindful of receptor reserve), native tissue systems (PFC), and phenotypic screens (behavioral convulsions) is essential to fully understand and evaluate lead compounds and enhance success in clinical development

Exploration of Allosteric Agonism Structure–Activity Relationships within an Acetylene Series of Metabotropic Glutamate Receptor 5 (mGlu₅) Positive Allosteric Modulators (PAMs): Discovery of 5‑((3-Fluorophenyl)ethynyl)‑<i>N</i>‑(3-methyloxetan-3-yl)picolinamide (ML254)

Positive allosteric modulators (PAMs) of metabotropic glutamate receptor 5 (mGlu₅) represent a promising therapeutic strategy for the treatment of schizophrenia. Both allosteric agonism and high glutamate fold-shift have been implicated in the neurotoxic profile of some mGlu₅ PAMs; however, these hypotheses remain to be adequately addressed. To develop tool compounds to probe these hypotheses, the structure–activity relationship of allosteric agonism was examined within an acetylenic series of mGlu₅ PAMs exhibiting allosteric agonism in addition to positive allosteric modulation (ago-PAMs). PAM <b>38t</b>, a low glutamate fold-shift allosteric ligand (maximum fold-shift ∼3.0), was selected as a potent PAM with no agonism in the in vitro system used for compound characterization and in two native electrophysiological systems using rat hippocampal slices. PAM <b>38t</b> (ML254) will be useful to probe the relative contribution of cooperativity and allosteric agonism to the adverse effect liability and neurotoxicity associated with this class of mGlu₅ PAMs