Principals in Programming Languages: Technical Results

This is the companion technical report for ``Principals in Programming Languages'' [20]. See that document for a more readable version of these results. In this paper, we describe two variants of the simply typed $\lambda$-calculus extended with a notion of {\em principal}. The results are languages in which intuitive statements like ``the client must call $\mathtt{open}$ to obtain a file handle'' can be phrased and proven formally. The first language is a two-agent calculus with references and recursive types, while the second language explores the possibility of multiple agents with varying amounts of type information. We use these calculi to give syntactic proofs of some type abstraction results that traditionally require semantic arguments

Structure-Based Prediction of G‑Protein-Coupled Receptor Ligand Function: A β‑Adrenoceptor Case Study

The spectacular advances in G-protein-coupled receptor (GPCR) structure determination have opened up new possibilities for structure-based GPCR ligand discovery. The structure-based prediction of whether a ligand stimulates (full/partial agonist), blocks (antagonist), or reduces (inverse agonist) GPCR signaling activity is, however, still challenging. A total of 31 β₁ (β₁R) and β₂ (β₂R) adrenoceptor crystal structures, including antagonist, inverse agonist, and partial/full agonist-bound structures, allowed us to explore the possibilities and limitations of structure-based prediction of GPCR ligand function. We used all unique protein–ligand interaction fingerprints (IFPs) derived from all ligand-bound β-adrenergic crystal structure monomers to post-process the docking poses of known β₁R/β₂R partial/full agonists, antagonists/inverse agonists, and physicochemically similar decoys in each of the β₁R/β₂R structures. The systematic analysis of these 1920 unique IFP–structure combinations offered new insights into the relative impact of protein conformation and IFP scoring on selective virtual screening (VS) for ligands with a specific functional effect. Our studies show that ligands with the same function can be efficiently classified on the basis of their protein–ligand interaction profile. Small differences between the receptor conformation (used for docking) and reference IFP (used for scoring of the docking poses) determine, however, the enrichment of specific ligand types in VS hit lists. Interestingly, the selective enrichment of partial/full agonists can be achieved by using agonist IFPs to post-process docking poses in agonist-bound as well as antagonist-bound structures. We have identified optimal structure–IFP combinations for the identification and discrimination of antagonists/inverse agonist and partial/full agonists, and defined a predicted IFP for the small full agonist norepinephrine that gave the highest retrieval rate of agonists over antagonists for <i>all</i> structures (with an enrichment factor of 46 for agonists and 8 for antagonists on average at a 1% false-positive rate). This β-adrenoceptor case study provides new insights into the opportunities for selective structure-based discovery of GPCR ligands with a desired function and emphasizes the importance of IFPs in scoring docking poses

Design, Synthesis, and Structure–Activity Relationships of Highly Potent 5‑HT₃ Receptor Ligands

The 5-HT₃ receptor, a pentameric ligand-gated ion channel (pLGIC), is an important therapeutic target. During a recent fragment screen, 6-chloro-<i>N</i>-methyl-2-(4-methyl-1,4-diazepan-1-yl)­quinazolin-4-amine (<b>1</b>) was identified as a 5-HT₃R hit fragment. Here we describe the synthesis and structure–activity relationships (SAR) of a series of (iso)­quinoline and quinazoline compounds that were synthesized and screened for 5-HT₃R affinity using a [³H]­granisetron displacement assay. These studies resulted in the discovery of several high affinity ligands of which compound <b>22</b> showed the highest affinity (p<i>K</i>_i > 10) for the 5-HT₃ receptor. The observed SAR is in agreement with established pharmacophore models for 5-HT₃ ligands and is used for ligand–receptor binding mode prediction using homology modeling and in silico docking approaches

Chemical Subtleties in Small-Molecule Modulation of Peptide Receptor Function: The Case of CXCR3 Biaryl-Type Ligands

The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure–activity relationship and structure–function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of <i>ortho</i>-substituents play an important role in determining agonist efficacies. Compounds <b>38</b> (VUF11222) and <b>39</b> (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling

Synthesis and Characterization of a Bidirectional Photoswitchable Antagonist Toolbox for Real-Time GPCR Photopharmacology

Noninvasive methods to modulate G protein-coupled receptors (GPCRs) with temporal and spatial precision are in great demand. Photopharmacology uses photons to control <i>in situ</i> the biological properties of photoswitchable small-molecule ligands, which bodes well for chemical biological precision approaches. Integrating the light-switchable configurational properties of an azobenzene into the ligand core, we developed a bidirectional antagonist toolbox for an archetypical family A GPCR, the histamine H₃ receptor (H₃R). From 16 newly synthesized photoswitchable compounds, VUF14738 (<b>28</b>) and VUF14862 (<b>33</b>) were selected as they swiftly and reversibly photoisomerize and show over 10-fold increased or decreased H₃R binding affinities, respectively, upon illumination at 360 nm. Both ligands combine long thermal half-lives with fast and high photochemical <i>trans</i>-/<i>cis</i> conversion, allowing their use in real-time electrophysiology experiments with oocytes to confirm dynamic photomodulation of H₃R activation in repeated second-scale cycles. VUF14738 and VUF14862 are robust and fatigue-resistant photoswitchable GPCR antagonists suitable for spatiotemporal studies of H₃R signaling

A Prospective Cross-Screening Study on G-Protein-Coupled Receptors: Lessons Learned in Virtual Compound Library Design

We present the systematic prospective evaluation of a protein-based and a ligand-based virtual screening platform against a set of three G-protein-coupled receptors (GPCRs): the β-2 adrenoreceptor (ADRB2), the adenosine A_2A receptor (AA2AR), and the sphingosine 1-phosphate receptor (S1PR1). Novel bioactive compounds were identified using a consensus scoring procedure combining ligand-based (frequent substructure ranking) and structure-based (Snooker) tools, and all 900 selected compounds were screened against all three receptors. A striking number of ligands showed affinity/activity for GPCRs other than the intended target, which could be partly attributed to the fuzziness and overlap of protein-based pharmacophore models. Surprisingly, the phosphodiesterase 5 (PDE5) inhibitor sildenafil was found to possess submicromolar affinity for AA2AR. Overall, this is one of the first published prospective chemogenomics studies that demonstrate the identification of novel cross-pharmacology between unrelated protein targets. The lessons learned from this study can be used to guide future virtual ligand design efforts

Catechol Pyrazolinones as Trypanocidals: Fragment-Based Design, Synthesis, and Pharmacological Evaluation of Nanomolar Inhibitors of Trypanosomal Phosphodiesterase B1

Trypanosomal phosphodiesterases B1 and B2 (TbrPDEB1 and TbrPDEB2) play an important role in the life cycle of <i>Trypanosoma brucei</i>, the causative parasite of human African trypanosomiasis (HAT), also known as African sleeping sickness. We used homology modeling and docking studies to guide fragment growing into the parasite-specific P-pocket in the enzyme binding site. The resulting catechol pyrazolinones act as potent TbrPDEB1 inhibitors with IC₅₀ values down to 49 nM. The compounds also block parasite proliferation (e.g., VUF13525 (<b>20b</b>): <i>T. brucei rhodesiense</i> IC₅₀ = 60 nM, <i>T. brucei brucei</i> IC₅₀ = 520 nM, <i>T. cruzi</i> = 7.6 μM), inducing a typical multiple nuclei and kinetoplast phenotype without being generally cytotoxic. The mode of action of <b>20b</b> was investigated with recombinantly engineered trypanosomes expressing a cAMP-sensitive FRET sensor, confirming a dose-response related increase of intracellular cAMP levels in trypanosomes. Our findings further validate the TbrPDEB family as antitrypanosomal target

Targeting a Subpocket in <i>Trypanosoma brucei</i> Phosphodiesterase B1 (TbrPDEB1) Enables the Structure-Based Discovery of Selective Inhibitors with Trypanocidal Activity

Several trypanosomatid cyclic nucleotide phosphodiesterases (PDEs) possess a unique, parasite-specific cavity near the ligand-binding region that is referred to as the P-pocket. One of these enzymes, <i>Trypanosoma brucei</i> PDE B1 (TbrPDEB1), is considered a drug target for the treatment of African sleeping sickness. Here, we elucidate the molecular determinants of inhibitor binding and reveal that the P-pocket is amenable to directed design. By iterative cycles of design, synthesis, and pharmacological evaluation and by elucidating the structures of inhibitor-bound TbrPDEB1, hPDE4B, and hPDE4D complexes, we have developed 4a,5,8,8a-tetrahydrophthalazinones as the first selective TbrPDEB1 inhibitor series. Two of these, <b>8</b> (NPD-008) and <b>9</b> (NPD-039), were potent (<i>K</i>_i = 100 nM) TbrPDEB1 inhibitors with antitrypanosomal effects (IC₅₀ = 5.5 and 6.7 μM, respectively). Treatment of parasites with <b>8</b> caused an increase in intracellular cyclic adenosine monophosphate (cAMP) levels and severe disruption of <i>T. brucei</i> cellular organization, chemically validating trypanosomal PDEs as therapeutic targets in trypanosomiasis