Joel Karpiak

Structure-based ligand discovery for the protein–protein interface of chemokine receptor CXCR4

G-protein–coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein–protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC50 values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein–protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

Structural basis of G protein–coupled receptor–G protein interactions

The interaction of G protein-coupled receptors (GPCRs) with heterotrimeric G proteins represents one of the most fundamental biological processes. However, the molecular architecture of the GPCR-G protein complex remains poorly defined. In the present study, we applied a comprehensive GPCR-G protein alpha subunit (Galpha) chemical cross-linking strategy to map a receptor-Galpha interface, both before and after agonist-induced receptor activation. Using the M(3) muscarinic acetylcholine receptor (M3R)-Galpha(q) system as a model system, we examined the ability of approximately 250 combinations of cysteine-substituted M3R and Galpha(q) proteins to undergo cross-link formation. We identified many specific M3R-Galpha(q) contact sites, in both the inactive and active receptor conformations, allowing us to draw conclusions regarding the basic architecture of the M3R-Galpha(q) interface and the nature of the conformational changes following receptor activation. As heterotrimeric G proteins as well as most GPCRs share a high degree of structural homology, our findings should be of broad general relevance.

Allosteric ligands for the pharmacologically dark receptors GPR68 and GPR65

At least 120 non-olfactory G-protein-coupled receptors in the human genome are ‘orphans’ for which endogenous ligands are unknown, and many have no selective ligands, hindering the determination of their biological functions and clinical relevance. Among these is GPR68, a proton receptor that lacks small molecule modulators for probing its biology. Using yeast-based screens against GPR68, here we identify the benzodiazepine drug lorazepam as a non-selective GPR68 positive allosteric modulator. More than 3,000 GPR68 homology models were refined to recognize lorazepam in a putative allosteric site. Docking 3.1 million molecules predicted new GPR68 modulators, many of which were confirmed in functional assays. One potent GPR68 modulator, ogerin, suppressed recall in fear conditioning in wild-type but not in GPR68-knockout mice. The same approach led to the discovery of allosteric agonists and negative allosteric modulators for GPR65. Combining physical and structure-based screening may be broadly useful for ligand discovery for understudied and orphan GPCRs.

Design, synthesis, crystal structures, and antimicrobial activity of sulfonamide boronic acids as β-lactamase inhibitors.

We investigated a series of sulfonamide boronic acids that resulted from the merging of two unrelated AmpC β-lactamase inhibitor series. The new boronic acids differed in the replacement of the canonical carboxamide, found in all penicillin and cephalosporin antibiotics, with a sulfonamide. Surprisingly, these sulfonamides had a highly distinct structure-activity relationship from the previously explored carboxamides, high ligand efficiencies (up to 0.91), and K(i) values down to 25 nM and up to 23 times better for smaller analogues. Conversely, K(i) values were 10-20 times worse for larger molecules than in the carboxamide congener series. X-ray crystal structures (1.6-1.8 Å) of AmpC with three of the new sulfonamides suggest that this altered structure-activity relationship results from the different geometry and polarity of the sulfonamide versus the carboxamide. The most potent inhibitor reversed β-lactamase-mediated resistance to third generation cephalosporins, lowering their minimum inhibitory concentrations up to 32-fold in cell culture.

In silico design of novel probes for the atypical opioid receptor MRGPRX2

The primate-exclusive MRGPRX2 G protein-coupled receptor (GPCR) has been suggested to modulate pain and itch. Despite putative peptide and small molecule MRGPRX2 agonists, selective nanomolar potency probes have not yet been reported. To identify a MRGPRX2 probe, we first screened 5,695 small molecules and found many opioid compounds activated MRGPRX2, including (−)- and (+)-morphine, hydrocodone, sinomenine, dextromethorphan and the prodynorphin-derived peptides, dynorphin A, dynorphin B, and α- and β-neoendorphin. We used these to select for mutagenesis-validated homology models and docked almost 4 million small molecules. From this docking, we predicted ZINC-3573, which represents a potent MRGPRX2-selective agonist, showing little activity against 315 other GPCRs and 97 representative kinases, and an essentially inactive enantiomer. ZINC-3573 activates endogenous MRGPRX2 in a human mast cell line inducing degranulation and calcium release. MRGPRX2 is a unique atypical opioid-like receptor important for modulating mast cell degranulation, which can now be specifically modulated with ZINC-3573.

Actin Is Required for IFT Regulation in Chlamydomonas reinhardtii

Assembly of cilia and flagella requires intraflagellar transport (IFT), a highly regulated kinesin-based transport system that moves cargo from the basal body to the tip of flagella [1]. The recruitment of IFT components to basal bodies is a function of flagellar length, with increased recruitment in rapidly growing short flagella [2]. The molecular pathways regulating IFT are largely a mystery. Because actin network disruption leads to changes in ciliary length and number, actin has been proposed to have a role in ciliary assembly. However, the mechanisms involved are unknown. In Chlamydomonas reinhardtii, conventional actin is found in both the cell body and the inner dynein arm complexes within flagella [3, 4]. Previous work showed that treating Chlamydomonas cells with the actin-depolymerizing compound cytochalasin D resulted in reversible flagellar shortening [5], but how actin is related to flagellar length or assembly remains unknown. Here we utilize small-molecule inhibitors and genetic mutants to analyze the role of actin dynamics in flagellar assembly in Chlamydomonas reinhardtii. We demonstrate that actin plays a role in IFT recruitment to basal bodies during flagellar elongation and that when actin is perturbed, the normal dependence of IFT recruitment on flagellar length is lost. We also find that actin is required for sufficient entry of IFT material into flagella during assembly. These same effects are recapitulated with a myosin inhibitor, suggesting that actin may act via myosin in a pathway by which flagellar assembly is regulated by flagellar length.

Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Significance Specific interactions between proteins control the function of essentially all cellular processes. Despite the importance of interaction specificity, it is unclear how structurally similar proteins achieve their unique recognition preferences. Here, we redesign the specificity of a protein binding domain and quantify the extent to which the designed specificity switch can be transferred to homologous domains. We show that identical mutations in structurally similar domains have a wide range of effects on specificity. We apply a structure-based computational model that recapitulates this context dependence. Our findings show how subtle structural differences between homologous domains contribute to their unique specificities. The differential responses to similar mutation observed here could help explain how families of recognition domains have evolved diverse new interactions. Reengineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.

Structure-inspired design of β-arrestin-biased ligands for aminergic GPCRs

Development of biased ligands targeting G protein-coupled receptors (GPCRs) is a promising approach for current drug discovery. Although structure-based drug design of biased agonists remains challenging even with an abundance of GPCR crystal structures, we present an approach for translating GPCR structural data into β-arrestin-biased ligands for aminergic GPCRs. We identified specific amino acid-ligand contacts at transmembrane helix 5 (TM5) and extracellular loop 2 (EL2) responsible for Gi/o and β-arrestin signaling, respectively, and targeted those residues to develop biased ligands. For these ligands, we found that bias is conserved at other aminergic GPCRs that retain similar residues at TM5 and EL2. Our approach provides a template for generating arrestin-biased ligands by modifying predicted ligand interactions that block TM5 interactions and promote EL2 interactions. This strategy could facilitate the structure-guided design of arrestin-biased ligands at other GPCRs, including polypharmacological biased ligands.

Small-Molecule Allosteric Modulators of the Protein Kinase PDK1 from Structure-Based Docking.

Finding small molecules that target allosteric sites remains a grand challenge for ligand discovery. In the protein kinase field, only a handful of highly selective allosteric modulators have been found. Thus, more general methods are needed to discover allosteric modulators for additional kinases. Here, we use virtual screening against an ensemble of both crystal structures and comparative models to identify ligands for an allosteric peptide-binding site on the protein kinase PDK1 (the PIF pocket). We optimized these ligands through an analog-by-catalog search that yielded compound 4, which binds to PDK1 with 8 μM affinity. We confirmed the docking poses by determining a crystal structure of PDK1 in complex with 4. Because the PIF pocket appears to be a recurring structural feature of the kinase fold, known generally as the helix αC patch, this approach may enable the discovery of allosteric modulators for other kinases.

Selectivity Challenges in Docking Screens for GPCR Targets and Anti-Targets

To investigate large library docking’s ability to find molecules with joint activity against on-targets and selectivity versus antitargets, the dopamine D2 and serotonin 5-HT2A receptors were targeted, seeking selectivity against the histamine H1 receptor. In a second campaign, κ-opioid receptor ligands were sought with selectivity versus the μ-opioid receptor. While hit rates ranged from 40% to 63% against the on-targets, they were just as good against the antitargets, even though the molecules were selected for their putative lack of binding to the off-targets. Affinities, too, were often as good or better for the off-targets. Even though it was occasionally possible to find selective molecules, such as a mid-nanomolar D2/5-HT2A ligand with 21-fold selectivity versus the H1 receptor, this was the exception. Whereas false-negatives are tolerable in docking screens against on-targets, they are intolerable against antitargets; addressing this problem may demand new strategies in the field.