Developing small-molecule (SM) therapeutics that target membrane proteins (MPs) is often challenging, because few biophysical methods can handle the detergents required to maintain target stability. Here, we report a surface plasmon resonance (SPR)-based methodology that enables the characterization of interactions between SMs and an ion channel receptor (MP1) in complex with a stabilizing antibody fragment (Fab) and surfactant. Briefly, a stable MP1-Fab complex was formed by coimmobilizing MP1 with an anti-MP1-Fab within the hydrogel film to study the interactions of MP1 with SMs. The critical micelle concentration (CMC) is the concentration at which 50% of the surfactant monomers are assembled into micelles. Micelles readily absorb compounds resulting in compound-loaded micelles that generate high nonspecific binding and hinder resolution of SM binding responses. This micelle-induced interference was avoided by utilizing a weak detergent at a concentration below its CMC, allowing for the resolution of compound binding to a solvent-exposed pocket. Additional Fab stabilization was required to rescue binding at a second pocket buried within the transmembrane region of MP1. The resulting SPR-based assay proved invaluable during hit-to-lead optimization by progressing structure-activity relationship (SAR) studies and resolving the mechanism of action (MoA).

NALCN (sodium leak channel, nonselective) is vital for regulating electrical activity in neurons and other excitable cells, and mutations in the channel or its auxiliary proteins lead to severe neurodevelopmental disorders. Here, we show that the neuronal SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex proteins syntaxin and SNAP25 (synaptosome-associated protein 25), which enable synaptic transmission in the nervous system, inhibit the activity of the NALCN channel complex in both heterologous systems and primary neurons. The existence of this interaction suggests that the neurotransmitter release machinery can regulate electrical signaling directly and therefore modulate the threshold for its own activity. We further find that reduction of NALCN currents is sufficient to promote cell survival in syntaxin-depleted cells. This suggests that disinhibited NALCN may cause the puzzling phenomenon of rapid neuronal cell death in the absence of syntaxin. This interaction could offer opportunities for future drug development against genetic diseases linked to both NALCN- and SNARE protein-containing complexes.

High-resolution structures of proteins and protein complexes are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. CryoEM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins, including that underlie human diseases are small (<50 kDa) and usually beyond its reach due to low signal-to- noise images and difficulties in particle alignment. Previously reported solutions (structure chaperones), which directly bind to and increase the overall size of the particle are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the chaperones themselves. Here we present an iterative, structure-guided protein engineering using disulfides, of the Fab (Antibody fragment) scaffold, and present a solution that transforms the typically flexible Fab into a conformationally rigid scaffold with a distinctive shape, and maintains affinity to antigen. The designs are transferable across fabs from different species and chimeras. When bound to proteins even as small as 6–20 kDa, Rigid Fabs allow their structure determination to high resolutions of ∼2.3–2.5 Å for the protein of interest. Since a Fab can be discovered for practically any protein, our Rigid Fab design presents a general approach to overcome the target size limitation of single particle cryoEM.

The voltage-gated sodium (Na V ) channel Na V 1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available Na V channel-blocking drugs are not selective among the nine Na V channel subtypes, Na V 1.1–Na V 1.9. Moreover, the two currently known classes of Na V 1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure–activity relationships of the acylsulfonamide class of Na V 1.7 inhibitors, exemplified by the clinical development candidate GDC-0310 , has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the Na V 1.7 channel, we pursued high-resolution ligand-bound Na V 1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the Na V 1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in Na V channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective Na V channel modulators targeting VSD4.

Integral membrane proteins (MP) are implicated in many disease processes and are the primary targets of numerous marketed drugs. Despite recent advances in the areas of MP solubilization, stabilization, and reconstitution, it remains a time-consuming task to identify the combination of constructs and purification conditions that will enable MP structure-function studies outside of the lipid bilayer. In this chapter, we describe a strategy for rapidly identifying and optimizing the solubilization and purification conditions for nearly any recombinant MP, based on the use of a noninvasive fluorescent probe (His-Glow) that specifically binds to the common hexahistidine affinity tag of expressed targets. This His-Glow approach permits fluorescent size-exclusion chromatography (FSEC) without the need for green fluorescent protein (GFP) fusion. A two-stage detergent screening strategy is employed at the solubilization stage, whereby appropriate detergent families are identified first, followed by optimization within these families. Screening up to 96 unique combinations of solubilization conditions and constructs can be achieved in less than 24 h. At the outset of each new project, we screen six different detergents for each construct and the subsequent implementation of a simple thermostability challenge further aids in the identification of constructs and conditions suitable for large-scale production. Our strategy streamlines the parallel optimization of appropriate production conditions for multiple MP targets to rapidly enable downstream biochemical, immunization, or structural studies.

The outer membrane is an essential structural component of Gram-negative bacteria that is composed of lipoproteins, lipopolysaccharides, phospholipids, and integral β-barrel membrane proteins. A dedicated machinery, called the Lol system, ensures proper trafficking of lipoproteins from the inner to the outer membrane. The LolCDE ABC transporter is the inner membrane component, which is essential for bacterial viability. Here, we report a novel pyrrolopyrimidinedione compound, G0507, which was identified in a phenotypic screen for inhibitors of Escherichia coli growth followed by selection of compounds that induced the extracytoplasmic σE stress response. Mutations in lolC, lolD, and lolE conferred resistance to G0507, suggesting LolCDE as its molecular target. Treatment of E. coli cells with G0507 resulted in accumulation of fully processed Lpp, an outer membrane lipoprotein, in the inner membrane. Using purified protein complexes, we found that G0507 binds to LolCDE and stimulates its ATPase activity. G0507 still binds to LolCDE harboring a Q258K substitution in LolC (LolCQ258K), which confers high-level resistance to G0507 in vivo but no longer stimulates ATPase activity. Our work demonstrates that G0507 has significant promise as a chemical probe to dissect lipoprotein trafficking in Gram-negative bacteria.