Protein-protein interactions are at the heart of all cellular processes. To understand how cells work, and to develop effective interventions when cellular processes go awry, researchers need effective tools to observe and monitor the interactions between proteins. Inevitably, attempts to observe cellular dynamics introduce an artificial element into the system, either by immobilizing, labeling, or otherwise disrupting the cell’s natural activities. But new innovations keep improving these efforts. The four companies highlighted in this article have devised new methods to get closer to watching proteins interact in real time.
Antibodies conjugated to DNA oligos boost specificity
Navinci (formerly Olink Biosciences) pioneered the in situ proximity ligation assay (PLA). Now, the company has a new assay system that is designed to offer increased sensitivity and specificity. Called the Naveni Proximity Ligation Platform, the system can allow for detection of interacting proteins even at low levels without artificial overexpression.
“What differentiates our method from a typical immunostaining is the fact that we apply two antibodies at a time,” says Agata Zieba-Wicher, PhD, R&D director, Navinci. “It’s ideal for the detection of interacting proteins, or protein phosphorylation, or proteins themselves as the single targets.”
First, primary antibodies bind to each protein of interest. Next come the “Navenibodies,” antibodies that bind to the primary antibodies. Navenibodies are conjugated to single-stranded DNA oligos. These oligos can hybridize to each other, and they are key to the system’s improved specificity.
Individually, the oligos form hairpins, and each hairpin can be digested by a different enzyme. When the digestion enzyme cuts open the first hairpin, its ends are released, but the newly freed fragment remains hybridized to the section that’s still attached to the antibody. When the other oligo gets digested, that hairpin is released, creating a single-stranded fragment that hybridizes to the loose ends of the first oligo. This forms a circle. That circular DNA is amplified, incorporating fluorescently labeled nucleotides for detection.
The oligos form the circle only if the two proteins of interest interact, bringing the Navenibodies within 40 nm of each other. In the event of off-target antibody binding, there’s low risk of false positives. Linking the antibody and the oligo together in the Navenibodies also minimizes the number of steps and increases the sensitivity of the assay. “Every single step on top of the previous ones will decrease the efficiency of the method,” Zieba-Wicher points out. “Now, we see an increase in signal strength up to 10 times that of the original PLA.”
One example of the Naveni platform at work is detecting incipient metastasis in cancer cells. Protein complexes that form between b-catenin and E-cadherin hold neighboring cells together. When they stop interacting, and the cells lose contact with each other, migration begins. The change in fluorescence indicated by the Navenibodies when b-catenin and E-cadherin are no longer interacting corresponds to the onset of migration. Because metastasis is triggered by a change in this interaction, not simply the proteins’ expression levels, simply tagging them isn’t enough.
“The expression of these proteins is not telling us everything,” Zieba-Wicher explains. “By looking at the interaction between two different proteins on two different cells, one can see when this transition happens.”
The method can also detect protein phosphorylation. When an antibody for total protein is used together with an antibody that detects, say, phosphotyrosine, only the phosphorylated protein will generate a signal, because that’s where both antibodies will interact.
“One can actually detect specific phosphorylation without having a specific antibody,” Zieba-Wicher says. “The quality of phospho antibodies is not always the best. There’s a lot of cross-reactivity. But this approach can be used with any target, as long as an antibody against total protein exists.”
Luciferase-based assays enable the study of dynamic interactions
In 2012, Promega introduced NanoLuc, a 19-kD luciferase derived from shrimp and engineered to glow 100 times brighter than previous luciferases. It has served as a luminescent donor for bioluminescence resonance energy transfer (BRET) assays that measure molecular proximity. For example, Promega has used NanoLuc to create two highly sensitive assays for detecting protein-protein interactions, NanoBRET (NanoLuc BRET) and NanoBiT (NanoLuc Binary Technology).
In NanoBRET, NanoLuc is fused to a protein of interest, and the interacting protein is fused to HaloTag, a bacterial enzyme bearing a red-shifted fluorophore. When the two molecules come together, energy transferred from NanoLuc to HaloTag activates the fluorophore, generating a signal.
“BRET is pretty similar to FRET,” says Erik Bonke, PhD, application specialist, Promega. “The difference is that instead of having two fluorophores, we have a luciferase as the donor.”
In an earlier version, called BRET1, the wavelengths of the donor and acceptor overlapped considerably, limiting the assay’s sensitivity. NanoBRET’s red-shifted acceptor fluorophore triples the separation between the donor and acceptor peak wavelengths. “In consequence, less donor signal bleeds through into the acceptor channel,” Bonke explains. “This allows us to massively improve signal-to-background ratios and increase assay sensitivity.”
NanoBRET works well in studies of dynamic interactions because the signal is reversible. “If the proteins disassociate again, we lose the BRET signal,” Bonke notes. “This technology really allows you to look at dynamics, because you can measure both association as well as dissociation.”
In validation tests, NanoBRET successfully detected interactions between the proteins FKBP and FKR at lower than in vivo concentrations. “We can actually go below the endogenous expression level and still detect the interaction of these two proteins,” Bonke asserts. “NanoBRET is a very robust and sensitive technology.”
In another demonstration, NanoBRET quantified the activity of a drug, IBET151, on the interaction between BRD4 and two different histone proteins. Fusion to HaloTag did not prevent the histones from incorporating normally into the chromatin, Bonke points out. When the experiment was run, the fluorescence data showed that IBET151 disrupted the BRD4-histone 4 interaction more potently than it disrupted the BRD4-histone 3 interaction.
NanoBiT uses a version of NanoLuc split into a large subunit and a small subunit. Each subunit is fused to a protein of interest so that when the proteins interact, the subunits connect to form a functional luciferase. The subunits were optimized for stability and low affinity.
“These two subunits in solution will not spontaneously complement,” Bonke stresses. “They will complement only when brought together and held into physical contact by other means.”
Like NanoBRET, the signal is reversible because the subunits can be separated. “This is not possible with split autofluorescent proteins like split GFP,” Bonke advises. “Once those fragments are assembled, fluorophore maturation means they won’t dissociate. With NanoBiT, you can measure both ways.”
Whereas NanoBRET requires specialized instrumentation to allow the fluorescent signal to be read, NanoBiT works with a conventional luminometer. Although this difference would seem to count in NanoBiT’s favor, there is also a difference that counts in NanoBRET’s favor. That is, with NanoBiT, the two fusion tags must physically interact, whereas with NanoBRET, they need only come near each other. So, depending on the protein-protein interaction being studied, these differences may determine which assay is preferable.
GTP Gi binding assay detects GPCR activation nonradioactively
G protein–coupled receptors (GPCRs) consitute a large and diverse family of transmembrane proteins, and they play important roles in numerous signaling pathways. PerkinElmer recently introduced new assay kits intended to advance therapeutic discovery of GPCRs and GPCR inhibitors. One of these new assays is the HTRF GTP Gi binding kit, the industry’s first TR-FRET-based assay for GTP binding.
The kit uses homogeneous time-resolved fluorescence (HTRF), which combines fluorescence resonance energy transfer technology (FRET) with time-resolved measurement for detecting molecular interactions. A key feature of HTRF is that it’s very stable over time, allowing repeated measurements over hours or days.
When a ligand binds the extracellular surface of the GPCR, the signaling cascade begins with the exchange of GDP for GTP. The HTRF GTP Gi binding assay kit uses a GTP analog tagged with a fluorescent donor molecule and an anti-Gi antibody labeled with a fluorescent acceptor. Upon activation of the GPCR, the labeled GTP analog binds to the G protein, bringing donor and acceptor together and generating the fluorescent signal. The signal intensity is proportional to the GPCR activity, providing a direct reading of GPCR activation. Compounds that inhibit activation will decrease the signal.
“This is extremely powerful, since GPCRs are involved in signaling pathways related to many diseases,” says Mathis Laffenetre, life sciences technology platform reagents leader, PerkinElmer. “In the case of this new assay kit, GTP Gi binding is, for example, associated with pain pathways, so such research could help in the development of new treatments for chronic pain conditions.”
As the first TR-FRET assay for GTP Gi binding, the kit provides an effective nonradioactive option for studying GPCR activation.
“Currently, the only widely used method to monitor GTP Gi binding is a radioactivity-based approach,” Laffenetre notes. “Now, scientists who are either untrained in radioactivity or do not wish to use radioactive materials can study GTP Gi binding using fluorescence-based technology.”
The GTP Gi assay can be combined with PerkinElmer’s other GPCR kits to fully characterize the GPCR being studied. The cAMP Gi kit, for instance, detects changes in cAMP accumulation with GPCR activation to reveal second messenger activity. Amplification of the signaling can occur at many points throughout the cascade. That’s why, Laffenetre explains, it’s important to measure both the source of the signal as well as the end results.
“GTP binding and cAMP assays are lenses that respectively look into the source of the signal and the river it becomes as it travels inside the cell and collects inputs from other biological systems,” he points out. “Using a dual GTP binding/cAMP system therefore allows for a more insightful picture of the signal because it accounts for that difference between the signal source and the end results.” A drug candidate that blocks the GPCR, he adds, might be less attractive to pursue if downstream amplifications account for a larger percentage of the final signal.
Nucleotide exchange can reveal turns along the Kras pathway
KRas mutations are common in cancers, but the protein has long been considered “undruggable” as it has no well-defined binding pocket that can be targeted with small molecules. Recently, however, new binding pockets have been identified, reviving interest in searching for potential inhibitors of KRas.
This year, Amgen’s Lumakras became the first KRas inhibitor to be approved by the FDA, and others may soon follow. But direct inhibition of KRas isn’t the only option: alternative approaches being explored include inhibition of exchange factor proteins or inhibition of interaction with effector proteins.
“We have produced proteins and validated a set of assays for the discovery and characterization of compounds that target different steps of the KRas pathway,” says Ekaterina Kuznetsova, PhD, research scientist, Reaction Biology.
KRas relies on effector proteins, including SOS1 and SOS2, which mediate the conversion of GDP to GTP. Reaction Bio’s suite of assays includes a nucleotide exchange assay, which detects SOS1/2-mediated exchange of fluorescently labeled GDP to GTP, as well as protein-protein interaction assays to screen for compounds that block the interaction between KRas and SOS1 or cRAF proteins.
In the nucleotide exchange assay, KRas is labeled with a fluorescent GDP molecule. When SOS1/2 exchanges GDP for unlabeled GTP, and KRas becomes activated, the fluorescence level decreases. Thus, measuring the fluorescence level amounts to measuring KRas activation.
The assay can be used to screen for compounds that inhibit activation of KRas or various KRas mutants. In the presence of inhibitors, the fluorescence level remains steady, indicating that the GDP-GTP exchange hasn’t taken place and that KRas remains “off.” The assay is also useful as a selectivity tool, to compare the effects of inhibitory compounds on different KRas mutants.
Reaction Bio also has HTRF-based assays to detect disruption of protein-protein interactions. Successful binding between GDP-bound KRas and SOS1 will generate a fluorescent signal, and the activity of inhibitors can be quantified by the drop in fluorescent signals. The cRAF assay uses GTP-bound KRas but works the same way. “We observe inhibition of this interaction both in wild-type KRas and G12 mutants,” Kuznetsova notes.
Reaction Bio also has a biophysical assay that uses surface plasmon resonance (SPR) to quantify binding affinity with KRas and SOS1. Wild-type or mutant KRas is bound to the chip, and the compound flows over it. “As it interacts with the protein, the measurements are performed in real time, so it enables you to calculate the rate of the association and dissociation,” Kuznetsova explains. “Based on those rates, it’s possible to calculate binding affinity.”
“You’re not limited to small-molecule binding,” Kuznetsova continues. “It’s also possible to look at peptide-binding and protein-protein interactions.” Binding SOS1 to the chip and flowing KRas plus the inhibitory compound BAY-293 enable quantification of binding inhibition at different concentrations of the compound.
The array of innovative tools and techniques allow researchers to observe, characterize, and quantify formerly inscrutable protein-protein interactions. As these tools generate new experiments and discoveries, more precise targeted therapeutics will surely follow.