Mapping the Surfaceome To Uncover Potential Drug Targets

Many of the most promising classes of drugs in development—from CAR T-cell therapies to antibody-drug conjugates—target proteins expressed on the cell surface.

In fact, it’s estimated that as many as 70% of drugs on the market today target cell-surface proteins. That’s not terribly surprising, since ensuring that a protein target is accessible to intervention is essential for developing a candidate into a successful therapy.

Even outside of the pharmaceutical industry, the biological importance of cell-surface proteins cannot be underplayed, given their key role in facilitating communication between a cell and its surroundings.

Given these factors, it seems clear that the ability to detect and identify all proteins that are accessible on the surface of the cell—described as the “surfaceome”—would be an important step for scientists in drug discovery and development programs.

Historically, no commercially available technology has been capable of the spatial precision needed to characterize the surfaceome; the overwhelming success of drugs targeting cell-surface proteins so far has been largely serendipitous. But technology has been evolving quickly, with the advent of spatial proteomics and single-cell tools that give scientists a greater ability to focus their studies on proteins found in specific locations.

These developments have been welcome complements to the older generation of tools, capable only of protein analysis in bulk cell populations. Still, these newer platforms vary greatly in the resolution and precision they deliver.

Challenges in targeting cell-surface proteins

Since accessibility of a protein is so important, it seems obvious that scientists would target those found on a cell’s surface. But identifying proteins within that sweet spot is very difficult.

For one thing, many proteins do not stay idly localized to a cell’s membrane; they move around, sometimes shuttling down into the cell or back out of it as they are influenced by environmental stimuli, disease conditions, and cell state. As a result, cell-surface proteins may change location, abundance, and even function. The dynamic cell membrane is a constantly shifting landscape that cannot be understood with a simple biological snapshot.

Another key challenge in characterizing the surfaceome stems from the relatively low abundance of these proteins, which represent just a small fraction of total cellular protein by mass.

Additionally, cell-surface proteins are difficult to isolate and analyze due to their heterogeneous and hydrophobic nature. Confirming their location requires pinpoint accuracy; the cell surface lipid bilayer is only ~5–10 nm thick. Therefore, proteins just inside the membrane could be easily mistaken for being on the surface, but from a drug-delivery perspective, they might be useless as therapeutic targets.

Despite these challenges, correctly identifying cell-surface proteins is vital for drug discovery programs. Protein targets believed to be accessible for therapeutic intervention but that ultimately aren’t can lead to failures in late-stage trials, costing millions in lost time and development investment.

The surfaceome represents the ideal pool of targets: proteins with the potential to serve as biological gateways for therapy.

Surfaceome analysis approaches

For the most part, conventional analytical tools cannot achieve the location precision needed to detect all proteins accessible on a cell surface. Commonly used approaches, such as laser capture microdissection and proximity labeling, have their own disadvantages. Laser microdissection, for example, can achieve single-cell precision, but it cannot be used to focus on an area as small as the cell surface. At the same time, proximity labeling can suffer from experimental artifacts and insufficient signal, while challenges associated with artificial binding can produce non-specific results.

Even with these newer spatial biology and single-cell platforms, protein analysis is usually limited to known targets. To characterize the surfaceome, which may contain many unexpected or even unknown proteins, a true discovery technique is required.

Another new approach leverages the strengths of these different technologies to achieve the precision needed to reliably identify cell-surface proteins. In this case, high-resolution microscopy is used to identify a biological region of interest with accuracy down to the tens of nanometers. Next, light emitted from a laser, in combination with a photoactivated reagent, photo-biotinylates proteins specifically within that region. These labeled proteins are then enriched and analyzed by mass spectrometry, enabling the discovery and identification of proteins from precisely the location of interest.

At this year’s United States meeting of the Human Proteome Organization, scientists presented results from a study in which this microscopy-guided proteomic approach was used for biomarker and target discovery. An analysis of HeLa cells identified at least 3500 proteins, of which more than 1600 were known cell-surface proteins. A Gene Ontology analysis found that over half of the most enriched proteins were localized to plasma-membrane compartments; an adjustment to the workflow protocol intended to increase specificity boosted that rate to about 70% of the most enriched proteins. The study also enabled scientists to discover novel cell-surface proteins that could be of interest for future target identification work.

Applications in drug discovery

In biopharmaceuticals, the ability to characterize cell-surface proteins will be most important for the identification of drug targets and discovery of biomarkers.

Target identification efforts can benefit from ensuring the physical accessibility of a cell-surface protein, a crucial component of selecting a strong target. Successfully identifying cell-surface proteins will eliminate the need to work out the challenges of intracellular delivery later down the line.

In biomarker discovery, the ability to select proteins based on their membrane localization will increase the likelihood that these biomarkers will be accessible for downstream clinical testing. Surface proteins also tend to be more biologically relevant for certain aspects of disease, with changes indicative of progression or successful intervention. Many of the best-known biomarkers used for precision medicine—matching patients with therapies most likely to be effective for their specific cases—are proteins found on the surface of cells.

Characterizing the surfaceome early in the drug discovery process will help to ensure that only the higher-confidence targets or biomarkers are advanced to the more costly stages of development. Having certainty that a protein of interest is found on the cell surface is useful, actionable information that can help minimize late-stage failures and potentially translate into meaningful improvements in efficacy.