**1. Introduction: targeting ePPIs to address disease burden**

The World Health Organization estimates that over 70% of deaths in 2016 worldwide were due to non-communicable diseases like cardiovascular disease (CVD) and cancer. This number is expected to grow to over 80% by 2060 [1]. Even if these diseases arise from environmental damage, the disease states usually depend on altered cellular communication, driven at the molecular level by altered ePPIs. For example, interactions between immune cells and arterial walls through adhesion proteins can initiate positive feedback loops which drive atherosclerotic plaque formation in CVD [2]. Similarly, while genetic mutations are the root cause of cancer, aberrant cell–cell interactions allow cancer cells to evade the immune system [3], migrate [4], siphon nutrients [5] and ignore signals to stop growing [6]. EPPIs also contribute to communicable diseases, which, highlighted by the

(ongoing as of this writing) COVID-19 pandemic, can rapidly increase human deaths with the introduction of a novel pathogen. As with many pathogens, the virus underlying the pandemic, SARS-CoV-2, exploits host cell-surface receptors to enter cells to replicate and spread [7, 8].

Because ePPIs are often central to the initiation and progression of diseases, they offer opportunities for molecular intervention using drugs. Greater understanding of the ePPIs underlying diseases allows them to be effectively targeted and manipulated to reverse disease phenotypes. For example, for CVD, several efforts to target different cytokines are showing promise in stemming the progression of atherosclerosis [9]. The development of cancer immunotherapies in the last decade has revolutionized cancer treatment. These treatments block ePPIs between immune checkpoint proteins such as CTLA-4 or PD-L1 and their binding partners to reinvigorate the body's defenses [10]. Even with SARS-CoV-2, an antibody cocktail (REGN-COV2) that blocks the ePPI between the virus spike protein and receptors on the cell, has been shown to stop viral entry and has gained emergency authorization for use in COVID-19 patients [11]. These examples highlight that identifying and targeting ePPIs can have strong therapeutic benefits in a variety of known and emerging diseases that make up a significant portion of human disease burden worldwide (**Figure 1**).

Despite the importance of ePPIs for both understanding and treating disease, our understanding of this field remains limited, especially compared to other classes of protein–protein interactions (PPIs). A main reason for this disparity is that common techniques for general PPI discovery are not well suited for ePPIs. Interactions between individual secreted or membrane proteins are typically weak, making them difficult to capture. Membrane proteins are biochemically recalcitrant and tend to misfold or aggregate outside of a native membrane context making them incompatible with many readouts designed for soluble proteins. Extracellular proteins also pick up many complex and heterogeneous post-translational modifications on their journey out of the cell, including specific disulfide bonds designed for the non-reducing extracellular environment. Since these can play roles in ePPIs but are not well characterized, they can be missed by common non-native expression

#### **Figure 1.**

*Examples of therapeutically relevant ePPIs. (A) The tumor microenvironment consists of a complex mix of cell types that communicate through ePPIs. One example is the expression of immune checkpoint proteins such as PD-L1 on cancer cells, which inhibits cytotoxic T-cell function, allowing the cancer cells to evade the immune system. Drugs targeting these ePPIs are the foundation for the cancer immunotherapies, which have provided significant benefits for cancer patients. Many other ePPIs in this space are under active investigation. (B) SAR-CoV-2 uses its spike protein to co-op the ACE2 receptor for viral entry into host cells and initiate viral replication and infection. Strategies for blocking this interaction are being explored to address the COVID-19 pandemic.*

*Unbiased Identification of Extracellular Protein–Protein Interactions for Drug Target… DOI: http://dx.doi.org/10.5772/intechopen.97310*

systems [12, 13]. Altogether, these biochemical features make most available technologies suboptimal and as a result, ePPIs are remarkably underrepresented in current databases.

Because of the difficulties with ePPI discovery, many new approaches have been developed to specifically identify human ePPIs that play roles in homeostasis and disease. While past low-throughput methods and focused studies have provided fundamental insights into specific receptors and pathways, the rapid explosion in sequencing, mass spectrometry (MS), targeted mutagenesis and high-throughput screening techniques has made the exhaustive identification of ePPIs a realistic goal. Here, we will address how new techniques deal with unique challenges associated with ePPIs and highlight the progress towards to the elucidation of a comprehensive network map of all human ePPIs.

### **2. Methods for detecting ePPIs**

From biophysical approaches to *in vivo* studies, a number of methods have been developed or are being improved that have the potential to enable unbiased ePPIs discovery. The majority of methods can be categorized into a few broad technological concepts: biochemical fractionation, affinity purification, protein-fragment complementation, proximity labeling, direct protein interaction detection and computational modeling. As is the case for other disciplines, deciphering the complexities of extracellular interactions requires a multipronged approach. Since the different approaches provide different types of information, these methodologies are complementary. Especially as these categories have matured, many new techniques bridge the different concepts to balance the various benefits and shortcomings and push for increased throughput. The specific method-of-choice will depend on the expertise, equipment and overall resources available in each laboratory (**Table 1**).


#### **Table 1.**

*Comparison of approaches for unbiased detecting ePPIs.*

#### **2.1 Biochemical fractionation**

#### *2.1.1 Concept description*

Biochemical fractionation is the splitting of a complex lysate, typically cell or tissue extract, into simpler mixtures to identify the simplest solution that retains a certain biochemical property. The measurable property could be complex cellular activities, such as the stimulation of cell migration, or simple ones such as binding to a target protein (**Figure 2**).

#### *2.1.2 Concept pros*

Biochemical fractionation does not require any knowledge of the components and can be an unbiased technique. It is a versatile concept since any biochemical property can be studied, from *in vivo* tissue level responses to molecular PPIs. Some degree of fractionation is easily combined with other techniques to reduce the starting complexity and to improve data interpretability.

#### *2.1.3 Concept cons*

The results from biochemical fractionations are dependent on the particular purification steps used and can be highly variable. Due to the multiple purification steps, this approach can also be labor and time intensive. The different purification steps can inactivate proteins by inducing misfolding or removing key co-factors. This is especially true for ePPIs that involve membrane proteins, which can lose activity if extracted from membranes [12, 13].
