Developing drugs for the ‘undruggable’

Over the last decade, PPIs have emerged as promising targets in the precision treatment of serious diseases in which therapeutic options are limited. This has opened up new avenues to target classically considered ‘undruggable’ proteins.

It has been estimated that there are up to 650,000 PPIs in the human interactome and only approximately 2% of these had been targeted with drugs by 2011 [1]. They underpin many important cellular processes – examples of disease-relevant PPIs include the associations between transcription factors and their co-activators, and other signaling proteins. However, due to difficulties with their study, most have remained elusive and not fully understood.

PPIs are attractive molecular targets for modern drug discovery, which is driven by the identification of novel therapeutics to selectively address disease-specific pathways [2]. The potential for drug development with PPIs is therefore immense; however, there are many challenges with the identification of small-molecule interference compounds, resulting in the need for novel screening approaches.

Addressing the ‘undruggable’

PPIs are classically considered to be undruggable [3]. This is due to the way in which the proteins interact – they don't have well-defined binding pockets, unlike enzyme–substrate interactions that are considered to be druggable. Instead, they have contact surfaces that are usually flat, featureless and relatively large, interacting through electrostatic and hydrophobic interactions, hydrogen bonds and Van der Waals forces over less well-defined, larger areas. These differences are demonstrated in Figure 1.

As they don't bind to small-molecule ligands (or substrates), there is no starting point provided for drug design to be based from, as is the case with enzyme–substrate interactions. PPIs provide further difficulties due to there being no natural measure for their occurrence, again unlike an enzyme–substrate interaction, there is no product formed as a result. This makes in vitro study and assay development difficult.

Even so, high-throughput screening also rarely identifies compounds that can fit PPIs. This is due to traditional drug compounds being too small to cover and bind to the involved proteins' surfaces. Larger biomolecules, such as other proteins or peptides, would be an ideal solution if not for their poor pharmacokinetic properties [3].

Despite these hurdles, it is crucial to explore PPIs as novel targets due to there being a greater number of them than single protein targets [5]. In theory, every disease could be tackled by PPI inhibitors and this is an especially attractive principle for diseases, such as Alzheimer's, that are hard to target in conventional approaches and lack ‘good’ enzymes, GPCRs or ion channels.

High-throughput screening

Renowned BioTechniques Editorial Board member, Igor Stagljar (a professor of Molecular Genetics and Biochemistry at the Donnelly Centre in the University of Toronto; ON, Canada), is at the forefront of PPI research and technology development [6].

Stagljar directs major proteomics projects to map how membrane proteins interact with each other and to identify novel PPI therapeutic targets. He has led the development of numerous technologies that have revolutionized research of how membrane proteins interact, including the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH).

Most recently, Stagljar's lab have further developed the MaMTH technology into a drug screening platform (MaMTH-DS) to assist in the development of therapeutics targeting membrane PPIs [7], as well as the split intein-mediated protein ligation (SIMPL) technology to improve the development of therapeutics against soluble PPIs [8]. Both screening platforms are live cell-based approaches, developed to address limitations with current screening methods.

The SIMPL approach involves fusing bait and prey proteins to intein N-terminal and C-terminal fragments, respectively, derived from a re-engineered split intein GP41-1. When the bait and prey proteins associate, the intein reconstructs, splicing the bait and prey peptides into a single protein that can then be detected by western blot and ELISA. With the additional development of a SIMPL ELISA platform, this allows for high-throughput PPI screening and inhibitor identification.

The MaMTH-DS method has been developed for the high-throughput identification of small molecules that target functional PPIs of receptor tyrosine kinases (RTKs) – transmembrane receptors with key pathological roles. RTK-targeting therapeutics are usually identified by in vitro kinase assays; however, there has been a need to diversify screening methods to identify more novel inhibitors for mutated RTKs that emerge as a result of drug resistance. Hence, the MaMTH-DS platform can be used alongside traditional drug screening approaches.

The success story

There is still a long way to go with exploring the potential of PPIs in drug development. Novel screening methods like Stagljar's will play a key role in how the future of this unfolds. However, there has already been a success story. In 2016, venetoclax became the first FDA-approved PPI inhibitor for use in treating patients with chronic lymphocytic leukemia [9].

Venetoclax is a small-molecule inhibitor that targets the interaction between the BCL-2 protein and pro-apoptotic proteins, such as BAK and BAX. In tumor cells that are ‘primed for death’, elevated levels of the BCL-2 protein (an anti-apoptotic protein) bind the pro-apoptotic proteins, preventing the cells from undergoing apoptosis, as demonstrated in Figure 2. When venetoclax interrupts this mechanism, binding to BCL-2, apoptosis can then be induced.

There are currently additional PPI-targeting therapeutics in preclinical and clinical trial stages, including two identified by Stagljar's MaMTH-DS technology for lung cancer patients who have become resistant to current therapies, one of which is expected to enter clinical trials at the end of 2020 [7]. Gilteritinib and midostaurin are already approved for use in patients with acute myeloid leukemia, so the team solely need to demonstrate efficacy in lung cancer patients to make them a viable new treatment option.

Determining the new normal

Cancer research is continually developing and striving for novel treatments in an effort to increase patient success rates and control cancers. But what other fields could benefit from targeting PPIs for novel therapeutic development? There has been recent work in the infectious diseases field with increased knowledge and characterization of PPIs between host and pathogen.

Specifically, in relation to malaria, there has been incredible progress in reducing the global malaria burden with improved prevention methods and reduced transmission rates. However, new threats include drug resistance and vaccine inefficacy.

To handle these, new targets for drug and vaccine development are sought after, so with increased understanding of the host–pathogen malaria interactome comes the possibility for new targets and treatments, which are currently under investigation [10].

Another infectious disease whose PPIs are under investigation for novel therapeutic targets is SARS-CoV-2 – the virus responsible for the global COVID-19 pandemic. In April 2020, a team of researchers published an article in Nature detailing how they cloned, tagged and expressed 26 of the 29 SARS-CoV-2 proteins that are present in human cells and identified the human proteins that physically associate with each of the SARS-CoV-2 proteins with the aim to reveal targets for drug repurposing [11].

They identified 332 PPIs between host and pathogen proteins and 66 druggable human proteins that are targeted by 69 compounds. When they screened a subset of the compounds, they discovered that two sets displayed antiviral activity. They believe that upon further study, and upon combination with drugs that directly target viral enzymes, a therapeutic treatment regimen for COVID-19 infection could be discovered through PPI inhibitor identification.

There is great promise for PPIs as targets in drug discovery. With improved screening capabilities, PPI identification and mechanism elucidation, comes the increased identification of novel therapeutic compounds. There is hope that utilizing PPIs in this way will become a ‘new normal’ step in drug discovery, making the ‘undruggable’, ‘druggable’.