The challenge of hard-to-drug targets

Billions of dollars and many research programs have been devoted to conventional drug discovery strategies, such as small molecules and monoclonal antibodies (mAbs). But they have not been able to successfully target these molecules. Today, the term ‘undruggable’ is being redefined. Advances in membrane protein solubilization, allosteric modulation, and therapeutic viral vectors have led to proof that these targets can be tackled.

VHH antibodies are emerging as a powerful solution, offering unparalleled stability, access to hidden epitopes, and even genetic delivery as “intrabodies” to hit intracellular targets. This blog will explore where challenges remain for hard-to-drug targets, the limitations of existing strategies, and how VHH antibodies unlock new possibilities.

Why do some targets remain “undruggable”? 

Despite recent advances achieved in drug discovery, many high-value targets remain intractable. This is due to their structural complexity, lack of easily accessible binding sites, or dynamic states. These proteins are not adequately targeted by conventional small molecules and mAbs, which limits the development of effective therapeutics.  Proteins lacking conventional binding pockets present a major challenge in drug discovery.

Small GTPases like KRAS, HRAS, and NRAS feature shallow and poorly defined pockets and are thus insensitive to conventional small-molecule inhibitors. Likewise, transcription factors like Myc, p53, ER, and AR possess highly disordered conformations with minimal binding sites, and it is hard to target them with both small molecules and biologics [1]

Another obstacle is the failure to target highly conserved or poorly immunogenic proteins. GPCRs and ion channels, which are essential for numerous physiological processes, are structurally complex and poorly immunogenic, and functional antibodies are hard to induce from animal immune responses.

Phosphatases, despite their importance to cellular signaling, are beset by high structural homology between species, making any immunization challenging. That’s why AstraZeneca chose Isogenica’s synthetic library approach to tackle the intracellular cardiac target phospholamban: 

Some proteins present additional challenges due to their intrinsic disorder. Many protein-protein interactions, such as those in the Bcl-2 family, lack a fixed conformation, making them difficult to target with traditional drugs [2]. Similarly, epigenetic modulators, DNA methyltransferases, and histone modifiers add another layer of complexity, often slowing drug development [3]. 

Beyond these biochemical hurdles, traditional biologics struggle with accessibility and tissue penetration. Large mAbs fail to reach intracellular or access cryptic epitopes and often cannot diffuse effectively into solid tumors due to both size and Fc-mediated effects. Advances in fragment-based drug discovery, AI-driven structure prediction, and nucleic acid medicine have improved targeting strategies. However, many difficult targets remain elusive, highlighting the need for next-generation biologics [4]

VHH antibodies as a potential tool for drugging challenging targets 

Many promising drug targets are inaccessible to conventional mAbs due to structural complexity and poor tissue penetration. In contrast, VHH antibodies overcome these limitations through their unique structural features. Their comparatively small 15 kDa size makes them able to penetrate deep tissues, while their extended CDR3 facilitates binding inside pockets and clefts that are not accessible to large antibodies. This is particularly important for binding to hidden surfaces inside membrane proteins like GPCRs and ion channels, making them valuable in oncology, neurology, and autoimmune disease treatment. 

In addition, VHHs are more resistant to extreme conditions, including high temperatures and low pH, and thus they are the most suitable for numerous applications – ranging from therapeutics to diagnostics.  While their small size can limit the number of sites available for chemical conjugation, their structural simplicity and stability make them ideal for genetic delivery approaches and intracellular expression. This enables their use in applications like gene therapy and targeted protein degradation which express the VHH in vivo either displayed on the surface of the cell or freely in the cytoplasm as “intrabodies”.

VHHs are capable of binding challenging targets and have proven instrumental in various therapeutic and structural applications. In the field of structural biology, they have long been used as crystallization chaperones, especially for GPCRs and membrane proteins, where they help stabilize dynamic conformations and enable high-resolution structural determination by X-ray crystallography and cryo-EM. Beyond that, VHHs continue to play a pivotal role in oncogenic pathway inhibition and immunotherapy engineering, demonstrating their therapeutic and mechanistic value across fields. 

VHHs are revolutionizing GPCR targeting 

A compelling example of VHHs unlocking hard-to-drug targets can be seen in their success with GPCR targeting – a challenging class of drug targets in modern medicine. GPCRs are involved in nearly every physiological process, from neurotransmission [5] to immune responses [6], yet their highly dynamic conformations make them notoriously difficult to drug with conventional biologics [1]. 

Recent studies have shown a marked increase in VHH-stabilized GPCR structures deposited in the PDB, particularly over the last 5 years. By stabilizing specific receptor states, our understanding of VHH targeting GPCRs has changed. Raynaud et al. [7] demonstrated that VHHs could lock GPCRs in active, intermediate, or inactive conformations, providing insights into receptor signaling.

This capability has enabled the development of high-affinity binders that selectively modulate GPCR activity with greater specificity than traditional mAbs or small molecules. Furthermore, VHHs have demonstrated the ability to modulate GPCR activity. A VHH called Nb60 acts as a negative allosteric modulator (NAM), stabilizing inactive receptor states and preventing activation by reinforcing structural constraints, such as the ionic lock in GPCRs  [6].

Other VHHs enhance receptor activation by mimicking G protein interactions. Nb80 stabilizes the β₂-adrenergic receptor (β₂AR) in an active-like conformation by mimicking G protein interactions. Its CDR3 inserts into the hydrophobic allosteric pocket, displacing the Gα subunit and disrupting the ionic lock between TM3 and TM6 – a structural constraint that maintains the receptor’s inactive state. This shift forces TM6 outward, a key feature of GPCR activation. Additionally, Nb80 enhances agonist binding by reducing ligand dissociation rates, underscoring its ability to modulate receptor activity with high specificity [9] [10].

Intrabodies are VHHs that are specifically designed to function inside cells, and can sometimes include G protein ‘mimics’ as described above. Table 1 showcases intrabodies that modulate GPCR signaling and trafficking, through their distinct mechanisms of action. The combination of small size, high specificity, and unique conformational stabilization makes VHHs a powerful tool in GPCR research and drug discovery in general.

Table 1. Intrabody examples that modulate protein activities. Adapted from Raynaud et al. (2022).

Integrating VHHs into Drug Discovery Pipelines 

Antibody discovery scientists are utilizing VHH antibodies to overcome the constraints of conventional mAbs in drug discovery with growing frequency. Their ability to target cryptic epitopes and penetrate difficult-to-reach tissues makes them ideal for GPCR, ion channel, and intracellular protein targeting—once considered undruggable. 

Isogenica’s synthetic VHH libraries represent a high-quality solution for those companies wishing to advance their drug pipelines. In contrast to traditional antibody discovery approaches, the libraries offer pre-humanized, high-affinity clones, and high-throughput functional screening has been made possible by integrating our workflows with Axxam’s in vitro HTS solutions. This methodology allows for earlier validation of binding specificity and function, enabling far more VHH antibodies to be assessed for critical but hard-to-engineer properties such as agonism.  

In addition to classical antibody uses, VHHs can be used to build sophisticated new therapies such as bi-specifics, CAR-T therapies, and intrabodies (including for targeted protein degradation), expanding their therapeutic reach. To support downstream development, Isogenica works within an ecosystem of CRO partners, streamlining the transition from discovery to preclinical validation. 

By incorporating VHH technology into discovery programs, biotech companies can open up new possibilities in precision medicine, with improved target engagement and therapeutic effect in oncology, immunology, and neurodegenerative disorders. 

Redefining What’s Druggable 

VHH antibodies have demonstrated a significant potential to tackle hard-to-drug targets. Their small size, enhanced stability, and unique binding capabilities allow for superior tissue penetration and access to previously undruggable molecules like GPCRs and intracellular proteins. With proven success in precision oncology, targeted drug delivery, and imaging, VHHs offer a powerful alternative to traditional biologics. As research advances, their role in next-generation therapeutics will continue to expand. To see how VHHs are being applied in real-world discovery programs—particularly for GPCRs and other complex targets—check out the Axxam–Isogenica webinar, where experts walk through case studies and screening strategies that redefine what’s druggable.

References

[1] Tao, Z., & Wu, X. (2023). Targeting Transcription Factors in Cancer: From “Undruggable” to “Druggable”. Methods in molecular biology (Clifton, N.J.), 2594, 107–131. https://doi.org/10.1007/978-1-0716-2815-7_9 

[2] Xie, X. et al (2023). Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials. Signal transduction and targeted therapy, 8(1), 335. https://doi.org/10.1038/s41392-023-01589-z 

[3] Feehley, T., O’Donnell, C. W., Mendlein, J., Karande, M., & McCauley, T. (2023). Drugging the epigenome in the age of precision medicine. Clinical epigenetics, 15(1), 6. https://doi.org/10.1186/s13148-022-01419-z Feehley, T., O’Donnell, C. W., Mendlein, J., Karande, M., & McCauley, T. (2023). Drugging the epigenome in the age of precision medicine. Clinical epigenetics, 15(1), 6. https://doi.org/10.1186/s13148-022-01419-z 

[4] Beckman, R. A., Weiner, L. M., & Davis, H. M. (2007). Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors. Cancer, 109(2), 170–179. https://doi.org/10.1002/cncr.22402 

[5] Rosenbaum, D. M., Rasmussen, S. G., & Kobilka, B. K. (2009). The structure and function of G-protein-coupled receptors. Nature, 459(7245), 356–363. https://doi.org/10.1038/nature08144  

[6] Wang, X., Iyer, A., Lyons, A. B., Körner, H., & Wei, W. (2019). Emerging Roles for G-protein Coupled Receptors in Development and Activation of Macrophages. Frontiers in immunology, 10, 2031. https://doi.org/10.3389/fimmu.2019.02031  

[7] Raynaud, P. et al. (2022). Intracellular VHHs to monitor and modulate GPCR signaling. Frontiers in endocrinology, 13, 1048601. https://doi.org/10.3389/fendo.2022.1048601 

[8] Staus, D. P., Strachan, R. T., Manglik, A., Pani, B., Kahsai, A. W., Kim, T. H., Wingler, L. M., Ahn, S., Chatterjee, A., Masoudi, A., Kruse, A. C., Pardon, E., Steyaert, J., Weis, W. I., Prosser, R. S., Kobilka, B. K., Costa, T., & Lefkowitz, R. J. (2016). Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation. Nature, 535(7612), 448–452. https://doi.org/10.1038/nature18636 

[9] Chen, Y., Fleetwood, O., Pérez-Conesa, S., & Delemotte, L. (2021). Allosteric Effect of Nanobody Binding on Ligand-Specific Active States of the β2 Adrenergic Receptor. Journal of chemical information and modeling, 61(12), 6024–6037. https://doi.org/10.1021/acs.jcim.1c00826  

[10] DeVree, B. T., Mahoney, J. P., Vélez-Ruiz, G. A., Rasmussen, S. G. F., Kuszak, A. J., Edwald, E., Fung, J.-J., Manglik, A., Masureel, M., Du, Y., Matt, R. A., Pardon, E., Steyaert, J., Kobilka, B. K., & Sunahara, R. K. (2016). Allosteric coupling from G protein to the agonist-binding pocket in GPCRs. Nature, 535(7610), 182–186. https://doi.org/10.1038/nature18324