Protein folding interference: a new path to hard-to-drug targets

Inducing protein degradation through folding interference

Protein folding is the process by which polypeptides, synthesised as a linear chain of amino acids by the ribosome, adopt a specific three-dimensional structure – the native state – in which they perform their physiological activity. A correct folding process is essential for preserving cellular function. Cells rigorously oversee this process with several quality-control systems that either fix unproperly folded proteins or dispose of them. Such intracellular disposal occurs through proteasome- or lysosome-facilitated degradation, maintaining balance and preventing the buildup of damaged or excess proteins.

The folding process for the nascent chain is characterised by iterative attempts, as the structure navigates a rough free-energy landscape with high-energy barriers, manoeuvring towards the native state. Medium and large sized proteins explore different metastable and partially-folded intermediate states during this process.¹ Such transitional conformations are capable of hosting druggable pockets but once buried into the native state upon folding completion they become inaccessible.

Sibylla Biotech leverages the idea that a small molecule binding the pocket on the intermediate structure creates interference with the process of folding, inducing the system in an improperly folded state. The protein thus cannot reach the native state and gets degraded through the physiological pathway. By executing such folding interference, small molecules can therefore selectively eliminate disease-causing proteins entirely by utilising the cell’s own disposal machinery.

Harnessing the power of protein folding interference to induce targeted protein degradation, Sibylla’s innovative Pharmacological Protein Inactivation by Folding Intermediates Targeting (PPI-FIT) technology² yields a pipeline of small molecules that act as folding interferers to address a range of challenging therapeutic areas.

An unprecedented therapeutic opportunity

Despite the diversity in mechanisms of action, all pharmaceutical drugs in the protein space target native proteins; however, an estimated 85 percent of the human proteome lacks traditional druggable pockets,³ rendering the majority of proteins refractory to classic small-molecule inhibition.

Despite the diversity in mechanisms of action, all pharmaceutical drugs in the protein space target native proteins; however, an estimated 85 percent of the human proteome lacks traditional druggable pockets.

Disease-causing proteins have historically been challenging to target because many lack well-defined binding pockets or active sites, which are typically required for conventional drugs such as small molecules to bind effectively. Instead, these proteins often have large, flat, or featureless surfaces for protein–protein interactions, making it difficult to design drugs with high specificity or affinity. Additionally, some disease-related proteins are intrinsically disordered, meaning they do not adopt a stable native structure, further complicating the ability to target them with traditional drug modalities.

This creates significant challenges in the treatment of many pathologies and presents a meaningful opportunity to develop therapies in indications with high unmet need, including neurodegenerative diseases, rare genetic diseases, immunology and inflammation, and oncology.

Although we cannot currently target intrinsically disordered proteins or multi-pass membrane proteins, our R&D team has found that at least 30 percent of soluble proteins and single-pass membrane proteins with known native structures form a folding intermediate. When selecting targets, the folding intermediate should contain a druggable pocket that is absent in the native state – with sufficient ligandability and suitable physicochemical properties – and it should have a lifetime compatible with small-molecule binding at therapeutically relevant concentrations. Although many in vitro refolding studies detect intermediates with very short lifetimes (milliseconds or less), which makes them poor pharmacological targets, in biological contexts intermediate lifetimes are governed by the rate of amino acid synthesis and typically extend to seconds, largely independent of the folding free energy barrier. Based on these criteria, we estimate roughly 5,000 potential targets.

This approach offers the possibility to access targets previously considered undruggable in their native conformation.

As the pocket in the folding intermediate state becomes buried and inaccessible once the folding process is complete, this approach offers the possibility to access targets previously considered undruggable in their native conformation. Moreover, the structural differentiation of these pockets – being inaccessible in the native state and lacking the high structural conservation typical of functional sites, such as ATP-binding sites in kinases or GTP-binding sites in GTPases – enables exploration of an under-explored chemical space. This could lead to the development of new chemical entities with a high degree of freedom to operate.

The pocket identified within an intermediate state should represent a bottleneck in the folding process. Targeting this state would trigger a degradation event that depends on the physiological protein degradation pathway and needs not engage a specific ligase. This unlocks a completely new mechanism of action, offering exciting and unprecedented therapeutic opportunities.

Sibylla has created a pipeline in the oncology space. Our lead asset is a folding interference small molecule that degrades Cyclin D1, a protein involved in the initiation of the G1 to S cell cycle progression through CDK 4/6 activation, in the inhibition of DNA mismatch repairs or the promotion of biosynthetic pathways.

Cyclin D1 is a validated driver of cancer cell proliferation and its overexpression is linked to resistance to chemotherapy and CDK 4/6 inhibitors.

Cyclin D1 is a validated driver of cancer cell proliferation⁴ and its overexpression is linked to resistance to chemotherapy and CDK 4/6 inhibitors.⁵ As a drug target, Cyclin D1 has been historically ‘undruggable’.⁶ By developing a folding interference small molecule with a degradation outcome, Sibylla could exploit a well-established oncogenic pathway and provide both next-generation options for cancers that are resistant to current standard of care treatment options and novel treatments in indications with unmet need.

Technology & platform highlights –a physics-based and AI-enhanced platform

Although it is now possible to infer the native structure from the primary sequence, ie, Alphafold, the way a protein folds was previously a black box. Sibylla’s technology, however, can simulate the folding of proteins, thereby accessing timescales not possible through standard molecular dynamics. Furthermore, Sibylla’s physics-based platform reliably identifies folding intermediates through means of enhanced sampling all-atom cotranslational or cotranslocational simulations that account for the sequential nature of folding inside the cells.

Distinct from ‘refolding’ simulations, where the target protein acquires the native shape in water from a denatured starting conformation, cotranslational and cotranslocational simulations consider key features of intracellular folding: the sequence of the process and interactions of the nascent chain with the ribosome or translocation machinery.

The computational platform employs state-of-the-art machine learning tailored to the unique mechanism of action to enhance the drug discovery processes, from binding site identification to rational hit discovery, or de novo design and lead optimisation. Continuously evolving with reliable in-house data, this platform efficiently steers the design of new chemical entities with optimal pharmacological profiles. By integrating AI-driven processes, Sibylla is accelerating its drug discovery pipeline. At the company, additional value is created by an interdisciplinary team of scientists in computational biophysics, medicinal chemistry, biology and translational medicine.

Potential future applications – expanding treatment options for patients

Interfering with protein folding to induce degradation could unlock entirely new therapeutic frontiers across fields such as oncology, neurology and rare diseases. By targeting folding intermediates, this approach offers the potential to address diseases currently considered untreatable due to the lack of druggable targets.

Unlocking the power of protein folding interference to expand druggable targets represents a paradigm shift, paving the way for innovative strategies in drug design and disease treatment that improve health outcomes in patients with a high unmet need. By focusing on intermediate folding states and opening a novel and unexplored approach to therapeutic intervention, Sibylla is advancing its mission to expand treatment options to relieve disease burdens for patients. While progressing its lead asset towards preclinical assessment and its pipeline into discovery and development, Sibylla is also engaged in early-stage collaborations with pharma companies, leveraging its platform to maximise the possibility of bringing value to patients.

Through the unique integration of physics, computation, AI, chemistry and biology into a unified discovery framework, Sibylla is laying the foundation to unlock the next generation of rational drug design, potentially transforming the druggability landscape of the human proteome, and redefining the boundaries of modern medicine.

References

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2. Spagnolli G, Massignan T, Astolfi A, et al. Pharmacological inactivation of the prion protein by targeting a folding intermediate. Commun Biol. 2021;4(1):62. Published 2021 Jan 12. doi:10.1038/s42003-020-01585-x
3. Neklesa TK, Winkler JD, Crews CM. Targeted protein degradation by PROTACs. Pharmacol Ther. 2017;174:138-144. doi:10.1016/j.pharmthera.2017.02.027
4. Otto T, Sicinski P. Cell cycle proteins as promising targets in cancer therapy. Nat Rev Cancer. 2017;17(2):93-115. doi:10.1038/nrc.2016.138
5. Álvarez-Fernández M, Malumbres M. Mechanisms of Sensitivity and Resistance to CDK4/6 Inhibition. Cancer Cell. 2020;37(4):514-529. doi:10.1016/j.ccell.2020.03.010
6. Xie X, Yu T, Li X, et al. Recent advances in targeting the “undruggable” proteins: from drug discovery to clinical trials. Sig Transduct Target Th 8, 335 (2023). https://doi.org/10.1038/s41392-023-01589-z