When Biology meets Physics - A marriage made inside the cell: Molecular Biocondensate

Biomolecular condensates are membraneless compartments formed by specialized biomolecule phase separation. This idea has given rise to a novel framework for understanding intracellular architecture and function, as well as a new method of biomolecular interaction distinct from the 'lock-and-key' paradigm. Biomolecular condensates have been found to play a role in a variety of cellular functions, including cell division and adhesion, mRNA processing and transport, transcription and translation regulation, protein degradation, gene silencing, chromatin compaction, and genome organization, since the first demonstration that P granules undergo liquid-liquid phase separation in Caenorhabditis elegans.

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The nucleolus was first discovered in 1835 and was named due to its speckled appearance in the nucleus. This fluid-filled structure contains essential components for protein synthesis, including rRNA and protein, and is where ribosomes, responsible for protein production, are created and assembled. Despite its crucial role, the nucleolus was not extensively studied until the 1950s, when micrometersions became better understood. The term "condensate" was coined almost six decades after its discovery.

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Many cellular activities have been connected to biomolecular condensates, including stress detection and response, biochemical reaction compartmentalization, mechanical control, and signaling. Their composition is often complicated, consisting of hundreds of distinct proteins and nucleic acids that create a large intermolecular network covering length scales ranging from nanometres to micrometres.

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Many biomolecular condensates are hypothesized to generate non-stoichiometric macromolecular assemblies in a concentration-dependent way by spontaneous or nucleated phase separation of a chosen set of proteins and/or nucleic acids. When the concentration of biomolecules surpasses the saturation concentration (Csat), phase separation occurs. Minor changes in biomolecule concentration that cross the phase boundary cause a rapid, switch-like reaction, resulting in either condensate formation or dissolution, altering the local concentration by many orders of magnitude. This mechanism is also quick and reversible, making it an excellent component for monitoring stress and other environmental changes.

Using dark-field microscopy , researchers tracked the motion of proteins in solution. They find clusters of proteins in solution are in low abundance overall. Researchers find clusters make up about 1% of the overall solution, even at protein concentrations just below the saturation level, where condensates form.

Temperature, stress, malnutrition, identification of foreign material, or other biological stimuli can all cause biomolecular condensates to react quickly and with a low energy threshold. Phase separation provides a reversible technique for boosting the local concentration of a certain component within the condensate while decreasing its concentration in the surrounding environment. These activities help to reduce cellular toxicity by sequestering excess materials in response to stress. Stress signals perceived in the cytoplasm, for example, cause the formation of stress granules, which compartmentalize untranslated RNA and RNA-binding proteins from the cytoplasm and nucleus.

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What is the function of condensates in diseases?

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Condensates play a critical role in the development of certain diseases known as "condensatopathies," which can lead to abnormal behavior. These diseases encompass various categories such as neurodegeneration like ALS, cancer such as prostate cancer, viral infections like respiratory syncytial virus (RSV), and cardiac illness. Genetic abnormalities have been linked to disorders affecting proteins found in biomolecular condensates, indicating that these mutations may disrupt cellular function and cause illness.

In cancer, two small molecules were found to have an impact on condensates, the anticancer drugs cisplatin and tamoxifen can partition into transcriptional condensates, altering their composition in cultured cells and in vitro reconstituted model condensates.

Pathogens, such as viruses, employ biomolecular condensates to hijack host cells and avoid the host's innate immunological self-defense systems. The roles of biomolecular condensates have been linked to several processes in the viral replication cycle, including viral entrance and egress, transcription, protein synthesis, and genome and virion assembly.

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So, Are condensates a platform for drug discovery and targets at the same time?

Biomolecular condensates are a unique way to regulate biological processes by compartmentalizing or excluding molecular components. These interactions are reversible and allow for spatiotemporal control. To prevent or reverse illness, condensate modifying therapies (c-mods) can affect the physical characteristics, macromolecular network, composition, dynamics, and/or function of certain biomolecular condensates. There are four main types of c-mods: dissolvers which dissolve condensates, inducers that cause condensate formation, localizers that change the sub-cellular localization of condensate community members, and morphers that modify the biophysical properties of existing condensates to change their morphology.

“I firmly believe that targeting condensates is going to change the way we think about developing drugs and really change the way we think about human disease,” said Isaac Klein, chief scientific officer of Dewpoint Therapeutics.

Dissolvers

Mitoxantrone, daunorubicin, quinacrine, and pyrvinium are all effective at dissolving persistent stress granules (SGs, condensate co-scaffold) in cells. Many proteins in SGs contain mutations that are genetically connected to driving pathobiological pathways of amyotrophic lateral sclerosis (ALS), a deadly neurodegenerative disease that primarily affects motor neurons.

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Inducers

Condensates can block metabolic processes by sequestering and inactivating biological components. Inducer c-mods may create a condensate to sequester disease-causing substances. Nusinersen, an antisense oligonucleotide (ASO) medication licensed for SMA, serves as a c-mod inducer. It inhibits SMN2 transcript splicing to restore functional SMN protein expression and downstream spliceosomal biogenesis. It's believed that Nusinersen induces nuclear condensates (Cajal bodies) to increase premRNA transcription, splicing, and nuclear export of mature mRNA for translation.

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Localizers

Condensate structure and function are intimately related to condensate community composition. Proteins that are incorrectly localized outside of their physiological condensate community or inside a non-native condensate can trigger pathological illness pathways. Localizer c-mods inhibit or rescue the spatial (re)positioning of a single biomolecule while preserving the majority of the condensate's integrity.

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Localizer c-mods are potential treatments for Acute Myeloid Leukemia (AML). A frameshift in the nucleolar protein NPM1—a monogenic cause of AML responsible for 30% of cases worldwide—initiates an abnormal nuclear export signal (NES). NPM1 mislocalization was recovered by small molecule localizer c-mods such as Avrainvillamide and Selinexor (KPT-330), which returned it to the nucleus and nucleolus while also correcting parts of the gain and/or loss of function.

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Morphers

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Morpher c-mods target condensate disease pathologies by changing the morphologies and material characteristics of condensates, affecting their activities.

Cyclopamine is a c-mod morpher that prevents viral replication by hardening a liquid-like replication condensate.

“By targeting the condensate, you can treat all of the patients regardless of the etiology of their disease. This is in direct contrast to the more common, more predominant drug discovery strategy which is ‘oh, they have a mutation, let’s target that.’ That’s predominantly failed,” said Klein.

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What is the advantage of using c-mods vs. other therapies?

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There are various advantages of using C-mods to treat illness. First, c-mods can be useful combination therapy, particularly for chemoresistance-prone medicines. Condensates can act as an integrating node in a few pathways that contribute to chemoresistance. As a result, c-mods have the potential to be more effective than traditional inhibitors, particularly when paired with chimeric protein degradation techniques such as PROTACs and molecular glues. Inducing condensates by concentrating the material targeted for proteostasis appears to be a normal reaction for a cell to establish successful compartmentalization and maximize ubiquitin ligase activity. Specific molecular regulators direct proteasomal or autophagic clearance of condensates enriched with a ubiquitinated molecular community.

Because condensate physiochemical characteristics respond to perturbations at quicker timescales, c-mods may be appealing candidates for acute therapy schemes.

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If C-mods interact with one or more components of a condensate community in different ways, they may have more than one mode of action for a target condensate. An ASO, for example, may interact with RNA, a peptide may disrupt an interaction between two condensate components, a small molecule may inhibit a protein-protein or protein-RNA interaction, a molecular glue may increase the proximity of two proteins, or a small molecule may inhibit a protein's enzymatic function.

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Companies working in the field.

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The field is still young, and many questions remain to be answered, because of that the very first companies working on the field were created between 2014 and 2020.

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Dewpoint officially launched in January 2019 based on the work of Anthony Hyman, the head of the team of Max Planck researchers that?discovered ?condensates. It’s since raised $287 million in three funding rounds and struck partnerships with Bayer, Merck & Co.,?Pfizer ?and biotech?Volastra Therapeutics . The company is led by Ameet Nathwani, a former Sanofi and Novartis executive.

Nereid Therapeutics ?debuted ?in November 2020 with $50 million in funding from investment firm ATP. Nereid was spun out of the research of Clifford Brangwynne, a former post-doctoral fellow in Hyman’s lab and also a pioneer in the field. (Brangwynne is now a Howard Hughes Medical Institute investigator at Princeton University.)

Transition Bio ?was?seeded ?in September 2020 and followed up earlier in 2022 with a?$50 million ?Series A round. The company’s founding research comes from Harvard University professor David Weitz and University of Cambridge professor Tuomas Knowles, who developed a way to study the properties of condensates and design drugs to target them. The startup is run by Greg Miller, who previously worked at Visterra, Concert Pharmaceuticals and Genzyme.

Aquinnah has existed longer than the others, forming in 2014 around?research ?from Ben Wolozin, a neuropharmacologist at the Boston University School of Medicine. The company has publicly disclosed just over $15 million in investments from?Pfizer, AbbVie ?and?Takeda . In February, it inked a collaboration with?Roche .

Faze was backed by Third Rock and the venture arms of Novartis, Eli Lilly and AbbVie.

The most interesting thing about this new field is that 7 of the top pharmaceutical companies and top investors are supporting this new technology and much more than that, they are co-discovering and co-developing a new pipeline with these companies.

Co-development agreements

The most recent agreement on the field is a deal worth $745 million for two small-molecule programs, with a lucrative option to expand into other modalities, which brings together the diabetes and metabolic disease expertise of Novo Nordisk A/S with an artificial intelligence technology platform from Dewpoint Therapeutics Inc.

Under the partnership, Dewpoint is eligible to receive up to $55 million in the near term which includes an upfront payment, research funding, and potential research milestones across two programs.?Dewpoint is also eligible to receive up to $690 million of clinical, commercial, and sales milestones plus royalties across two commercial products. If Novo Nordisk decides to pursue other modalities for the condensates, Dewpoint will be eligible to receive up to $107.5 million in total milestones per product.

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Investment Tip.

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Dewpoint Therapeutics, a trailblazer in this field, is potentially gearing up for an IPO. With a remarkable $287 million raised and strategic partnerships in place with potential + USD 1 billion, Dewpoint's advancements could shape the future of drug discovery.

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Final Thoughts:

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Nonetheless, the dynamic nature of biomolecular condensates makes characterization difficult; most structural biology approaches and in vitro, reconstitution studies are insufficient unless complemented with quantitative biophysical techniques. As a result, more sophisticated tools are required to non-invasively label and modulate condensates (such as optogenetic-based techniques), visualize them in real-time at high resolution and throughput (using advanced super-resolution microscopy), and probe their materials and partitioning properties in situ. Predictive computational models will be required to streamline and optimize these efforts. Such advancements will presumably give the long-desired causation proof of the role of condensate malfunction in particular illnesses, which has so far remained elusive.

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“Progress in science depends on new techniques, new discoveries, and new ideas, probably in that order.” Nobel prize winner Sydney Brenner?

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