Trivalent PROTACs Enhance Protein Degradation Through Cooperativity and Avidity

Abstract Bivalent small-molecule degraders, or proteolysis targeting chimeras (PROTACs), work by simultaneously binding a target protein and E3 ubiquitin ligase to produce a ternary complex. To drive target ubiquitination and degradation at low catalytic concentrations, degraders must form appropriately positioned complexes of sufficient stability, aided by intra-complex interactions. We hypothesized these molecular recognition features could be enhanced by increasing binding valency. Here we present trivalent PROTACs as a strategy to boost protein degradation. Our design for a trivalent PROTAC consisted of two BET bromodomain inhibitors and an E3 ligase ligand, each separately tethered via a branched linker. In screening, we identified SIM1, a VHL-based PROTAC, as a highly potent BET degrader, capable of low picomolar degradation for all family members, with preference for BRD2. In functional comparison studies to bivalent PROTACs or inhibitors, SIM1 showed more sustained anti-cancer activity across numerous therapeutically relevant cell lines. Biophysical, biochemical, and cellular mechanistic studies showed SIM1 induces conformational changes upon binding to the BET protein to simultaneously engage with high avidity both its bromodomains in a cis intramolecular fashion. The resulting 1:1:1 complex showed positive cooperativity, high stability and prolonged cellular residence time. We provide proof-of-concept for augmenting the binding valency of proximity-induced modalities as a strategy to leverage both cooperativity and avidity within the ternary complex to advance functional outcomes.

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Trivalent PROTACs Enhance Protein Degradation Through Cooperativity and Avidity

10.26434/chemrxiv.13218695 ◽

2020 ◽

Author(s):

Satomi Imaide ◽

Kristin M. Riching ◽

Vesna Vetma ◽

Claire Whitworth ◽

Scott J. Hughes ◽

...

Keyword(s):

Protein Degradation ◽

Conformational Changes ◽

Ternary Complex ◽

High Avidity ◽

Proof Of Concept ◽

Complex Interactions ◽

Anti Cancer ◽

Sufficient Stability ◽

Bet Protein ◽

Molecular Recognition Features

<p><b>Bivalent small-molecule degraders, or proteolysis targeting chimeras (PROTACs), work by simultaneously binding a target protein and E3 ubiquitin ligase to produce a ternary complex. To drive target ubiquitination and degradation at low catalytic concentrations, degraders must form appropriately positioned complexes of sufficient stability, aided by intra-complex interactions. We hypothesized these molecular recognition features could be enhanced by increasing binding valency. Here we present trivalent PROTACs as a strategy to boost protein degradation. Our design for a trivalent PROTAC consisted of two BET bromodomain inhibitors and an E3 ligase ligand, each separately tethered via a branched linker. In screening, we identified SIM1, a VHL-based PROTAC, as a highly potent BET degrader, capable of low picomolar degradation for all family members, with preference for BRD2. In functional comparison studies to bivalent PROTACs or inhibitors, SIM1 showed more sustained anti-cancer activity across numerous therapeutically relevant cell lines. Biophysical, biochemical, and cellular mechanistic studies showed SIM1 induces conformational changes upon binding to the BET protein to simultaneously engage with high avidity both its bromodomains in a cis intramolecular fashion. The resulting 1:1:1 complex showed positive cooperativity, high stability and prolonged cellular residence time. We provide proof-of-concept for augmenting the binding valency of proximity-induced modalities as a strategy to leverage both cooperativity and avidity within the ternary complex to advance functional outcomes.</b></p>

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Trivalent PROTACs enhance protein degradation via combined avidity and cooperativity

10.33774/chemrxiv-2021-7lbk4-v2 ◽

2021 ◽

Author(s):

Satomi Imaide ◽

Kristin M. Riching ◽

Nikolai Makukhin ◽

Vesna Vetma ◽

Claire Whitworth ◽

...

Keyword(s):

Protein Degradation ◽

Functional Outcomes ◽

Ternary Complex ◽

High Avidity ◽

Bet Inhibitor ◽

Anti Cancer ◽

Work Drive ◽

Cancer Activity ◽

In Vivo Pharmacokinetics

Bivalent PROTACs work drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhanced degradation. Here, we designed trivalent PROTACs consisting of a bivalent BET inhibitor and an E3 ligand, tethered via a branched linker. We identified VHL-based SIM1 as a low picomolar BET degrader, with preference for BRD2. Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anti-cancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.

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Minimal mechanistic component of proteasome activation and prevention of impairment by pathological oligomers

10.21203/rs.3.rs-900719/v1 ◽

2021 ◽

Author(s):

Janelle Chuah ◽

Tifffany Thibaudeau ◽

David Smith

Keyword(s):

Small Molecules ◽

Conformational Changes ◽

Bound State ◽

Proof Of Concept ◽

Α Subunit ◽

Gate Opening ◽

Allosteric Mechanism ◽

Dependent Mechanism ◽

Degradation Of Peptides

Abstract Impairment of proteasomal function has been implicated in neurodegenerative diseases, justifying the need to understand how the proteasome is activated for protein degradation. Here, using biochemical and structural (cryo-EM) strategies in both archaeal and mammalian proteasomes, we further determine the HbYX(-motif)-dependent mechanism of proteasomal activation used by multiple proteasome-activating complexes including the 19S Particle. We identify multiple proteasome α subunit residues involved in HbYX-dependent activation, a point mutation that activates the proteasome by partially mimicking a HbYX-bound state, and conformational changes involved in gate-opening with a 2.0A structure. Through an iterative process of peptide synthesis, we successfully design a HbYX-like dipeptide mimetic as a robust tool to elucidate how the motif autonomously activates the proteasome. The mimetic induces near complete gate-opening at saturating concentration, activating mammalian proteasomal degradation of peptides and proteins. Findings using our peptide mimetic suggest the HbYX-dependent mechanism requires cooperative binding in at least two intersubunit pockets of the α ring. Collectively, the results presented here unambiguously demonstrate the lone role of the HbYX tyrosine in the allosteric mechanism of proteasome activation and offer proof of concept for the robust potential of HbYX-like small molecules to activate the proteasome.

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Improved Accuracy for Modeling PROTAC-Mediated Ternary Complex Formation and Targeted Protein Degradation via New In Silico Methodologies

Journal of Chemical Information and Modeling ◽

10.1021/acs.jcim.0c00897 ◽

2020 ◽

Vol 60 (10) ◽

pp. 5234-5254 ◽

Cited By ~ 1

Author(s):

Michael L. Drummond ◽

Andrew Henry ◽

Huifang Li ◽

Christopher I. Williams

Keyword(s):

Complex Formation ◽

Protein Degradation ◽

In Silico ◽

Ternary Complex ◽

Ternary Complex Formation ◽

Improved Accuracy

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Shedding Light on the Interaction of Human Anti-Apoptotic Bcl-2 Protein with Ligands through Biophysical and in Silico Studies

International Journal of Molecular Sciences ◽

10.3390/ijms20040860 ◽

2019 ◽

Vol 20 (4) ◽

pp. 860 ◽

Cited By ~ 2

Author(s):

Joao Ramos ◽

Jayaraman Muthukumaran ◽

Filipe Freire ◽

João Paquete-Ferreira ◽

Ana Otrelo-Cardoso ◽

...

Keyword(s):

Conformational Changes ◽

In Silico ◽

Nucleotide Polymorphisms ◽

Ligand Interaction ◽

Zinc Database ◽

In Silico Studies ◽

Anti Cancer ◽

Binding Groove ◽

The Stability ◽

Dynamics Simulations

Bcl-2 protein is involved in cell apoptosis and is considered an interesting target for anti-cancer therapy. The present study aims to understand the stability and conformational changes of Bcl-2 upon interaction with the inhibitor venetoclax, and to explore other drug-target regions. We combined biophysical and in silico approaches to understand the mechanism of ligand binding to Bcl-2. Thermal shift assay (TSA) and urea electrophoresis showed a significant increase in protein stability upon venetoclax incubation, which is corroborated by molecular docking and molecular dynamics simulations. An 18 °C shift in Bcl-2 melting temperature was observed in the TSA, corresponding to a binding affinity multiple times higher than that of any other reported Bcl-2 inhibitor. This protein-ligand interaction does not implicate alternations in protein conformation, as suggested by SAXS. Additionally, bioinformatics approaches were used to identify deleterious non-synonymous single nucleotide polymorphisms (nsSNPs) of Bcl-2 and their impact on venetoclax binding, suggesting that venetoclax interaction is generally favored against these deleterious nsSNPs. Apart from the BH3 binding groove of Bcl-2, the flexible loop domain (FLD) also plays an important role in regulating the apoptotic process. High-throughput virtual screening (HTVS) identified 5 putative FLD inhibitors from the Zinc database, showing nanomolar affinity toward the FLD of Bcl-2.

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Radiosynthesis of [18F]-Labelled Pro-Nucleotides (ProTides)

Molecules ◽

10.3390/molecules25030704 ◽

2020 ◽

Vol 25 (3) ◽

pp. 704

Author(s):

Alessandra Cavaliere ◽

Katrin C. Probst ◽

Stephen J. Paisey ◽

Christopher Marshall ◽

Abdul K. H. Dheere ◽

...

Keyword(s):

Total Synthesis ◽

Pet Imaging ◽

Early Stage ◽

Proof Of Concept ◽

Synthesis Time ◽

Positron Emission ◽

Anti Cancer ◽

Radiochemical Yields ◽

Radiochemical Synthesis

Phosphoramidate pro-nucleotides (ProTides) have revolutionized the field of anti-viral and anti-cancer nucleoside therapy, overcoming the major limitations of nucleoside therapies and achieving clinical and commercial success. Despite the translation of ProTide technology into the clinic, there remain unresolved in vivo pharmacokinetic and pharmacodynamic questions. Positron Emission Tomography (PET) imaging using [18F]-labelled model ProTides could directly address key mechanistic questions and predict response to ProTide therapy. Here we report the first radiochemical synthesis of [18F]ProTides as novel probes for PET imaging. As a proof of concept, two chemically distinct radiolabelled ProTides have been synthesized as models of 3′- and 2′-fluorinated ProTides following different radiosynthetic approaches. The 3′-[18F]FLT ProTide was obtained via a late stage [18F]fluorination in radiochemical yields (RCY) of 15–30% (n = 5, decay-corrected from end of bombardment (EoB)), with high radiochemical purities (97%) and molar activities of 56 GBq/μmol (total synthesis time of 130 min.). The 2′-[18F]FIAU ProTide was obtained via an early stage [18F]fluorination approach with an RCY of 1–5% (n = 7, decay-corrected from EoB), with high radiochemical purities (98%) and molar activities of 53 GBq/μmol (total synthesis time of 240 min).

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FTIR spectroscopy reveals the concentration dependence of cellular modifications induced by anticancer drugs

Spectroscopy An International Journal ◽

10.1155/2010/461542 ◽

2010 ◽

Vol 24 (1-2) ◽

pp. 45-49 ◽

Cited By ~ 9

Author(s):

Régis Gasper ◽

Tatjana Mijatovic ◽

Robert Kiss ◽

Erik Goormaghtigh

Keyword(s):

Prostate Cancer ◽

Molecular Biology ◽

Anticancer Drugs ◽

Large Scale ◽

Proof Of Concept ◽

Prostate Cancer Cells ◽

Chemical Libraries ◽

Anti Cancer ◽

Cellular Modifications ◽

Large Scale Screening

Large-scale screening to determine the mechanisms of anti-cancer actions of chemical libraries still presents technical challenges that are beyond the capabilities of conventional methods used in cellular or molecular biology. We recently demonstrated in a proof-of-concept study that infrared (IR) spectrum of cells exposed to anticancer drugs could be used to classify their mechanisms of actions. This study highlighted the fact that molecules inducing unique metabolic modifications could be selected for further pharmacological improvements. We show in this paper that drug concentration is an important parameter to be taken into account when analyzing mechanisms of anti-cancer actions by means of FTIR. The data indeed demonstrated that distinct spectral modifications occur in human PC-3 prostate cancer cells when exposed to ouabain at 10 × IC50versus 1 × IC50. Longer incubation times at 1 × IC50never resulted in spectral modifications fitting with those observed at 10 × IC50.

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Bis-biguanide dihydrochloride inhibits intracellular replication of M. tuberculosis and controls infection in mice

Scientific Reports ◽

10.1038/srep32725 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 5

Author(s):

Hongbo Shen ◽

Feifei Wang ◽

Gucheng Zeng ◽

Ling Shen ◽

Han Cheng ◽

...

Keyword(s):

High Throughput Screening ◽

Cancer Drug ◽

Drug Resistant ◽

Proof Of Concept ◽

Intracellular Replication ◽

Anti Cancer ◽

Mdr Tb ◽

Approved Drugs ◽

Effective Drugs ◽

Slow Growing

Abstract While there is an urgent need to develop new and effective drugs for treatment of tuberculosis (TB) and multi-drug resistant TB (MDR-TB), repurposing FDA (U.S. Food and Drug Administration) -approved drugs for development of anti-TB agents may decrease time and effort from bench to bedside. Here, we employed host cell-based high throughput screening (HTS) assay to screen and characterize FDA-approved, off-patent library drugs for anti-Mycobacterium tuberculosis (MTB) activities. The cell-based HTS allowed us to identify an anti-cancer drug of bis-biguanide dihydrochloride (BBD) as potent anti-mycobacteria agent. Further characterization showed that BBD could inhibit intracellular and extracellular growth of M. smegmatis and slow-growing M. bovis BCG. BBD also potently inhibited replication of clinically-isolated MTB and MDR-TB strains. The proof-of-concept study showed that BBD treatment of MTB-infected mice could significantly decrease CFU counts in the lung and spleen. Notably, comparative evaluation showed that MTB CFU counts in BBD-treated mice were lower than those in rifampicin-treated mice. No apparent BBD side effects were found in BBD-treated mice. Thus, our findings support further studies to develop BBD as a new and effective drug against TB and MDR-TB.

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