Heterobifunctional Molecules Research Articles

Design of Murine Double Minute 2 Proteolysis Targeting Chimera Degraders with a Built-In Tumor-Targeting Ability.

Proteolysis targeting chimeras (PROTACs) are heterobifunctional molecules to induce the proteasomal degradation of target proteins. Currently, there are no tumor-targeting PROTACs for modulating oncogenic murine double minute 2 (MDM2). AS1411 is a tumor-targeting aptamer that specifically recognizes nucleolin (NCL) overexpressed on the surface of tumor cells. We recently repurposed AS1411 as an MDM2 recruiter since it could form an NCL-bridged ternary complex with MDM2. In this study, we design a PROTAC molecule AS1411-VH032 via conjugating AS1411 with a recruiter of von Hippel-Lindau (VHL) ligase VH032. AS1411-VH032 facilitates tumor-selective degradation of MDM2, leading to tumor shrinkage with no detectable toxicity. Besides being a molecular target, MDM2 also serves as an E3 ligase harnessed by PROTACs. Thus, we developed an AS1411-based homo-PROTAC homoAS1411, which induces tumor-specific suicide degradation of MDM2 and prevents tumor progression without causing side effects. Both AS1411-VH032 and homoAS1411 are promising MDM2 degraders with built-in tumor-targeting ability, which balances the antitumor efficacy with a favorable safety profile.

PROTAC-mediated activation, rather than degradation, of a nuclear receptor reveals complex ligand-receptor interaction network.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules containing a ligand for a protein of interest linked to an E3 ubiquitin ligase ligand that induce protein degradation through E3 recruitment to the target protein. Small changes in PROTAC linkers can have drastic consequences, including loss of degradation activity, but the structural mechanisms governing such changes are unclear. To study this phenomenon, we screened PROTACs of diverse targeting modalities and identified dTAG-13 as an activator of the xenobiotic-sensing pregnane X receptor (PXR), which promiscuously binds various ligands. Characterization of dTAG-13 analogs and precursors revealed interplay between the PXR-binding moiety, linker, and E3 ligand that altered PXR activity without inducing degradation. A crystal structure of PXR ligand binding domain bound to a precursor ligand showed ligand-induced binding pocket distortions and a linker-punctured tunnel to the protein exterior at a region incompatible with E3 complex formation, highlighting the effects of linker environment on PROTAC activity.

Rationalize the Functional Roles of Protein-Protein Interactions in Targeted Protein Degradation by Kinetic Monte-Carlo Simulations.

Targeted protein degradation is a promising therapeutic strategy to tackle disease-causing proteins that lack binding pockets for traditional small-molecule inhibitors. Its first step is to trigger the proximity between a ubiquitin ligase complex and a target protein through a heterobifunctional molecule, such as proteolysis targeting chimeras (PROTACs), leading to the formation of a ternary complex. The properties of protein-protein interactions play an important regulatory role during this process, which can be reflected by binding cooperativity. Unfortunately, although computer-aided drug design has become a cornerstone of modern drug development, the endeavor to model targeted protein degradation is still in its infancy. The development of computational tools to understand the impacts of protein-protein interactions on targeted protein degradation, therefore, is highly demanded. To reach this goal, we constructed a non-redundant structural benchmark of the most updated ternary complexes and applied a kinetic Monte-Carlo method to simulate the association between ligases and PROTAC-targeted proteins in the benchmark. Our results show that proteins in most complexes with positive cooperativity tend to associate into native-like configurations more often. In contrast, proteins very likely failed to associate into native-like configurations in complexes with negative cooperativity. Moreover, we compared the protein-protein association through different interfaces generated from molecular docking. The native-like binding interface shows a higher association probability than all the other alternative interfaces only in the complex with positive cooperativity. These observations support the idea that the formation of ternary complexes is closely regulated by the binary interactions between proteins. Finally, we applied our method to cyclin-dependent kinases 4 and 6 (CDK4/6). We found that their interactions with the ligase are not as similar as their structures. Altogether, our study paves the way for understanding the role of protein-protein interactions in PROTACE-induced ternary complex formation. It can potentially help in searching for degraders that selectively target specific proteins.

Targeted Protein Localization by Covalent 14-3-3 Recruitment.

14-3-3 proteins have a unique ability to bind and sequester a multitude of diverse phosphorylated signaling proteins and transcription factors. Many previous studies have shown that interactions of 14-3-3 with specific phosphorylated substrate proteins can be enhanced through small-molecule natural products or fully synthetic molecular glue interactions. However, enhancing 14-3-3 interactions with both therapeutically intractable transcription factor substrates and potential neo-substrates to sequester and inhibit their function remains elusive. One of the 14-3-3 proteins, 14-3-3σ or SFN, has cysteine C38 at the substrate-binding interface, near the sites where previous 14-3-3 molecular glues have been found to bind. In this study, we screen a fully synthetic cysteine-reactive covalent ligand library to identify molecular glues that enhance the interaction of 14-3-3σ with not only druggable transcription factors such as estrogen receptor (ERα) but also challenging oncogenic transcription factors such as YAP and TAZ, which are part of the Hippo transducer pathway. We identify a hit EN171 that covalently targets both C38 and C96 on 14-3-3 to enhance 14-3-3 interactions with ERα, YAP, and TAZ, leading to impaired estrogen receptor and Hippo pathway transcriptional activity. We further demonstrate that EN171 could not only be used as a molecular glue to enhance native protein interactions but could also be used as a covalent 14-3-3 recruiter in heterobifunctional molecules to sequester nuclear neo-substrates such as BRD4 and BLC6 into the cytosol. Overall, our study reveals a covalent ligand that acts as a novel 14-3-3 molecular glue for challenging transcription factors such as YAP and TAZ and demonstrates that these glues can be potentially utilized in heterobifunctional molecules to sequester nuclear neo-substrates out of the nucleus and into the cytosol to enable targeted protein localization.

Functional Investigations of p53 Acetylation Enabled by Heterobifunctional Molecules.

Post-translational modifications (PTMs) dynamically regulate the critical stress response and tumor suppressive functions of p53. Among these, acetylation events mediated by multiple acetyltransferases lead to differential target gene activation and subsequent cell fate. However, our understanding of these events is incomplete due to, in part, the inability to selectively and dynamically control p53 acetylation. We recently developed a heterobifunctional small molecule system, AceTAG, to direct the acetyltransferase p300/CBP for targeted protein acetylation in cells. Here, we expand AceTAG to leverage the acetyltransferase PCAF/GCN5 and apply these tools to investigate the functional consequences of targeted p53 acetylation in human cancer cells. We demonstrate that the recruitment of p300/CBP or PCAF/GCN5 to p53 results in distinct acetylation events and differentiated transcriptional activities. Further, we show that chemically induced acetylation of multiple hotspot p53 mutants results in increased stabilization and enhancement of transcriptional activity. Collectively, these studies demonstrate the utility of AceTAG for functional investigations of protein acetylation.

Proteolysis Targeting Chimeras (PROTACs): An Innovative Strategy for Targeted Protein Degradation and Disease Treatment

Review Proteolysis Targeting Chimeras (PROTACs): An Innovative Strategy for Targeted Protein Degradation and Disease Treatment Jun Xia, James K.S. Norris, May-Li MacKinnon and Sam Butterworth * Division of Pharmacy and Optometry, Faculty of Biology Medicine and Health, The University of Manchester, Manchester M13 9PL, UK * Correspondence: sam.butterworth@manchester.ac.uk Received: 18 May 2024; Revised: 9 Jule 2024; Accepted: 9 Jule 2024; Published: 26 August 2024 Abstract: Protein ubiquitination is a highly conserved post-translational modification through which cells initiate the proteasomal degradation of undesired, aberrant, or damaged proteins. Protein ubiquitination plays a crucial role in protein homeostasis and regulates a wide range of essential physiological processes including DNA repair, immunological response, cell survival and apoptosis. Dysregulation of ubiquitination is associated with various pathologies including cancers, neurodegenerative diseases, and immune disorders. The ubiquitin-proteasome system (UPS) machinery has been utilized in therapeutic research as it can be manipulated to induce the degradation of undruggable proteins in a superior manner to traditional drug modalities. One such a method of specific protein degradation is the use of heterobifunctional molecules such as proteolysis targeting chimeras (PROTACs). This literature review will focus on the composition, mechanism of action and developmental milestones of PROTACs, comparing these against traditional drug discovery and treatment approaches. In addition, the potential benefits of PROTAC usage will be highlighted by analyzing their practical applications in drug therapies.

BCR::ABL1 Proteolysis-targeting chimeras (PROTACs): The new frontier in the treatment of Ph+ leukemias?

BCR::ABL1 tyrosine kinase inhibitors (TKIs) have turned chronic myeloid leukemia (CML) from a lethal condition into a chronic ailment. With optimal management, the survival of CML patients diagnosed in the chronic phase is approaching that of age-matched controls. However, only one-third of patients can discontinue TKIs and enter a state of functional cure termed treatment-free remission (TFR), while the remainder require life-long TKI therapy to avoid the recurrence of active leukemia. Approximately 10% of patients exhibit primary or acquired TKI resistance and eventually progress to the blast phase. It is thought that recurrence after attempted TFR originates from CML stem cells (LSCs) surviving despite continued suppression of BCR::ABL1 kinase. Although kinase activity is indispensable for induction of overt CML, kinase-independent scaffold functions of BCR::ABL1 are known to contribute to leukemogenesis, raising the intriguing but as yet hypothetical possibility, that degradation of BCR::ABL1 protein may accomplish what TKIs fail to achieve - eliminate residual LSCs to turn functional into real cures. The advent of BCR::ABL1 proteolysis targeting chimeras (PROTACs), heterobifunctional molecules linking a TKI-based warhead to an E3 ligase recruiter, has moved clinical protein degradation into the realm of the possible. Here we examine the molecular rationale as well as pros and cons of degrading BCR::ABL1 protein. We review reported BCR::ABL1 PROTACs, point out limitations of available data and compoundsand suggest directions for future research. Ultimately, clinical testing of a potent and specific BCR::ABL1 degrader will be required to determine the efficacy and tolerability of this approach.

The emerging role of targeted protein degradation to treat and study cancer.

The evolution of cancer treatment has provided increasingly targeted strategies both in the upfront and relapsed disease settings. Small-molecule inhibitors and immunotherapy have risen to prominence with chimeric antigen receptor T-cells, checkpoint inhibitors, kinase inhibitors, and monoclonal antibody therapies being deployed across a range of solid organ and haematological malignancies. However, novel approaches are required to target transcription factors and oncogenic fusion proteins that are central to cancer biology and have generally eluded successful drug development. Thalidomide analogues causing protein degradation have been a cornerstone of treatment in multiple myeloma, but a lack of in-depth mechanistic understanding initially limited progress in the field. When the protein cereblon (CRBN) was found to mediate thalidomide analogues' action and CRBN's neo-targets were identified, existing and novel drug development accelerated, with applications outside multiple myeloma, including non-Hodgkin's lymphoma, myelodysplastic syndrome, and acute leukaemias. Critically, transcription factors were the first canonical targets described. In addition to broadening the application of protein-degrading drugs, resistance mechanisms are being overcome and targeted protein degradation is widening the scope of druggable proteins against which existing approaches have been ineffective. Examples of targeted protein degraders include molecular glues and proteolysis targeting chimeras (PROTACs): heterobifunctional molecules that bind to proteins of interest and cause proximity-induced ubiquitination and proteasomal degradation via a linked E3 ligase. Twenty years since their inception, PROTACs have begun progressing through clinical trials, with early success in targeting the oestrogen receptor and androgen receptor in breast and prostate cancer respectively. This review explores important developments in targeted protein degradation to both treat and study cancer. It also considers the potential advantages and challenges in the translational aspects of developing new treatments. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Deciphering the mechanism of E3 ubiquitin ligases in plant responses to abiotic and biotic stresses and perspectives on PROTACs for crop resistance.

With global climate change, it is essential to find strategies to make crops more resistant to different stresses and guarantee food security worldwide. E3 ubiquitin ligases are critical regulatory elements that are gaining importance due to their role in selecting proteins for degradation in the ubiquitin-proteasome proteolysis pathway. The role of E3 Ub ligases has been demonstrated in numerous cellular processes in plants responding to biotic and abiotic stresses. E3 Ub ligases are considered a class of proteins that are difficult to control by conventional inhibitors, as they lack a standard active site with pocket, and their biological activity is mainly due to protein-protein interactions with transient conformational changes. Proteolysis-targeted chimeras (PROTACs) are a new class of heterobifunctional molecules that have emerged in recent years as relevant alternatives for incurable human diseases like cancer because they can target recalcitrant proteins for destruction. PROTACs interact with the ubiquitin-proteasome system, principally the E3 Ub ligase in the cell, and facilitate proteasome turnover of the proteins of interest. PROTAC strategies harness the essential functions of E3 Ub ligases for proteasomal degradation of proteins involved in dysfunction. This review examines critical advances in E3 Ub ligase research in plant responses to biotic and abiotic stresses. It highlights how PROTACs can be applied to target proteins involved in plant stress response to mitigate pathogenic agents and environmental adversities.

Nano-Proteolysis Targeting Chimeras (Nano-PROTACs) in Cancer Therapy.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have the capability to induce specific protein degradation. While playing a revolutionary role in effectively degrading the protein of interest (POI), PROTACs encounter certain limitations that impede their clinical translation. These limitations encompass off-target effects, inadequate cell membrane permeability, and the hook effect. The advent of nanotechnology presents a promising avenue to surmount the challenges associated with conventional PROTACs. The utilization of nano-proteolysis targeting chimeras (nano-PROTACs) holds the potential to enhance specific tissue accumulation, augment membrane permeability, and enable controlled release. Consequently, this approach has the capacity to significantly enhance the controllable degradation of target proteins. Additionally, they enable a synergistic effect by combining with other therapeutic strategies. This review comprehensively summarizes the structural basis, advantages, and limitations of PROTACs. Furthermore, it highlights the latest advancements in nanosystems engineered for delivering PROTACs, as well as the development of nano-sized PROTACs employing nanocarriers as linkers. Moreover, it delves into the underlying principles of nanotechnology tailored specifically for PROTACs, alongside the current prospects of clinical research. In conclusion, the integration of nanotechnology into PROTACs harbors vast potential in enhancing the anti-tumor treatment response and expediting clinical translation.

Targeting a lineage-specific PI3Kɣ-Akt signaling module in acute myeloid leukemia using a heterobifunctional degrader molecule.

Dose-limiting toxicity poses a major limitation to the clinical utility of targeted cancer therapies, often arising from target engagement in nonmalignant tissues. This obstacle can be minimized by targeting cancer dependencies driven by proteins with tissue-restricted and/or tumor-restricted expression. In line with another recent report, we show here that, in acute myeloid leukemia (AML), suppression of the myeloid-restricted PIK3CG/p110γ-PIK3R5/p101 axis inhibits protein kinase B/Akt signaling and compromises AML cell fitness. Furthermore, silencing the genes encoding PIK3CG/p110γ or PIK3R5/p101 sensitizes AML cells to established AML therapies. Importantly, we find that existing small-molecule inhibitors against PIK3CG are insufficient to achieve a sustained long-term antileukemic effect. To address this concern, we developed a proteolysis-targeting chimera (PROTAC) heterobifunctional molecule that specifically degrades PIK3CG and potently suppresses AML progression alone and in combination with venetoclax in human AML cell lines, primary samples from patients with AML and syngeneic mouse models.

Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

Folate-PEG-PROTAC Micelles for Enhancing Tumor-Specific Targeting Proteolysis In Vivo.

Proteolysis targeting chimeras (PROTACs) technology is rapidly developed as a novel and selective medicinal strategy for the degradation of cellular proteins in cancer therapy. However, the applications of PROTACs as heterobifunctional molecules are largely limited by high molecular weight, low bioavailability, poor permeability, insufficient targeting, and low efficacy in vivo. Herein, self-assembling micelles of FA-PEG-PROTAC are designed for cancer cell selective targeting and reductive-response proteolysis in tumor-bearing mice. FA-PEG-PROTAC is prepared by conjugating folic acid (FA)-PEG with EGFR-targeting PROTAC via a disulfide bond. The FA-PEG-PROTAC micelles, formed by self-assembling, are demonstrated to significantly improve tumor targeting efficacy and exhibit excellent anti-tumor efficacy in the mouse xenograft model compared to the traditional PROTACs. The strategy of applying self-assembled FA-PEG-PROTAC micelles in tumor therapy can not only improve targeted proteolysis efficiency but also broaden applications in the development of PROTAC-based drugs.

In vitro and in vivo proof-of-concept studies of extracellular protein degradation using novel M6PR-targeting chimeras (MTACs).

Abstract The mannose-6-phosphate-receptor (M6PR) traffics proteins from extracellular and intracellular locales to late endosomes/lysosomes for degradation and is ubiquitously expressed. This natural endogenous function of the mannose-6-phosphate-receptor (M6PR) may be harnessed for degradation of soluble circulating proteins and cell specific degradation of membrane proteins. Here we describe the design and development of novel M6PR targeting chimeras (MTACs). MTACs are heterobifunctional molecules containing Avilar’s novel, potent, small molecule M6PR-binding ligands conjugated to ligands that bind a target protein of interest for degradation. For proof-of-concept studies, MTACs were designed to target the extracellular protein IgG, the second most abundant circulating protein with a long plasma half-life. Reduction of pathogenic IgG autoantibodies is a clinically validated strategy for treating multiple debilitating autoimmune diseases. In vitro characterization of tridentate and monodentate MTACs revealed potent biochemical binding to M6PR and IgG, and MTAC-mediated cellular uptake and degradation of IgG via the endolysosomal pathway in K562 and LNCaP cells. In vivo MTAC-mediated depletion of human IgG was demonstrated using a heterologous rat PK/PD model.

Repurposing AS1411 for constructing ANM-PROTACs

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules consisting of two ligands joined by a linker, enabling them to simultaneously bind with an E3 ligase and a protein of interest (POI) and trigger proteasomal degradation of the POI. Limitations of PROTAC include lack of potent E3 ligands, poor cell selectivity, and low permeability. AS1411 is an antitumor aptamer specifically recognizing a membrane-nucleus shuttling nucleolin (NCL). Here, we repurpose AS1411 as a ligand for an E3 ligase mouse double minute 2 homolog (MDM2) via anchoring the NCL-MDM2 complex. Then, we construct an AS1411-NCL-MDM2-based PROTAC (ANM-PROTAC) by conjugating AS1411 with large-molecular-weight ligands for "undruggable" oncogenic STAT3, c-Myc, p53-R175H, and AR-V7. We show that the ANM-PROTAC efficiently penetrates tumor cells, recruits MDM2 and degrades the POIs. The ANM-PROTAC achieves tumor-selective distribution and exhibits excellent antitumor activity with no systemic toxicity. This is a PROTAC with built-in tumor-targeting and cell-penetrating capacities.

Abstract 4518: Proteolysis targeting chimera (PROTAC)-driven internalization of antibodies targeting oncogenic cell surface receptors

Abstract Antibody-drug conjugates (ADCs) are increasingly being used in clinic, and many novel ADCs are being tested in clinical trials. Most ADCs act by delivering cytotoxic payloads into target cells. The activity of most ADCs is predicated upon internalization to release the cytotoxic payloads intracellularly to act on the malignant cells. The degree of ADC internalization is incomplete or absent for many cell surface targets, limiting the activity of ADCs. PROTACs are heterobifunctional molecules that result in the polyubiquitylation of their targets by inducing proximity with E3 ligases. The proteasome then recognizes the poly-ubiquitin chains and degrades these targets. We hypothesized that PROTACs that act on the same targets as ADCs could improve the internalization and efficacy of these ADCs. Using multiple cell lines derived from breast, lung or pancreatic cancers that express the oncogenic cell surface proteins EGFR (ERBB1), HER2 (ERBB2) or MET (HGF), we measured antibody internalization with live cell imaging. Antibodies were labeled with a pH sensitive reagent that fluoresces red (640 nm) upon exposure to acidic environments. Fluorescence was measured over time to create an area under the curve which was compared between the cell lines that were treated with and without PROTACs. Antibody internalization was observed in all cell lines that expressed the targets recognized by the antibodies. The addition of PROTACs targeting the same oncogenic cell surface proteins as the antibodies significantly increased antibody internalization beyond that observed without PROTACs. Internalization of antibodies was confirmed by measuring intracellular immunoglobulins by Western blot. Antibody internalization with PROTACs was dose-dependent, increased over time and was blocked by either the clatharin inhibitor Dyngo4a or the proteasome inhibitor MG132. In the case of HER2 for which there are FDA-approved anti-HER2 ADCs, the cytotoxicity of trastuzumab emtansine (TDM1) significantly increased with the addition of a HER2-targeting PROTAC across a range of doses. PROTACs that target the same oncogenic cell surface proteins as antibodies improve antibody internalization and ADC cytotoxicity, in a process dependent upon clatharin and the proteasome. This novel application of PROTACs may impact the use of ADCs, and provides a rationale to combine these agents in clinical trials. Citation Format: Ezequiel J. Tolosa, Lin Yang, Jennifer Ayers-Ringler, Jayapal Mallareddy, Janet Schaefer-Klein, Farhad Kosari, Amar Natarajan, Aaron S. Mansfield. Proteolysis targeting chimera (PROTAC)-driven internalization of antibodies targeting oncogenic cell surface receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4518.

Abstract 2110: Characterization of the correlation between BTK degradation and tumor growth inhibition of the BTK target protein degraders using PK/PD modeling

Abstract Introduction: Chimeric degradation activation compound (CDACs) are heterobifunctional molecules causing target protein degradation by simultaneously binding to the target protein as well as an E3-ubiquitin ligase. As an emerging new therapeutic modality, it is critical to understand the contribution of compound-specific parameters (e.g. drug exposure and binding potency to the target protein) and the system-specific parameters (e.g. target protein turnover) of CDACs to its in vivo effect. Method: To identify the key parameters for guiding compound optimization and to find the correlation between BTK degradation and the extent of tumor growth inhibition, a mechanistic PK/PD model was built (Phoenix WinNonlin 8.1) on a dataset generated from a BTK CDAC (Compound A). The PK/PD model was simplified mathematically compared to the full mechanistic models published previously1, since the binding affinity of Compound A to BTK was much higher than the binding affinity of CDAC to E3 ligase. This model can be linked to a tumor growth inhibition (TGI) mathematical model to find the correlation between target degradation and efficacy.Result: In mouse Rec-1 xenograft model, the BTK degradation and TGI was studied. The mice were dosed with Compound A orally at 0.3, 1, or 3 mg/kg, dose-dependent PK and BTK degradation in tumor was observed. After a single dose administration of Compound A, ~50% BTK degradation was observed in tumor at 24 h post dosing and the tumor BTK rebound to baseline at 72 hours post dosing at 3 mg/kg. The tumor BTK degradation was deeper after repeated dosing. A PK/PD model was built based on the data in this PK/PD study. The calculated BTK turnover half-life in Rec-1 model is about 16 h. In the efficacy study, Compound A was dosed orally once a day to the mice at 0.3, 1, or 3 mg/kg. The 3 mg/kg group achieved ~90% tumor growth inhibition in Rec-1 xenograft model. The PK/PD model was then linked with a mathematical model describing TGI2. A threshold level of BTK degradation in tumor to achieve tumor stasis was identified to be 96% for Rec-1 tumor model. At 90% tumor growth inhibition, average of 70% BTK was predicted to be degraded in tumor at steady state. The model was subsequently used to characterize the PK/PD relationship of BGB-16673. The efficacy of BGB-16673 was predicted using the model and it agreed with the observed value.Conclusions: The current model deepened our understanding of the PK/PD relationship of CDACs. The approach can be used to simulate different scenarios, e.g. different BTK turnover, potency or PK exposure. It can also be used in compound selection and optimization to predict repeated dose PD and efficacy from a single dose PK/PD data.1. J Pharmacokinet Pharmacodyn. 2021 Feb;48(1):149-163.2. Cancer Res. 2004 Feb 1;64(3):1094-101. Citation Format: Yue Wu, Shuran Li, Fan Wang, Nan Hu, Longbo Yin, Xiaomin Song, Jiye Zhang, Aiying Xu, Shasha Yang. Characterization of the correlation between BTK degradation and tumor growth inhibition of the BTK target protein degraders using PK/PD modeling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2110.

Structure-Guided Design and Optimization of Covalent VHL-Targeted Sulfonyl Fluoride PROTACs.

Proteolysis-targeting chimeras (PROTACs) are heterobifunctional molecules that have emerged as a therapeutic modality to induce targeted protein degradation (TPD) by harnessing cellular proteolytic degradation machinery. PROTACs which ligand the E3 ligase in a covalent manner have attracted intense interest; however, covalent PROTACs with a broad protein of interest (POI) scope have proven challenging to discover by design. Here, we report the structure-guided design and optimization of Von Hippel-Lindau (VHL) protein-targeted sulfonyl fluorides which covalently bind Ser110 in the HIF1α binding site. We demonstrate that their incorporation in bifunctional degraders induces targeted protein degradation of BRD4 or the androgen receptor without further linker optimization. Our study discloses the first covalent VHL ligands which can be implemented directly in bifunctional degrader design, expanding the substrate scope of covalent E3 ligase PROTACs.

PROTAC-Design-Evaluator (PRODE): An Advanced Method for In-Silico PROTAC Design.

PROTAC (proteolysis-targeting chimeras) is a rapidly evolving technology to target undruggable targets. The mechanism by which this happens is when a bifunctional molecule binds to a target protein and also brings an E3 ubiquitin ligase in proximity to trigger ubiquitination and degradation of the target protein. Yet, in-silico-driven approaches to design these heterobifunctional molecules that have the desired functional properties to induce proximity between the target protein and E3 ligase remain to be established. In this paper, we present a novel in-silico method for PROTAC design and to demonstrate the validity of our approach, we show that for a BRD4-VHL-PROTAC-mediated ternary complex known in the literature, we are able to reproduce the PROTAC binding mode, the structure of the ternary complex formed therein, and the free energy (ΔG) thermodynamics favoring ternary complexation through theoretical/computational methodologies. Further, we demonstrate the use of thermal titration molecule dynamics (TTMD) to differentiate the stability of PROTAC-mediated ternary complexes. We employ the proposed methodology to design a PROTAC for a new system of FGFR1-MDM2 to degrade the FGFR1 (fibroblast growth factor receptor 1) that is overexpressed in cancer. Our work presented here and named as PROTAC-Designer-Evaluator (PRODE) contributes to the growing literature of in-silico approaches to PROTAC design and evaluation by incorporating the latest in-silico methods and demonstrates advancement over previously published PROTAC in-silico literature.

A chemical magnet: Approaches to guide precise protein localization

Small molecules that chemically induce proximity between two proteins have been widely used to precisely modulate protein levels, stability, and activity. Recently, several studies developed novel strategies that employ heterobifunctional molecules that co-opt shuttling proteins to control the spatial localization of a target protein, unlocking new potential within this domain. Together, these studies lay the groundwork for novel targeted protein relocalization modalities that can rewire the protein circuitry and interactome to influence biological outcomes.