Bifunctional Small Molecules Research Articles

Discovery of small molecules for autophagy-lysosome degradation of immune checkpoint proteins

Targeted protein degradation (TPD) technologies, particularly proteolysis targeting chimeras (PROTACs), have emerged as a promising branch of targeted therapy. Current ubiquitin-proteasome-dependent TPD technologies are limited to targeting intracellular proteins. Although the blockade of immune checkpoints has achieved great clinical success, most immune checkpoints are transmembrane proteins, which are difficult to be ubiquitinated and degraded by PROTACs. Herein, we developed a novel discovery strategy of bifunctional small molecules, which could mediate autophagy-lysosome degradation of immune checkpoints. F-1 was demonstrated to activate the autophagy-lysosome system, and conjugation of F-1 with inhibitors targeting programmed cell death-ligand 1 (PD-L1) or V-domain Ig suppressor of T-cell activation (VISTA) generated a new class of small molecules that effectively induce the degradation of PD-L1 or VISTA in tumor cells. The most promising PD-L1 degrader B3 significantly induced PD-L1 degradation in RKO cells through the autophagy-lysosome system and exhibited good tumor-inhibiting effects in vivo. Our work could expand the development of degraders targeting immune checkpoints and provide a promising discovery strategy for future autophagy-lysosome targeting degradation technology.

Translational modelling to predict human pharmacokinetics and pharmacodynamics of a Bruton's tyrosine kinase-targeted protein degrader BGB-16673.

Bifunctional small molecule degraders, which link the target protein with E3 ubiquitin ligase, could lead to the efficient degradation of the target protein. BGB-16673 is a Bruton's tyrosine kinase (BTK) degrader. A translational PK/PD modelling approach was used to predict the human BTK degradation of BGB-16673 from preclinical in vitro and in vivo data. A simplified mechanistic PK/PD model was used to establish the correlation between the in vitro and in vivo BTK degradation by BGB-16673 in a mouse model. Human and mouse species differences were compared using the parameters generated from in vitro human or mouse blood, and human or mouse serum spiked TMD-8 cells. Human PD was then predicted using the simplified mechanistic PK/PD model. BGB-16673 showed potent BTK degradation in mouse whole blood, human whole blood, and TMD-8 tumour cells in vitro. Furthermore, BGB-16673 showed BTK degradation in a murine TMD-8 xenograft model in vivo. The PK/PD model predicted human PD and the observed BTK degradation in clinical studies both showed robust BTK degradation in blood and tumour at clinical dose range. The presented simplified mechanistic model with reduced number of model parameters is practically easier to be applied to research projects compared with the full mechanistic model. It can be used as a tool to better understand the PK/PD behaviour for targeted protein degraders and increase the confidence when moving to the clinical stage.

Paralogue-Selective Degradation of the Lysine Acetyltransferase EP300.

The transcriptional coactivators EP300 and CREBBP are critical regulators of gene expression that share high sequence identity but exhibit nonredundant functions in basal and pathological contexts. Here, we report the development of a bifunctional small molecule, MC-1, capable of selectively degrading EP300 over CREBBP. Using a potent aminopyridine-based inhibitor of the EP300/CREBBP catalytic domain in combination with a VHL ligand, we demonstrate that MC-1 preferentially degrades EP300 in a proteasome-dependent manner. Mechanistic studies reveal that selective degradation cannot be predicted solely by target engagement or ternary complex formation, suggesting additional factors govern paralogue-specific degradation. MC-1 inhibits cell proliferation in a subset of cancer cell lines and provides a new tool to investigate the noncatalytic functions of EP300 and CREBBP. Our findings expand the repertoire of EP300/CREBBP-targeting chemical probes and offer insights into the determinants of selective degradation of highly homologous proteins.

Quantitative Measurement of Rate of Targeted Protein Degradation.

Targeted protein degradation (TPD) is a therapeutic approach that leverages the cell's natural machinery to degrade targets instead of inhibiting them. This is accomplished by using mono- or bifunctional small molecules designed to induce the proximity of target proteins and E3 ubiquitin ligases, leading to ubiquitination and subsequent proteasome-dependent degradation of the target. One of the most significant attributes of the TPD approach is its proposed catalytic mechanism of action, which permits substoichiometric exposure to achieve the desired pharmacological effects. However, apart from one in vitro study, studies supporting the catalytic mechanism of degraders are largely inferred based on potency. A more comprehensive understanding of the degrader catalytic mechanism of action can help aspects of compound development. To address this knowledge gap, we developed a workflow for the quantitative measurement of the catalytic rate of degraders in cells. Comparing a selective and promiscuous BTK degrader, we demonstrate that both compounds function as efficient catalysts of BTK degradation, with the promiscuous degrader exhibiting faster rates due to its ability to induce more favorable ternary complexes. By leveraging computational modeling, we show that the catalytic rate is highly dynamic as the target is depleted from cells. Further investigation of the promiscuous kinase degrader revealed that the catalytic rate is a better predictor of optimal degrader activity toward a specific target compared to degradation magnitude alone. In summary, we present a versatile method for mapping the catalytic activity of any degrader for TPD in cells.

Electrophilic proximity-inducing synthetic adapters enhance universal T cell function by covalently enforcing immune receptor signaling

Proximity-induction of cell-cell interactions via small molecules represents an emerging field in basic and translational sciences. Covalent anchoring of these small molecules represents a useful chemical strategy to enforce proximity; however, it remains largely unexplored for driving cell-cell interactions. In immunotherapeutic applications, bifunctional small molecules are attractive tools for inducing proximity between immune effector cells like Tcells and tumor cells to induce tumoricidal function. We describe a two-component system composed of electrophilic bifunctional small molecules and paired synthetic antigen receptors (SARs) that elicit Tcell activation. The molecules, termed covalent immune recruiters (CIRs), were designed to affinity label and covalently engage SARs. We evaluated the utility of CIRs to direct anti-tumor function of human Tcells engineered with three biologically distinct classes of SAR. Irrespective of the electrophilic chemistry, tumor-targeting moiety, or SAR design, CIRs outperformed equivalent non-covalent bifunctional adapters, establishing a key role for covalency in maximizing functionality. We determined that covalent linkage enforced early Tcell activation events in a manner that was dependent upon each SARs biology and signaling threshold. These results provide a platform to optimize universal SAR-T cell functionality and more broadly reveal new insights into how covalent adapters modulate cell-cell proximity-induction.

Abstract PO-010: Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma

Abstract Background: Non-Hodgkin lymphoma (NHL) represents a biologically and clinically diverse group of hematologic malignancies originating from B cells, T cells, and/or natural killer cells, with those of B-cell origin constituting ∼80–85% of all NHL cases. The BCL6 transcription factor is a key oncogenic driver of B-cell lymphomagenesis, and deregulated BCL6 expression is a common feature of diffuse large B-cell lymphoma (DLBCL), the most common type of NHL. BCL6 translocations have been observed in follicular lymphomas, the second most prevalent NHL, with studies suggesting that these translocations may portend a higher risk of transformation into aggressive lymphoma. BCL6 is also a lineage-defining transcription factor of T follicular helper cells, the cell of origin for angioimmunoblastic T-cell lymphoma (AITL). Moreover, BCL6 expression has been identified in biopsies of patients with AITL, and its continued expression was required for tumor growth in a mouse model of AITL, supporting BCL6 as a cancer driver in this type of NHL as well. ARV-393, an orally administered PROteolysis TArgeting Chimera (PROTAC) BCL6 degrader, is a bifunctional small molecule consisting of a BCL6-binding domain attached by a linker to a cereblon E3 ubiquitin ligase–binding domain. ARV-393 induces ubiquitination and subsequent degradation of BCL6 by the proteasome. In preclinical studies, ARV-393 induced rapid and robust degradation (>90%) of BCL6 in NHL cell lines and demonstrated dose-responsive tumor growth inhibition leading to tumor stasis or regression in xenograft models, supporting further investigation in patients with NHL. This multicenter, first-in-human, phase 1 study will evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and preliminary antitumor activity of ARV-393 in patients with advanced B-cell NHL or AITL. Methods: Eligible patients (aged ≥18 years) have relapsed/refractory mature B-cell NHL and ≥2 prior systemic therapies, or histologically confirmed AITL that has recurred or progressed following institutional standard-of-care therapy. Patients must also have ≥1 measurable lesion at study entry, Eastern Cooperative Oncology Group performance status of 0 or 1, freshly biopsied or archival tumor tissue available, adequate organ function, no active central nervous system involvement, and no prior allogeneic stem cell transplant or solid organ transplantation. Autologous stem cell transplant must not have occurred ≤100 days and previous CAR T-cell therapy ≤60 days prior to cycle 1 day 1 of ARV-393 treatment. ARV-393 will be administered orally in 28-day cycles with dose escalation according to a Bayesian optimal interval design. The primary objectives are to evaluate the safety and tolerability of ARV-393, determine maximum tolerated dose if necessary, and identify the recommended phase 2 dose(s) and dosing schedule. Secondary objectives are to characterize the pharmacokinetic profile of ARV-393 and evaluate its preliminary anti-tumor activity. Enrollment for this study will open in 2024. Citation Format: Paolo Caimi, Scott Huntington, Sean Landrette, Sheryl M Gough, Vivian Dai, Bryan Jackson, Juan Chavez, Sarah Tannenbaum-Dvir, Matthew Matasar. Trial in Progress: Phase 1 Study of ARV-393, a PROTAC BCL6 Degrader, in Advanced Non-Hodgkin Lymphoma [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-010.

Targeted Protein O-GlcNAcylation Using Bifunctional Small Molecules.

Protein O-linked β-N-acetylglucosamine modification (O-GlcNAcylation) plays a crucial role in regulating essential cellular processes. The disruption of the homeostasis of O-GlcNAcylation has been linked to various human diseases, including cancer, diabetes, and neurodegeneration. However, there are limited chemical tools for protein- and site-specific O-GlcNAc modification, rendering the precise study of the O-GlcNAcylation challenging. To address this, we have developed heterobifunctional small molecules, named O-GlcNAcylation TArgeting Chimeras (OGTACs), which enable protein-specific O-GlcNAcylation in living cells. OGTACs promote O-GlcNAcylation of proteins such as BRD4, CK2α, and EZH2 in cellulo by recruiting FKBP12F36V-fused O-GlcNAc transferase (OGT), with temporal, magnitude, and reversible control. Overall, the OGTACs represent a promising approach for inducing protein-specific O-GlcNAcylation, thus enabling functional dissection and offering new directions for O-GlcNAc-targeting therapeutic development.

Abstract 1687: Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring

Abstract Hematopoiesis relies on an intricate balance between stem-cell self-renewal and terminal maturation of blood cells. Disrupting this balance can lead to the development of deadly blood cancers, like acute myeloid leukemia (AML). Increasing evidence suggests that acquiring hematopoietic stem cell (HSC) gene expression programs in AML confers a particularly poor prognosis and increases risk of relapse with conventional therapy. Acquisition of these features in AML is commonly driven by dysregulation of MECOM, a transcription factor and master regulator of HSC self-renewal. To define the role of MECOM in AMLs, we engineered cell-line models of high-risk AML by endogenously tagging MECOM with an FKBP12F36V degron. These models enable targeted degradation of MECOM which we employed in concert with multi-omic readouts to assess changes in nascent transcription and chromatin accessibility to identify direct transcriptional targets of MECOM. We demonstrate that MECOM degradation results in a rapid increase in chromatin accessibility at MECOM-bound sites and increased expression of cognate genes to establish a pro-differentiation gene program. These findings suggest a previously unappreciated mechanism for MECOM in repressing differentiation-promoting cis-regulatory elements (cisREs) and their cognate target genes in AML. We then employed a pooled CRISPR inhibition (CRISPRi) screen to functionally characterize the role that each cisRE plays in promoting high-risk features in AML. Utilizing CRISPRi to repress MECOM-bound cisREs in concert with dTAG-mediated MECOM degradation, we examined if repression of over 500 individual MECOM-bound cisREs is sufficient to maintain AML progenitors in the absence of MECOM. This functional dissection revealed that the individual repression of three cisREs that regulate CEBPA, GFI1B, and RUNX1 is sufficient to maintain AML progenitor cells in the absence of MECOM. This result implicates MECOM as a direct repressor of known regulators of myeloid differentiation. Given these findings, we hypothesized that small molecule-based recruitment of a transcriptional coactivator to MECOM could rewire its repressive function to activate myeloid differentiation and subsequent leukemia cell death. To test this approach, we treated a MECOM-FKBP12F36V AML degron model with the bifunctional small molecule NICE-01 (AP1867-PEG2-JQ1) to recruit an epigenetic activator (BRD4) to MECOM to functionally rewire this transcriptional network. NICE-01 treatment induced significant differentiation of AML progenitor cells relative to JQ1 treatment alone. Our work highlights the utility of targeted protein degradation to mechanistically interrogate the function of a key driver of high-risk AMLs and suggests the potential for small molecule-rewiring of stem cell gene regulatory networks to confer therapeutic benefit. Citation Format: Travis J. Fleming, Michael Gundry, Richard Voit, Mateusz Antoszewski, William J. Gibson, Ananthan Sadagopan, Vijay G. Sankaran. Targeted degradation of MECOM in high-risk acute myeloid leukemia reveals a novel repressive function that is amenable to therapeutic small-molecule rewiring [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 1687.

Abstract 3307: c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC

Abstract The interaction between E3 ligases and chaperones is crucial for proteostasis, regulating more than 10% of total proteasomes and over 30% of human E3 ligases (Kevei, 2017). Chaperones also can recognize their substrates and direct them to degradation by the ubiquitin-proteasome system (UPS). Developing a targeted protein degradation (TPD) technology that utilizes these advantageous attributes of chaperones, termed chaperone-mediated protein degradation (CMPD) technology, would broaden the range of disease-causing target proteins and would efficiently degrade them by recruiting various intracellular E3 ligases through interaction with chaperones, compare to other TPD approaches. The c-mesenchymal-epithelial transition (c-MET) is recognized as primary cause of drug-resistance to EGFR inhibitors in non-small cell lung cancer (NSCLC). Herein, to degrade the c-MET, we synthesized bifunctional small molecule compound inducing c-MET and chaperones in proximity, termed CMPD-MET degrader, and evaluate it in MET-mutant NSCLC cell lines and mouse xenograft models. The efficacy and potency of CMPD-MET degrader were assessed using H596 and H1437 cells in vitro. Cell proliferation was measured using MTT assay and c-Met degradation assay was conducted, along with the examination of proteasome-dependent degradation via western blot. An in vivo H596 xenograft model gauged impact of CMPD-MET degrader via I.P. administration. In NSCLC with MET Exon 14 skipping mutation cell line H596 and H1437, treatment a OZD-MET degrader resulted inhibition of cell proliferation with IC50< 5 µM and subsequent proteasome-dependent c-MET degradation with DC50 ranging from 4-6 µM for 24h. The degradation of c-MET levels significantly induced by OZD-MET degrader, especially, the c-Met was degraded by 70.4% and 51.7% at 5 µM for 24h in H596 and H1437 cells, respectively. Also, OZ-MET degrader effectively suppressed c-MET mediated signaling and showed cleavage of PARP-1 in a dose-dependent manner. OZD-MET degrader induced selective degradation of c-MET showing at least more than about 5-fold selectivity over other chaperones-regulated client proteins.In the H596 xenograft model, OZD-MET degrader was administered in 5 dosage groups (minimum dose is 2.5 mg/kg) resulted in dramatic reduction in tumor size compared to the vehicle-treated group. Remarkably, administration of OZD-MET degrader revealed more than 90% degradation of c-Met in tumor tissues compared to the vehicle groups via western blots and fluorescence image. In contrast, c-MET inhibitor treated group showed no effect on degradation of c-MET in tumor tissues. In summary, we developed a novel c-Met targeting chaperone-mediated protein degrader, showed highly potent and selective proteasome-dependent c-MET degradation and notable inhibition of tumor size in xenograft models. These results provide a strong foundation for further evaluation in clinical trials. Citation Format: Han Jung Chae, Jisun Kim, SunHye Lee, Ho Yoo, Hyun-Kyoung Kim, Young-Jin Yu. c-Met targeting chaperone-mediated protein degrader for therapeutic intervention in NSCLC [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 3307.

Abstract B113: Discovery of PRT3789, a first-in-class potent and selective SMARCA2 degrader in clinical trials for the treatment of patients with SMARCA4 mutated cancers

Abstract SWI/SNF complexes play an important role in controlling gene expression by remodeling chromatin. SMARCA2 (BRM) and SMARCA4 (BRG1) are the core catalytic subunits of the SWI/SNF complexes. SMARCA4 expression is lost in some cancers due to its gene mutations, and SMARCA4-deficient cancer cells are highly dependent on its paralog gene SMARCA2 for their survival. SMARCA4 mutation is associated with worse patient prognosis and reduced responsiveness to inhibitors of KRAS G12C or immune checkpoint, compared to patients with SMARCA4 WT cancers. Furthermore, SMARCA4 mutation is often mutually exclusive with other targetable oncogenic alterations such as EGFR, ALK, MET, RET and ROS1, making SMARCA4 mutated cancers a clinically unmet disease. We have identified PRT3789, a potent and selective SMARCA2 degrader, that selectively targets SMARCA4-deficient cancer cells. In the present studies, we discuss the mechanism and activity of PRT3789 in SMARCA4-deficient tumor models as well as our clinical methods to assess target engagement in PRT3789 treated patients. PRT3789 is a bifunctional small molecule comprised of a novel SMARCA2-bromodomain binder linked to a VHL E3 ubiquitin ligase-binding moiety. PRT3789 has been shown to effectively catalyze the polyubiquitination of specific lysine residues of SMARCA2, resulting in SMARCA2 selective degradation over SMARCA4. The selectivity of PRT3789-induced SMARCA2 degradation was confirmed by cellular assays, protein mass spectrometry as well as ex vivo treatment of human PBMCs. PRT3789 selectively inhibits cell proliferation of SMARCA4-deficient cancer cells, but not SMARCA4 WT cells, when tested in a panel of cancer cell lines. Cell lines with specific SMARCA4 missense mutations also responded to PRT3789, suggesting that they are LOF mutations. Mechanistically, degradation of SMARCA2 leads to disruption of SWI/SNF complexes, dysregulation of chromatin remodeling and gene expression, resulting in inhibition of oncogenic pathways such as cell-cycle and DNA replication gene signatures. After treatment with PRT3789, SMARCA4-deleted cancer cells undergo G1 cell-cycle arrest and apoptosis. In vivo efficacy studies were conducted with genomically selected cell line-derived and patient-derived NSCLC xenograft models in mice. PRT3789 administration significantly inhibits the growth of SMARCA4-deleted NSCLC but demonstrates no effects on SMARCA4 WT tumor growth. In preclinical models, the effects on tumor growth were synergistic when PRT3789 was combined with other SOC therapies including gemcitabine, docetaxel, KRAS G12Ci as well as investigational drug candidates such as MCL1i, CDK4/6i, and CDK9i. PRT3789 also showed combination effects with anti-PD1 mAb in efficacy models in vivo. In the first-in-human Phase I dose-escalation study, PRT3789 target engagement will be assessed by determining the levels of SMARCA2 protein in human PBMCs following treatment with PRT3789. We aim to further investigate the safety, pharmacokinetics (PK) as well as anti-tumor activity of PRT3789 in the ongoing clinical trial (NCT05639751). Citation Format: Koichi Ito, Michael Hulse, Anjana Agarwal, Jack Carter, Monisha Sivakumar, Komali Vykuntam, Min Wang, Miles Cowart, Joy Cote, William Gowen-MacDonald, Brian Vidal Torres, Justin Kurian, Neha Bhagwat, Norman Fultang, Alexander Grego, Andrew Moore, Ashley Schwab, Jessica Burtell, Olusola Peace Osinubi, Jacob Spruance, Liang Lu, Philip Pitis, Corey Basch, Klare Bersch, Chaofeng Dai, Raul Leal, Artem Shvartsbart, Ganfeng Cao, Bo Shen, Patrick Wen, Joseph Rager, Ross Kuskovsky, Bob Landman, Tom Emm, Stefan Ruepp, Chunhua Qin, Gina Paris, Jennifer Xavier, Rachel Chiaverelli, Sang Hyun Lee, Sandy Geeganage, Hong Lin, Diane Heiser, Bruce Ruggeri, Naveen Babbar, Andrew Combs, Peggy Scherle. Discovery of PRT3789, a first-in-class potent and selective SMARCA2 degrader in clinical trials for the treatment of patients with SMARCA4 mutated cancers [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B113.

Bifunctional Small Molecules That Induce Nuclear Localization and Targeted Transcriptional Regulation.

The aberrant localization of proteins in cells is a key factor in the development of various diseases, including cancer and neurodegenerative disease. To better understand and potentially manipulate protein localization for therapeutic purposes, we engineered bifunctional compounds that bind to proteins in separate cellular compartments. We show these compounds induce nuclear import of cytosolic cargoes, using nuclear-localized BRD4 as a "carrier" for co-import and nuclear trapping of cytosolic proteins. We use this system to calculate kinetic constants for passive diffusion across the nuclear pore and demonstrate single-cell heterogeneity in response to these bifunctional molecules with cells requiring high carrier to cargo expression for complete import. We also observe incorporation of cargo into BRD4-containing condensates. Proteins shown to be substrates for nuclear transport include oncogenic mutant nucleophosmin (NPM1c) and mutant PI3K catalytic subunit alpha (PIK3CAE545K), suggesting potential applications to cancer treatment. In addition, we demonstrate that chemically induced localization of BRD4 to cytosolic-localized DNA-binding proteins, namely, IRF1 with a nuclear export signal, induces target gene expression. These results suggest that induced localization of proteins with bifunctional molecules enables the rewiring of cell circuitry, with significant implications for disease therapy.

Mitochondrial Protease Targeting Chimeras for Mitochondrial Matrix Protein Degradation.

Targeted protein degradation (TPD) is an emerging technique for protein regulation. Currently, all TPD developed in eukaryotic cells relies on either ubiquitin-proteasome or lysosomal systems, thus are powerless against target proteins in membrane organelles lacking proteasomes and lysosomes, such as mitochondria. Here, we developed a mitochondrial protease targeting chimera (MtPTAC) to address this issue. MtPTAC is a bifunctional small molecule that can bind to mitochondrial caseinolytic protease P (ClpP) at one end and target protein at the other. Mechanistically, MtPTAC activates the hydrolase activity of ClpP while simultaneously bringing target proteins into proximity with ClpP. Taking mitochondrial RNA polymerase (POLRMT) as a model protein, we have demonstrated the powerful proteolytic ability and antitumor application prospects of MtPTAC, both in vivo and in vitro. This is the first modularly designed TPD that can specifically hydrolyze target proteins inside mitochondria.

Design, Synthesis and Biological Evaluation of α-Synuclein Proteolysis-Targeting Chimeras.

α-Synuclein aggregation under pathological conditions is one of the causes of related neurodegenerative diseases. PROTACs (proteolysis targeting chimeras) are bifunctional small molecules that induce a post-translational erasure of proteins via the ubiquitination of target proteins by E3 ubiquitin ligase and subsequent proteasomal degradation. However, few research studies have been conducted for targeted protein degradation of α-synuclein aggregates. In this article, we have designed and synthesized a series of small-molecule degraders 1-9 based on a known α-synuclein aggregation inhibitor sery384. In silico docking studies of sery384 with α-synuclein aggregates were accomplished to ensure that the compounds bound to α-synuclein aggregates specifically. The protein level of α-synuclein aggregates was determined to evaluate the degradation efficiency of PROTAC molecules on α-synuclein aggregates in vitro. The results show that compound 5 had the most significant degradation effect, with DC50 of 5.049 μM, and could induce the degradation of α-synuclein aggregates in a time- and dose-dependent manner in vitro. Furthermore, compound 5 could inhibit the elevation of the ROS level caused by overexpression and aggregation of α-synuclein and protect H293T cells from α-synuclein toxicity. Conclusively, our results provide a new class of small-molecule degraders and an experimental basis for the treatment of α-synuclein related neurodegenerative diseases.

Recent Developments in Medicinal Chemistry and Therapeutic Potential of Anti-Cancer PROTACs-Based Molecules.

PROTACs is an emerging technique that addresses the disease causing proteins by targeting protein degradation. PROTACs molecules are bifunctional small molecules that simultaneously bind to the protein of interest (POIs) and an E3 ligase followed by ubiquitination and degradation of the protein of interest by the proteasome. PROTACs technology offers many advantages over classical inhibition such as PROTACs molecules can target intracellular proteins regardless of their function and have good tissue distribution. They are capable to target mutated and overexpressed proteins, thus potent molecules with the high degradation selectivity can be designed. Moreover, PROTACs molecules can target the undruggable proteome which makes up almost 85% of human proteins. Several PROTACs-based compounds have exhibited high therapeutic potency and some of them are currently under clinical trials. Current article gives a comprehensive overview of the current development of PROTACs-based anticancer compounds along with the structure-activity relationship of the reported molecules. The development of PROTACs-based compounds and related research regarding medicinal chemistry is one of the most active and hot topics for research. It is believed that the current review article can be helpful to understand the logical design of more efficacious PROTACs-based molecules with less toxicity and more selectivity.

Structural-based design of HD-TAC7 PROteolysis TArgeting chimeras (PROTACs) candidate transformations to abrogate SARS-CoV-2 infection

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for about 672 million infections and 6.85 million deaths worldwide. Upon SARS-CoV-2 infection, Histone deacetylases (HDACs) hyperactivate the pro-inflammatory response resulting in stimulation of Acetyl-Coenzyme A and cholesterol for viral entry. HDAC3 inhibition results in the anti-inflammatory activity and reduction of pro-inflammatory cytokines that may restrict COVID-19 progression. Here, we have designed 44 conformational ensembles of previously known HD-TAC7 by enumerating torsions of dihedral angles tested for their binding preferences against HDAC3. Through scrutinizing their placements at active site and binding affinities, three hits were isolated. Cereblon (CRBN) is a well-known E3 ligase that facilitates Proteolysis Targeting Chimeras (PROTACs) targeting. Three entities, including HDAC3-binding moiety (4-acetamido-N-(2-amino-4 fluorophenyl) benzamide), a 6-carbon linker, and CRBN binding ligand (pomalidomide) were assembled to design 4 PROTACs followed by energy minimization and docking against HDAC3 and CRBN, respectively. Subsequent molecular dynamics (MD) and free energy analyses corroborated similar binding trends and favorable energy values. Among all cases, Met88, GLu106, Pro352, Trp380 and Trp388 residues of CRBN, and Pro23, Arg28, Lys194, Phe199, Leu266, Thr299 and Ile346 residues of HDAC3 were engaged in PROTAC binding. Thus, conformational dynamics of both HDAC3 and CRBN moieties are essential for the placement of PROTAC, resulting in target degradation. Overall, the proposed bifunctional small molecules may effectively target HDAC3, stimulating innate immune response to restrict COVID-19 hyperinflammation. This study supports the basis for designing new PROTACs by limiting the conformational search space that may prove more efficient for targeting the protein of interest.Communicated by Ramaswamy H. Sarma

Selective androgen receptor degrader (SARD) to overcome antiandrogen resistance in castration-resistant prostate cancer.

In patients with castration-resistant prostate cancer (CRPC), clinical resistances such as androgen receptor (AR) mutation, AR overexpression, and AR splice variants (ARVs) limit the effectiveness of second-generation antiandrogens (SGAs). Several strategies have been implemented to develop novel antiandrogens to circumvent the occurring resistance. Here, we found and identified a bifunctional small molecule Z15, which is both an effective AR antagonist and a selective AR degrader. Z15 could directly interact with the ligand-binding domain (LBD) and activation function-1 region of AR, and promote AR degradation through the proteasome pathway. In vitro and in vivo studies showed that Z15 efficiently suppressed AR, AR mutants and ARVs transcription activity, downregulated mRNA and protein levels of AR downstream target genes, thereby overcoming AR LBD mutations, AR amplification, and ARVs-induced SGAs resistance in CRPC. In conclusion, our data illustrate the synergistic importance of AR antagonism and degradation in advanced prostate cancer treatment.

Designing Janus catalysts for renewable energy-relevant bifunctional small molecule activation

Developing bifunctional catalysts is critical for ensuring renewable energy applications via small molecule activation.

HZ-R061, a Selective STAT3 Degrader with Activity in Preclinical Model of Hematologic Malignances

The Janus kinase/Signal transducer and activator of transcription (JAK-STAT) pathway is one of the core cancer pathways that integrates signals from cytokines, hormones and growth factors to induce or repress gene expression in cells . The pathway consists of four JAK kinases and seven STAT transcription factors (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6). STAT3 is the best-studied family member of the JAK-STAT pathway in cancer, and is a known oncogene in various types of tumor. So STAT3 is an attractive cancer therapeutic target, great efforts have been made to discover effective STAT3 inhibitor. However most of reported STAT3 inhibitors got failed for further development due to the low selectivity, weak binding affinity or poor drugability. STAT3 is considered as an undruggable target for long time. Recently, the proteolysis targeting chimeras (PROTAC) strategy has gained momentum for its promise in the discovery and development of an entirely new type of therapeutics for the treatment of human diseases. PROTAC molecule is a bifunctional small molecule designed to recruit cellular degradation machinery to induce targeted protein degradation in a catalytical way. PROTAC can be a new strategy to effectively target undruggable targets such as STAT3. HZ-R061 is a novel STAT3 selective degrader which is discovered and developed through our DaTProD® platform which was set up to promote PROTAC development. HZ-R061 can selective and effective degrade STAT3 in various types of cells including MOLM16, A549, SU-DHL1, karpas299, LY3, THP1 and human PBMC with DC50 values at nanomolar level. In in-vitro anti-proliferative assay, HZ-R061 exhibits potent anti-proliferative activity against CTCL and AML cell lines. Furthermore, MOLM16, SU-DHL1 and Karpas299 xenograft models were used for in-vivo anti-tumor evaluation. After IV administration of HZ-R061 (QW), the growth of tumor was significantly suppressed in all three models. As STAT3 is a master regulator in tumor microenvironment, inhibition of STAT3 could remodel TME towards in favor of anti-cancer. The combination of HZ-R061 with PD-(L)1 mAb was also conducted in CT26 model. HZ-R061 exhibits significantly synergistic effect in combination with PD-(L)1 mAb. Additionally, HZ-R061 also potently decreases phosphorylation level of STAT3 in IL-6 stimulated THP-1 cells with IC50 value of 3.8nM, which is 50 folds stronger than JAK inhibitor Ruxolitinib. HZ-R061 also shows strong effect in in-vitro Th17 differentiation assay and EAE model. These results suggest that HZ-R061 is also a promising drug candidate for various Th17 driven auto-immune diseases including MS, RA, SSc and IPF. In conclusion, we discovered a pre-clinical effective STAT3 degrader HZ-R061, which has potential for further development in tumor and auto-immune diseases.

Design, Synthesis, and Bioactivity of Novel Bifunctional Small Molecules for Alzheimer's disease.

The abnormal phosphorylation of the τ-protein is a typical early pathological feature of Alzheimer’s disease (AD). The excessive phosphorylation of the τ-protein in the brain causes the formation of neurofibrillary tangles (NFTs) and increases the neurotoxicity of amyloid-β (Aβ). Thus, targeting the τ-protein is considered a promising strategy for treating AD. Herein, we designed and synthesized a series of molecules containing bifunctional groups to recognize the τ-protein and the E3 ligase. The molecules were examined in vitro, and their effects were tested on PC12 cells. In addition, we further studied the pharmacokinetics of compound I3 in healthy rats. Our data showed that compound I3 could effectively degrade τ-protein, reduce Aβ-induced cytotoxicity, and regulate the uneven distribution of mitochondria, which may open a new therapeutic strategy for the treatment of AD.

Measurement of PROTAC ternary complex formation using the switchSENSE Y-structure and FRET signals

Proteolysis targeting chimeras (PROTACs) are essential bifunctional small molecules that engage the formation of a ternary complex consisting of an E3 ubiquitin ligase, a target protein of interest and the PROTAC itself. Using switchSENSE® technology and the novel DNA Y-structure, an E3 ligase as well as a target protein can be functionalized on each separate end of two FRET pair color-coded Y-arms. The Y-structure closes upon PROTAC binding and the subsequent ternary complex formation bringing together the green donor and the red acceptor dye into a closer, FRET sensitive, distance.