From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9

Petrilli, Whitney L.; Adam, Gregory C.; Erdmann, Roman S.; Abeywickrema, Pravien; Agnani, Vijayalakshmi; Ai, Xi; Baysarowich, Jennifer; Byrne, Noel; Caldwell, John P.; Chang, Wonsuk; DiNunzio, Edward; Feng, Zhe; Ford, Rachael E.; Ha, Sookhee; Huang, Yunxin; Hubbard, Brian K.; Johnston, Jennifer M.; Kavana, Michael; Lisnock, JeanMarie; Liang, Rui; Lu, Jun; Lu, Zhijian; Meng, Juncai; Orth, Peter; Palyha, Oksana; Parthasarathy, Gopal; Salowe, Scott P.; Sharma, Sujata; Shipman, Jennifer M.; Soisson, S.M.; Strack, Alison M.; Youm, Hyewon; Zhao, Kake; Zink, Deborah L.; Zokian, Hratch; Addona, George H.; Akinsanya, Karen; Tata, James R.; Xiong, Yusheng; Imbriglio, Jason E.

doi:10.1016/j.chembiol.2019.10.002

Cited by 50 publications

(37 citation statements)

References 40 publications

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“…In previous reports, the researchers discovered of the cryptic peptide-binding sites on PCSK9 and they can be used for the design of small molecular antagonists [23,36]. In most instances, small molecular drugs have very short half-life period and major side effects.…”

Section: Discussionmentioning

confidence: 99%

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

Wang

Zhang

et al. 2020

Clinical & Translational Med

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BackgroundThe advent of proprotein convertase subtilisin/kexin type 9 (PCSK9)–inhibiting drugs have provided an effective, but extremely expensive treatment for the management of low density lipoprotein (LDL). Our aim was to explore a cost‐effective application of camelid anti‐PCSK9 single domain antibodies (sdAbs), which are high variable regions of the camelid heavy chain antibodies (VHHs), as a human PCSK9 (hPCSK9) inhibitor. One female llama was immunized with hPCSK9. Screening of high affinity anti‐PCSK9 VHHs was carried out based on surface plasmon resonance (SPR) technology. We reported a lysate kinetic analysis method improving the screening efficiency. To increase the serum half‐life and targeting properties, the constant region fragment of the human immunoglobulin gamma sub‐type 4 (IgG4 Fc) was incorporated to form a novel llama‐human chimeric molecule (VHH‐hFc). ResultsThe PCSK9 inhibiting effects of the VHH proteins were analyzed in two human liver hepatocellular cells (HepG2 and Huh7) and in the hPCSK9 transgenic Sprague–Dawley (SD) rat model. The hPCSK9 antagonistic potency of the bivalent VHH‐hFc exceeded the monovalent VHH (P < 0.001) in hepatocarcinoma cells. Furthermore, the llama‐human chimeric VHH‐Fc protein had a similar reduction (~ 40%) of the LDL‐c and total cholesterol when compared to the approved evolocumab in transgenic SD rat model, but with low cost. More surprisingly, the chimeric heavy chain antibodies could be persevered for 3 months at room temperature with little loss of the affinity. ConclusionsDue to the high yield and low cost of Pichia pastoris, lipid‐lowering effect and strong stability, the llama‐human chimeric antibody (VHH‐Fc) offers a potent therapeutic candidate for the control of the serum lipid level.

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Section: Discussionmentioning

confidence: 99%

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

Wang

Zhang

et al. 2020

Clinical & Translational Med

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“…The best hit was found to be an allosteric interactor, which was optimized to generate a binder exposing a moiety suitable for functionalization. Of note, protacs based on E3 ligases did not induce degradation; instead, the Boc3Arg ligand could induce a remarkable decrease in PCSK9 endogenous levels, even though complete target depletion was not reached [142].…”

Section: Ubiquitin-independent Protacsmentioning

confidence: 97%

“…Oncology: breast cancer [138] Ubiquitin-independent degrader eDHFR and GST-α1 GST-π E3-independent Research use [140] Ubiquitin-independent degrader GST proteins, eDHFR E3-independent Oncology [141] Ubiquitin-independent degrader Proprotein convertase substilisin-like/kexin type 9(PCSK9) E3-independent (20S targeting) Vascular disease [142] Table 1. Conts.…”

Section: Concluding Remarks: An Exciting Third Generation Of Protein-mentioning

confidence: 99%

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

2020

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The induction of protein degradation in a highly selective and efficient way by means of druggable molecules is known as targeted protein degradation (TPD). TPD emerged in the literature as a revolutionary idea: a heterobifunctional chimera with the capacity of creating an interaction between a protein of interest (POI) and a E3 ubiquitin ligase will induce a process of events in the POI, including ubiquitination, targeting to the proteasome, proteolysis and functional silencing, acting as a sort of degradative knockdown. With this programmed protein degradation, toxic and disease-causing proteins could be depleted from cells with potentially effective low drug doses. The proof-of-principle validation of this hypothesis in many studies has made the TPD strategy become a new attractive paradigm for the development of therapies for the treatment of multiple unmet diseases. Indeed, since the initial protacs (Proteolysis targeting chimeras) were posited in the 2000s, the TPD field has expanded extraordinarily, developing innovative chemistry and exploiting multiple degradation approaches. In this article, we review the breakthroughs and recent novel concepts in this highly active discipline.

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“…Based on this concept, cell lysates with and without Erlotinib were incubated at different temperatures followed by centrifugation to separate soluble-(protein-ligand complex) and insoluble-protein (denatured and aggregated). The relationship between aggregation phenomena and its impact by temperature with or without compounds is verified by analyzing the soluble fraction by western blotting [29][30][31]. To investigate optimal conditions for POLA2 aggregation, we first performed temperature-dependent CETSA at 39-67 • C (Figure S3c).…”

Section: Identification Of New Erlotinib Binding Proteinsmentioning

confidence: 99%

DNA Polymerase Alpha Subunit B Is a Binding Protein for Erlotinib Resistance in Non-Small Cell Lung Cancer

Kim

Lee

et al. 2020

Cancers

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Erlotinib inhibits epithelial growth factor receptor (EGFR) kinase activity and is used to treat non-small cell lung cancer (NSCLC). Despite its high efficacy, recurrence can occur in patients who become resistant to the drug. To address the underlying mechanism of Erlotinib resistance, we investigated additional mechanisms related to mode-of-drug-action, by multiple protein-binding interactions, besides EGFR by using drug affinity responsive target stability (DARTS) and liquid chromatography-mass spectrometry (LC-MS/MS) methods with non-labeled Erlotinib. DNA polymerase alpha subunit B (POLA2) was identified as a new Erlotinib binding protein that was validated by the DARTS platform, complemented with cellular thermal shift assays. Genetic knock-down of POLA2 promoted the anti-proliferative effect of the drug in the Erlotinib-resistant cell line H1299 with high POLA2 expression, whereas the overexpression of POLA2 restored anti-proliferative effects in the Erlotinib-sensitive cell line HCC827 with low POLA2 expression. Importantly, POLA2 expression levels in four NSCLC cell lines were positively correlated with anti-proliferative Erlotinib efficacy (Pearson correlation coefficient, R = 0.9886). These results suggest that POLA2 is a novel complementary target protein of Erlotinib, and could clinically provide validity as a surrogate marker for drug resistance in patients with NSCLC.

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From Screening to Targeted Degradation: Strategies for the Discovery and Optimization of Small Molecule Ligands for PCSK9

Cited by 50 publications

References 40 publications

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

The novel llama‐human chimeric antibody has potent effect in lowering LDL‐c levels in hPCSK9 transgenic rats

The Potential of Proteolytic Chimeras as Pharmacological Tools and Therapeutic Agents

DNA Polymerase Alpha Subunit B Is a Binding Protein for Erlotinib Resistance in Non-Small Cell Lung Cancer

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