Proteolysis Targeting Chimeric Molecules Research Articles

PROTAC EZH2 degrader-1 overcomes the resistance of podophyllotoxin derivatives in refractory small cell lung cancer with leptomeningeal metastasis

BackgroundLeptomeningeal metastasis (LM) of small cell lung cancer (SCLC) is a highly detrimental occurrence associated with severe neurological disorders, lacking effective treatment currently. Proteolysis-targeting chimeric molecules (PROTACs) may provide new therapeutic avenues for treatment of podophyllotoxin derivatives-resistant SCLC with LM, warranting further exploration.MethodsThe SCLC cell line H128 expressing luciferase were mutated by MNNG to generate H128-Mut cell line. After subcutaneous inoculation of H128-Mut into nude mice, H128-LM and H128-BPM (brain parenchymal metastasis) cell lines were primarily cultured from LM and BPM tissues individually, and employed to in vitro drug testing. The SCLC-LM mouse model was established by inoculating H128-LM into nude mice via carotid artery and subjected to in vivo drug testing. RNA-seq and immunoblotting were conducted to uncover the molecular targets for LM.ResultsThe SCLC-LM mouse model was successfully established, confirmed by in vivo live imaging and histological examination. The upregulated genes included EZH2, SLC44A4, VEGFA, etc. in both BPM and LM cells, while SLC44A4 was particularly upregulated in LM cells. When combined with PROTAC EZH2 degrader-1, the drug sensitivity of cisplatin, etoposide (VP16), and teniposide (VM26) for H128-LM was significantly increased in vitro. The in vivo drug trials with SCLC-LM mouse model demonstrated that PROTAC EZH2 degrader-1 plus VM26 or cisplatin/ VP16 inhibited H128-LM tumour significantly compared to VM26 or cisplatin/ VP16 alone (P < 0.01).ConclusionThe SCLC-LM model effectively simulates the pathophysiological process of SCLC metastasis to the leptomeninges. PROTAC EZH2 degrader-1 overcomes chemoresistance in SCLC, suggesting its potential therapeutic value for SCLC LM.

EGFR degraders in non-small-cell lung cancer: Breakthrough and unresolved issue.

The epidermal growth factor receptor (EGFR) has been well validated as a therapeutic target for anticancer drug discovery. Osimertinib has become the first globally accessible third-generation EGFR inhibitor, representing one of the most advanced developments in non-small-cell lung cancer (NSCLC) therapy. However, a tertiary Cys797 to Ser797 (C797S) point mutation has hampered osimertinib treatment in patients with advanced EGFR-mutated NSCLC. Several classes of fourth-generation EGFR inhibitors were consequently discovered with the aim of overcoming the EGFRC797S mutation-mediated resistance. However, no clinical efficacy data of the fourth-generation EGFR inhibitors were reported to date, and EGFRC797S mutation-mediated resistance remains an "unmet clinical need." Proteolysis-targeting chimeric molecules (PROTACs) obtained from EGFR-TKIs have been developed to target drug resistance EGFR in NSCLC. Some PROTACs are from nature products. These degraders compared with EGFR inhibitors showed better efficiency in their cellular potency, inhibition, and toxicity profiles. In this review, we first introduce the structural properties of EGFR, the resistance, and mutations of EGFR, and then mainly focus on the recent advances of EGFR-targeting degraders along with its advantages and outstanding challenges.

Design, synthesis, and evaluation of VHL-based EZH2 degraders for breast cancer

EZH2 (enhancer of zeste homolog 2) is one of the most important histone methyltransferases (HMTs), and overexpression of EZH2 can lead to proliferation, migration and angiogenesis of tumor cells. But most of EZH2 inhibitors are only effective against some hematologic malignancies and have poor efficacy against solid tumors. Here, we report the design, synthesis, and evaluation of highly potent proteolysis targeting chimeric (PROTACs) small molecules targeting EZH2. We developed a potent and effective EZH2 degrader P4, which effectively induced EZH2 protein degradation and inhibited breast cancer cell growth. Further studies showed that P4 can significantly decrease the degree of H3K27me3 in MDA-MB-231 cell line, induce apoptosis and G0/G1 phase arrest in Pfeiffer and MDA-MB-231 cell lines. Therefore, P4 is a potential anticancer molecule for breast cancer treatment.

Chemical Specification of E3 Ubiquitin Ligase Engagement by Cysteine-Reactive Chemistry.

Targeted protein degradation relies on small molecules that induce new protein-protein interactions between targets and the cellular protein degradation machinery. Most of these small molecules feature specific ligands for ubiquitin ligases. Recently, the attachment of cysteine-reactive chemical groups to pre-existing small molecule inhibitors has been shown to drive specific target degradation. We demonstrate here that different cysteine-reactive groups can specify target degradation via distinct ubiquitin ligases. By focusing on the bromodomain ligand JQ1, we identify cysteine-reactive functional groups that drive BRD4 degradation by either DCAF16 or DCAF11. Unlike proteolysis-targeting chimeric molecules (PROTACs), the new compounds use a single small molecule ligand with a well-positioned cysteine-reactive group to induce protein degradation. The finding that nearly identical compounds can engage multiple ubiquitination pathways suggests that targeting cellular pathways that search for and eliminate chemically reactive proteins is a feasible avenue for converting existing small molecule drugs into protein degrader molecules.

Proteolysis Targeting Chimera (PROTAC) as a promising novel therapeutic modality for the treatment of triple-negative breast cancer (TNBC).

Breast cancer (BC) is considered one of the most prevalent malignancies impacting women worldwide, constituting 15% of all new cancer cases. It is classified according to its molecular targets into three subtypes; HR+/ERBB2-, ERBB2+/HR+, or HR- and triple-negative breast cancer (TNBC). TNBC is considered aggressive cancer with bad prognosis and poor clinical outcomes. It lacks estrogen, progesterone, and ERBB2 receptors rendering its treatment more challenging. Owing to its unresponsiveness to hormonal therapy, quick tumor growth, and the high probability to have spread at the time of discovery, it is more likely to recur following therapy. Proteolysis Targeting Chimeric molecules (PROTACs) are hetero-bifunctional molecules that are able to hijack the ubiquitin-proteasome system, a major physiological proteolytic mechanism. This leads to ubiquitination through endogenous E3 ligases and subsequent degradation through 26-S proteasome. Since its discovery in 2001, PROTACs have been considered an important therapeutic modality due to their ability to target and degrade undruggable proteins. In vitro studies have been conducted to investigate the possibility of using PROTACs as a therapeutic modality in TNBC. Data available propose great potential of PROTACs technology as a major candidate in TNBC management. Further in vitro and in vivo clinical trials are required to establish a definitive decision. In this review, we aim to give a brief overview of TNBC and PROTACs and highlight the rationale behind using PROTACs as a therapeutic modality in patients with TNBC.

Structure-Based Discovery of Selective Histone Deacetylase 8 Degraders with Potent Anticancer Activity.

Inducing protein degradation by proteolysis targeting chimeras has gained tremendous momentum as a promising novel therapeutic strategy. Here, we report the design, synthesis, and biological characterization of highly potent proteolysis targeting chimeric small molecules targeting the epigenetic regulator histone deacetylase 8 (HDAC8). We developed potent and effective HDAC8 degraders, as exemplified by SZUH280 (16e), which effectively induced HDAC8 protein degradation and inhibited cancer cell growth even at low micromolar concentrations. Our preliminary mechanistic studies revealed that SZUH280 hampers DNA damage repair in cancer cells, promoting cellular radiosensitization. In mice, a single SZUH280 dose induced rapid and prolonged HDAC8 protein degradation in xenograft tumor tissues. Moreover, SZUH280 alone or in combination with irradiation resulted in long-lasting tumor regression in an A549 tumor mouse model. Our findings qualify a new chemical tool for HDAC8 knockdown and may lead to the development of a new class of cancer therapeutics.

Proteolysis Targeting Chimeric Molecules: Tuning Molecular Strategies for a Clinically Sound Listening.

From seminal evidence in the early 2000s, the opportunity to drive the specific knockdown of a protein of interest (POI) through pharmacological entities called Proteolysis Targeting Chimeric molecules, or PROTACs, has become a possible therapeutic option with the involvement of these compounds in clinical trials for cancers and autoimmune diseases. The fulcrum of PROTACs pharmacodynamics is to favor the juxtaposition between an E3 ligase activity and the POI, followed by the ubiquitination of the latter and its degradation by the proteasome system. In the face of an apparently modular design of these drugs, being constituted by an E3 ligase binding moiety and a POI-binding moiety connected by a linker, the final structure of an efficient PROTAC degradation enhancer often goes beyond the molecular descriptors known to influence the biological activity, specificity, and pharmacokinetics, requiring a rational improvement through appropriate molecular strategies. Starting from the description of the basic principles underlying the activity of the PROTACs to the evaluation of the strategies for the improvement of pharmacodynamics and pharmacokinetics and rational design, this review examines the molecular elements that have been shown to be effective in allowing the evolution of these compounds from interesting proof of concepts to potential aids of clinical interest.

Beyond Proteolysis-Targeting Chimeric Molecules: Designing Heterobifunctional Molecules Based on Functional Effectors.

In recent years, with the successful development of proteolysis-targeting chimeric molecules (PROTACs), the potential of heterobifunctional molecules to contribute to reenvisioning drug design, especially small-molecule drugs, has been increasingly recognized. Inspired by PROTACs, diverse heterobifunctional molecules have been reported to simultaneously bind two or more molecules and bring them into proximity to interaction, such as ribonuclease-recruiting, autophagy-recruiting, lysosome-recruiting, kinase-recruiting, phosphatase-recruiting, glycosyltransferase-recruiting, and acetyltransferase-recruiting chimeras. On the basis of the heterobifunctional principle, more opportunities for advancing drug design by linking potential effectors to a protein of interest (POI) have emerged. Herein, we introduce heterobifunctional molecules other than PROTACs, summarize the limitations of existing molecules, list the main challenges, and propose perspectives for future research directions, providing insight into alternative design strategies based on substrate-proximity-based targeting.

Recent insight into the role of RING-finger E3 ligases in glioma.

The ubiquitin proteasome system (UPS) serves as the major posttranslational modification system for the maintenance of protein homeostasis. The ubiquitin ligases (E3s) are responsible for the recognition and recruitment of specific substrate proteins for polyubiquitination. Really interesting new gene (RING) finger E3s account for the majority of E3s. The human genome encodes more than 600 RING E3s, which are divided into three subclasses: single polypeptide E3s, cullin-RING ligases (CRLs) and other multisubunit E3s. The abnormal regulation of RING E3s has been reported to disrupt normal biological processes and induce the occurrence of many human malignancies. Glioma is the most common type of malignant primary brain tumor. In the last few decades, patient prognosis has improved as novel targeted therapeutic agents have developed. In this review, we will summarize the current knowledge about the dysregulation of RING E3s and the altered stability of their substrates in glioma. We will further introduce and discuss the current status and future perspectives of the application of small inhibitors and proteolysis-targeting chimeric molecules (PROTACs) interfering with RING E3s as potential anticancer agents for glioma.

AML-397: Preclinical Efficacy of a PROTAC-based MDM2 Degrader in AML models

Mouse double minute 2 homolog (MDM2) is a E3 ubiquitin ligase that promotes p53 tumor suppressor degradation. MDM2 has emerged as a therapeutic target in the treatment of TP53 wild-type expressing acute myeloid leukemia (AML) where MDM2 is overexpressed. Several MDM2 inhibitors are currently in clinical trials. However, p53 stabilization upregulates MDM2, which limits the clinical efficacy of the inhibitors. Proteolysis Targeting Chimeric (PROTAC) molecules recruit targeted proteins for degradation. We developed a MDM2 degrader, MI-265, that binds to MDM2 and targets it for degradation. We tested MI-265 in a panel of 101 primary AML samples in ex vivo cultures. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. After treatment with drugs for 72 hrs, cell death and apoptosis induction were measured by flow-cytometry for DAPI-Annexin V binding. The AML cells were obtained from newly diagnosed AML (N=86) or relapse/refractory AML (N=15). The median IC50 for MI-265 was 120 pM. In contrast, median IC50 for the inhibitor MI-1061 was 20,000-fold higher at 2.3 μM. Sensitivity and resistance to MDM2-degrader and inhibitor were similar in most of the samples suggesting that the mechanism of action of both drugs are also similar. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%), and other mutations (10%). Based on classical karyotype, 30% of them had a normal karyotype and 55% had abnormal karyotype. The MDM2 degrader-resistant LSCs (IC50 greater than 1 μM) were analyzed for TP53 mutation status and 8 of 14 were found to harbor mutations in TP53. The degradation of MDM2 and overexpression of p53 was confirmed by immunoblotting after a 4-hr exposure of LSCs to degrader. Moreover, treatment of normal CD34-positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. Evaluation of efficacy in patient-derived xenografts is ongoing. In conclusion, MI-265 represents a highly potent and efficacious MDM2 degrader, and is currently undergoing extensive preclinical evaluation for the treatment of acute leukemias.

Selective Degradation of Target Proteins by Chimeric Small-Molecular Drugs, PROTACs and SNIPERs.

New therapeutic modalities are needed to address the problem of pathological but undruggable proteins. One possible approach is the induction of protein degradation by chimeric drugs composed of a ubiquitin ligase (E3) ligand coupled to a ligand for the target protein. This article reviews chimeric drugs that decrease the level of specific proteins such as proteolysis targeting chimeric molecules (PROTACs) and specific and nongenetic inhibitor of apoptosis protein (IAP)-dependent protein erasers (SNIPERs), which target proteins for proteasome-mediated degradation. We cover strategies for increasing the degradation activity induced by small molecules, and their scope for application to undruggable proteins.

Improved Anti-Leukemic Pre-Clinical Efficacy of a Protac Based MDM2 Degrader in a Large AML Cohort

Mouse double minute 2 homolog (MDM2) is a E3 ubiquitin ligase that promotes p53 tumor suppressor degradation. Many tumors harbor mutations in TP53 that don't bind MDM2, nevertheless, MDM2 has emerged as a therapeutic target in the treatment of TP53 wild-type cancers. Although in acute myeloid leukemia (AML) incidence of TP53 mutations is low (15-20%) inactivation of wild-type p53 protein frequently occurs through overexpression of its negative regulator MDM2. Thus MDM2 is an attractive target for novel therapeutics in AML tailored to treat TP53-wild type AML overexpressing MDM2. Several MDM2 inhibitors are currently in clinical trials. However, p53 stabilization upregulates MDM2, which limits the clinical efficacy of the inhibitors. Proteolysis Targeting Chimeric (PROTAC) molecules recruit targeted proteins for degradation and therefore can improve efficacy by removing the targeted protein. We developed a MDM2 degrader, MI-265, that binds to MDM2 and targets it for degradation thus removing the repression of p53, which results in apoptosis induction in leukemic cells. We tested MI-265 in a panel of 96 primary AML samples in ex-vivo cultures. AML blood and bone marrow samples were collected after obtaining University of Michigan IRB approved informed consent from AML patients at Michigan Medicine. Leukemia stem cells (LSC) were enriched by positive selection with CD34 antibody. Cells were plated in LSC conducive medium containing growth factors. Cell death and apoptosis induction was measured by flow-cytometry for DAPI-Annexin V binding after incubation with MDM2-inhibitor and MDM2-degrader for 3 days in the presence of drug. The AML cells were obtained from newly diagnosed AML (N=82) or relapse/refractory AML (N=14). The median IC50s was 120 pM (range 32 pm to 23 nM). In contrast median IC50 for the inhibitor MI-1061 was 20,000 fold higher at 2.3 µM (range 3 nM to 455 µM). Sensitivity and resistance to MDM2-degrader and inhibitor were similar in most of the samples suggesting that the mechanism of action of both drugs are also similar. The AML cells were heterogeneous for the expression of mutant FLT-3 (36%), NPM1 (22%), IDH1/2 (16%), CEBPalpha (2%), DNMT3A (2%) and other mutations (10%). Based on classical karyotype 30% of them had a normal karyotype and 55% had abnormal karyotype (15% data missing). The MDM2-degrader resistant LSCs (IC50 greater than 1 µM) were analyzed for TP53 mutation status and 8 of 14 were found to harbor mutations in TP53. The degradation of MDM2 and overexpression of p53 was confirmed by immunoblotting after a 4 hr exposure of LSCs to degrader. Moreover, treatment of normal CD34 positive hematopoietic cells in apoptosis assay and colony formation assays suggests lack of toxicity to normal hematopoiesis. In conclusion, MI-265 represents a highly potent and efficacious MDM2 degrader, and is currently undergoing extensive preclinical evaluation for the treatment of acute leukemias. Disclosures Peterson: Penrose TherapeuTx: Employment. Wang:Oncopia Therapeutics: Consultancy, Equity Ownership, Patents &amp; Royalties. Talpaz:Imago BioSciences: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; CTI BioPharma: Research Funding; Samus Therapeutics: Research Funding; Novartis: Research Funding; Constellation: Research Funding; Incyte: Research Funding.

Proteolysis targeting chimeric molecules as therapy for multiple myeloma: efficacy, biomarker and drug combinations.

Proteolysis targeting chimeric molecule ARV 825 causes ubiquitination of bromodomains resulting in their efficient degradation by proteasome activity. Bromodomain degradation down-regulates MYC transcription contributing to growth inhibition of various human cancers. We examined the therapeutic potential of ARV 825 against multiple myeloma (MM) cells both in vitro and in vivo. In a dose-dependent manner, ARV 825 inhibited proliferation of 13 human MM cell lines and three fresh patient samples, and was associated with cell cycle arrest and apoptosis. ARV 825 rapidly and efficiently degraded BRD 2 and BRD 4. Sensitivity of MM cells to ARV 825 was positively correlated with cereblon levels. RNA sequencing analysis showed important genes such as CCR1, RGS, MYB and MYC were down-regulated by ARV 825. A total of 170 small molecule inhibitors were screened for synergy with ARV 825. Combination of ARV 825 with inhibitor of either dual PI3K/mTOR, CRM1, VEGFR, PDGFRα/b, FLT3, IGF-1R, protein kinase C, CBP-EP300 or JAK1/2 showed synergistic activity. Importantly, ARV 825 significantly inhibited the growth of MM xenografts and improved mice survival. Taken together, our results, in conjunction with recently published findings, provide a rationale for investigating the efficacy of ARV 825 for MM, use of cereblon as a biomarker for therapy of MM patients, and the combination of ARV 825 with small molecule inhibitors to improve the outcome of MM patients.

PROTACs: An Emerging Targeting Technique for Protein Degradation in Drug Discovery.

Proteolysis-targeting chimeric molecules (PROTACs) represent an emerging technique that is receiving much attention for therapeutic intervention. The mechanism is based on the inhibition of protein function by hijacking a ubiquitin E3 ligase for protein degradation. The hetero-bifunctional PROTACs contain a ligand for recruiting an E3 ligase, a linker, and another ligand to bind with the protein targeted for degradation. Thus, PROTACs have profound potential to eliminate "undruggable" protein targets, such as transcription factors and non-enzymatic proteins, which are not limited to physiological substrates of the ubiquitin-proteasome system. These findings indicate great prospects for PROTACs in the development of therapeutics. However, there are several limitations related to poor stability, biodistribution, and penetrability in vivo. This review provides an overview of the main PROTAC-based approaches that have been developed and discusses the promising opportunities and considerations for the application of this technology in therapies and drug discovery.

Emerging small molecule approaches to enhance the antimyeloma benefit of proteasome inhibitors.

Multiple myeloma (MM) is a clonal plasma cell malignancy which, despite recent treatment advances, remains incurable in the vast majority of the over 118,000 patients in the USA afflicted with this disease. Treatment of MM has dramatically improved in the past decade with the introduction of new drugs into therapeutic strategies in both the frontline and relapse settings that has led to a significant improvement in the median overall survival (OS). These drugs have been incorporated into clinical guidelines and transformed the treatment approach to MM. Numerous classes of antimyeloma agents, i.e., alkylators, steroids, proteasome inhibitors, immunomodulatory agents, deactylase inhibitors, and monoclonal antibodies, are now FDA-approved and can be combined in doublet or triplet regimens. Moreover, many patients do not respond to therapy and those that do eventually relapse. Emerging therapies that may overcome drug resistance and improve MM treatment include that inhibit regulatory and Ub-processing components of the proteasome, a specialized variant of the proteasome known as the immunoproteasome, proteolysis-targeting chimeric molecules (PROTACS and Degronomids). Emerging strategies also include accessory plasmacytoid dendritic cells (pDCs), vaccines, checkpoint inhibitors, and chimeric antigen receptor-engineered T (CAR-T) cells. Advances in understanding proteasome and plasma cell biology may allow for earlier treatment of MM patients using rationally informed combination therapies with curative potential.

New strategy for renal fibrosis: Targeting Smad3 proteins for ubiquitination and degradation

PurposeSmad3 is a critical signaling protein in renal fibrosis. Proteolysis targeting chimeric molecules (PROTACs) are small molecules designed to degrade target proteins via ubiquitination. They have three components: (1) a recognition motif for E3 ligase; (2) a linker; and (3) a ligand for the target protein. We aimed to design a new PROTAC to prevent renal fibrosis by targeting Smad3 proteins and using hydroxylated pentapeptide of hypoxia-inducible factor-1α as the recognition motif for von Hippel–Lindau (VHL) ubiquitin ligase (E3). MethodsComputer-aided drug design was used to find a specific ligand targeting Smad3. Surface plasmon resonance (SPR) was used to verify and optimize screening results. Synthesized PROTAC was validated by two-stage mass spectrometry. The PROTAC’s specificity for VHL (E3 ligase) was proved with two human renal carcinoma cell lines, 786-0 (VHL−) and ACHN (VHL+), and its anti-fibrosis effect was tested in renal fibrosis cell models. ResultsThirteen small molecular compounds (SMCs) were obtained from the Enamine library using GLIDE molecular docking program. SPR results showed that #8 SMC (EN300-72284) combined best with Smad3 (KD=4.547×10−5M). Mass spectrometry showed that synthesized PROTAC had the correct peptide molecular weights. Western blot showed Smad3 was degraded by PROTAC with whole-cell lysate of ACHN but not 786-0. Degradation, but not ubiquitination, of Smad3 was inhibited by proteasome inhibitor MG132. The upregulation of fibronectin and Collagen I induced by TGF-β1 in both renal fibroblast and mesangial cells were inhibited by PROTAC. ConclusionThe new PROTAC might prevent renal fibrosis by targeting Smad3 for ubiquitination and degradation.

Degradation of Akt using protein-catalyzed capture agents.

Abnormal signaling of the protein kinase Akt has been shown to contribute to human diseases such as diabetes and cancer, but Akt has proven to be a challenging target for drugging. Using iterative in situ click chemistry, we recently developed multiple protein-catalyzed capture (PCC) agents that allosterically modulate Akt enzymatic activity in a protein-based assay. Here, we utilize similar PCCs to exploit endogenous protein degradation pathways. We use the modularity of the anti-Akt PCCs to prepare proteolysis targeting chimeric molecules that are shown to promote the rapid degradation of Akt in live cancer cells. These novel proteolysis targeting chimeric molecules demonstrate that the epitope targeting selectivity of PCCs can be coupled with non-traditional drugging moieties to inhibit challenging targets.

Protein Knockdown Technology: Application of Ubiquitin Ligase to Cancer Therapy.

Selective degradation of pathogenic proteins by small molecules in cells is a novel approach for development of therapeutic agents against various diseases, including cancer. We and others have developed a protein knockdown technology with a series of hybrid small compounds, called SNIPERs (Specific and Nongenetic IAP-dependent Protein ERasers); and peptidic chimeric molecules, called PROTACs (proteolysis-targeting chimeric molecules), which induce selective degradation of target proteins via the ubiquitin-proteasome pathway. These compounds include two different ligands connected by a linker; one is a ligand for a ubiquitin ligase and the other is a ligand for the target protein, which are expected to crosslink these proteins in cells. Theoretically, any cytosolic protein can be targeted for degradation by this technology. To date, several SNIPERs and PROTACs against various oncogenic proteins have been developed, which specifically induce polyubiquitylation and proteasomal degradation of the oncogenic proteins, resulting in cell death, growth arrest, or impaired migration of cancer cells. Thus, this protein knockdown technology has a great potential for cancer therapy.

Two-headed PROTAC: an effective new tool for targeted protein degradation.

Two-headed PROTAC: an effective new tool for targeted protein degradation.

Targeted Degradation of Proteins by PROTACs

In recent years, small interference RNAs (siRNAs) have greatly enhanced our understanding of protein functions by allowing knockdown of targeted proteins at the mRNA level. Similarly, in an effort to achieve degradation of targeted proteins at the post-translational level, chimeric small molecules called "PROTACs" (PROteolysis TArgeting Chimeric molecules) have been developed. The PROTAC approach utilizes chimeric small molecules which recruit targeted proteins to the ubiquitin-proteasome pathway, a major intracellular protein degradation system. Unlike conventional small molecules that bind to protein and inhibit its function, the PROTAC approach induces destruction of target protein via the ubiquitin-proteasome system. This article presents a typical strategy for PROTAC design and preparation and biological characterization. Curr. Protoc. Chem Biol. 2:71-87. © 2010 by John Wiley & Sons, Inc.