Small‐Molecule PROTACS: New Approaches to Protein Degradation

The current inhibitor‐based approach to therapeutics has inherent limitations owing to its occupancy‐based model: 1) there is a need to maintain high systemic exposure to ensure sufficient in vivo inhibition, 2) high in vivo concentrations bring potential for off‐target side effects, and 3) there is a need to bind to an active site, thus limiting the drug target space. As an alternative, induced protein degradation lacks these limitations. Based on an event‐driven model, this approach offers a novel catalytic mechanism to irreversibly inhibit protein function by targeting protein destruction through recruitment to the cellular quality control machinery. Prior protein degrading strategies have lacked therapeutic potential. However, recent reports of small‐molecule‐based proteolysis‐targeting chimeras (PROTACs) have demonstrated that this technology can effectively decrease the cellular levels of several protein classes.     

Small‐Molecule Control of Intracellular Protein Levels through Modulation of the Ubiquitin Proteasome System

Traditionally, biological probes and drugs have targeted the activities of proteins (such as enzymes and receptors) that can be readily controlled by small molecules. The remaining majority of the proteome has been deemed “undruggable”. By using small‐molecule modulators of the ubiquitin proteasome, protein levels, rather than protein activity, can be targeted instead, thus increasing the number of druggable targets. Whereas targeting of the proteasome itself can lead to a global increase in protein levels, the targeting of other components of the UPS (e.g., the E3 ubiquitin ligases) can lead to an increase in protein levels in a more targeted fashion. Alternatively, multiple strategies for inducing protein degradation with small‐molecule probes are emerging. With the ability to induce and inhibit the degradation of targeted proteins, small‐molecule modulators of the UPS have the potential to significantly expand the druggable portion of the proteome beyond traditional targets, such as enzymes and receptors.     

Modular PROTAC Design for the Degradation of Oncogenic BCR‐ABL

Proteolysis Targeting Chimera (PROTAC) technology is a rapidly emerging alternative therapeutic strategy with the potential to address many of the challenges currently faced in modern drug development programs. PROTAC technology employs small molecules that recruit target proteins for ubiquitination and removal by the proteasome. The synthesis of PROTAC compounds that mediate the degradation of c‐ABL and BCR‐ABL by recruiting either Cereblon or Von Hippel Lindau E3 ligases is reported. During the course of their development, we discovered that the capacity of a PROTAC to induce degradation involves more than just target binding: the identity of the inhibitor warhead and the recruited E3 ligase largely determine the degradation profiles of the compounds; thus, as a starting point for PROTAC development, both the target ligand and the recruited E3 ligase should be varied to rapidly generate a PROTAC with the desired degradation profile.     

Metal–Organic‐Framework‐Templated Polyelectrolyte Nanocapsules for the Encapsulation and Delivery of Small‐Molecule–Polymer Conjugates

Herein, we report a strategy for exploiting nanoscale metal–organic frameworks (nano‐MOFs) as templates for the layer‐by‐layer (LbL) assembly of polyelectrolytes. Because small‐molecule drugs or imaging agents cannot be efficiently encapsulated by polyelectrolyte nanocapsules, we investigated two promising and biocompatible polymers (comb‐shaped polyethylene glycol (PEG) and hyperbranched polyglycerol‐based PEG) for the conjugation of model drugs and imaging agents, which were then encapsulated inside the nano‐MOF‐templated nanocapsules. Furthermore, we also systemically explored the release kinetics of the encapsulated conjugates, and examined how the encapsulation and/or release processes could be controlled by varying the composition and architecture of the polymers. We envision that our nano‐MOFs‐templated nanocapsules, through combining with small‐molecule–polymer conjugates, will represent a new type of delivery system that could open up new opportunities for biomedical applications.     

Insight into the Inhibition of Drug‐Resistant Mutants of the Receptor Tyrosine Kinase EGFR

Targeting acquired drug resistance represents the major challenge in the treatment of EGFR‐driven non‐small‐cell lung cancer (NSCLC). Herein, we describe the structure‐based design, synthesis, and biological evaluation of a novel class of covalent EGFR inhibitors that exhibit excellent inhibition of EGFR‐mutant drug‐resistant cells. Protein X‐ray crystallography combined with detailed kinetic studies led to a deeper understanding of the mode of inhibition of EGFR‐T790M and provided insight into the key principles for effective inhibition of the recently discovered tertiary mutation at EGFR‐C797S.     

Fragment‐Based De Novo Design Reveals a Small‐Molecule Inhibitor of Helicobacter Pylori HtrA

Sustained identification of innovative chemical entities is key for the success of chemical biology and drug discovery. We report the fragment‐based, computer‐assisted de novo design of a small molecule inhibiting Helicobacter pylori HtrA protease. Molecular binding of the designed compound to HtrA was confirmed through biophysical methods, supporting its functional activity in vitro. Hit expansion led to the identification of the currently best‐in‐class HtrA inhibitor. The results obtained reinforce the validity of ligand‐based de novo design and binding‐kinetics‐guided optimization for the efficient discovery of pioneering lead structures and prototyping drug‐like chemical probes with tailored bioactivity.     

Deactivation of Mcl‐1 by Dual‐Function Small‐Molecule Inhibitors Targeting the Bcl‐2 Homology 3 Domain and Facilitating Mcl‐1 Ubiquitination

By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. Noxa^BH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, Bim^BH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.     

A Small‐Molecule Drug Conjugate for the Treatment of Carbonic Anhydrase IX Expressing Tumors

Antibody–drug conjugates are a very promising class of new anticancer agents, but the use of small‐molecule ligands for the targeted delivery of cytotoxic drugs into solid tumors is less well established. Here, we describe the first small‐molecule drug conjugates for the treatment of carbonic anhydrase IX expressing solid tumors. Using ligand–dye conjugates we demonstrate that such molecules can preferentially accumulate inside antigen‐positive lesions, have fast targeting kinetics and good tumor‐penetrating properties, and are easily accessible by total synthesis. A disulfide‐linked drug conjugate with the maytansinoid DM1 as the cytotoxic payload and a derivative of acetazolamide as the targeting ligand exhibited a potent antitumor effect in SKRC52 renal cell carcinoma in vivo. It was furthermore superior to sunitinib and sorafenib, both small‐molecule standard‐of‐care drugs for the treatment of kidney cancer.     

Probing the Small‐Molecule Inhibition of an Anticancer Therapeutic Protein‐Protein Interaction Using a Solid‐State Nanopore

Nanopore sensing is an emerging technology for the single‐molecule‐based detection of various biomolecules. In this study, we probed the anticancer therapeutic p53 transactivation domain (p53TAD)/MDM2 interaction and its inhibition with a small‐molecule MDM2 antagonist, Nutlin‐3, using low‐noise solid‐state nanopores. Although the translocation of positively charged MDM2 through a nanopore was detected at the applied negative voltage, this MDM2 translocation was almost completely blocked upon formation of the MDM2/GST‐p53TAD complex owing to charge conversion. In combination with NMR data, the nanopore measurements showed that the addition of Nutlin‐3 rescued MDM2 translocation, indicating that Nutlin‐3 disrupted the MDM2/GST‐p53TAD complex, thereby releasing MDM2. Taken together, our results reveal that solid‐state nanopores can be a valuable platform for the ultrasensitive, picomole‐scale screening of small‐molecule drugs against protein–protein interaction (PPI) targets.     

Synthesis,characterization, and evaluation of enzymatically degradable poly(N‐isopropylacrylamide‐co‐acrylic acid) hydrogels for colon‐specific drug delivery

The enzymatically degradable poly(N‐isopropylacrylamide‐co‐acrylic acid) hydrogels were prepared using 4,4^′‐bis(methacryloylamino)azobenzene (BMAAB) as the crosslinker. It was found that the incorporated N‐isopropylacrylamide (NIPAAm) monomer did not change the enzymatic degradation of hydrogel, but remarkably enhanced the loading of protein drug. The hydrogels exhibited a phase transition temperature between 4°C (refrigerator temperature) and 37°C (human body temperature). Bovine serum albumin (BSA) as a model drug was loaded into the hydrogels by soaking the gels in a pH 7.4 buffer solution at 4°C, where the hydrogel was in a swollen status. The high swelling of hydrogels at 4°C enhanced the loading of BSA (loading capability, ca. 144.5 mg BSA/g gel). The drug was released gradually in the pH 7.4 buffer solution at 37°C, where the hydrogel was in a shrunken state. In contrast, the enzymatic degradation of hydrogels resulted in complete release of BSA in pH 7.4 buffer solution containing the cecal suspension at 37°C (cumulative release: ca. 100 mg BSA/g gel after 4 days). Copyright © 2008 John Wiley & Sons, Ltd.     

Near‐Infrared‐Controlled,Targeted Hydrophobic Drug‐Delivery System for Synergistic Cancer Therapy

Hydrophobicity has been an obstacle that hinders the use of many anticancer drugs. A critical challenge for cancer therapy concerns the limited availability of effective biocompatible delivery systems for most hydrophobic therapeutic anticancer drugs. In this study, we have developed a targeted near‐infrared (NIR)‐regulated hydrophobic drug‐delivery platform based on gold nanorods incorporated within a mesoporous silica framework (AuMPs). Upon application of NIR light, the photothermal effect of the gold nanorods leads to a rapid rise in the local temperature, thus resulting in the release of the entrapped drug molecules. By integrating chemotherapy and photothermotherapy into one system, we have studied the therapeutic effects of camptothecin‐loaded AuMP‐polyethylene glycol‐folic acid nanocarrier. Results revealed a synergistic effect in vitro and in vivo, which would make it possible to enhance the therapeutic effect of hydrophobic drugs and decrease drug side effects. Studies have shown the feasibility of using this nanocarrier as a targeted and noninvasive remote‐controlled hydrophobic drug‐delivery system with high spatial/temperal resolution. Owing to these advantages, we envision that this NIR‐controlled, targeted drug‐delivery method would promote the development of high‐performance hydrophobic anticancer drug‐delivery system in future clinical applications.