A small-molecule PI3Kα activator for cardioprotection and neuroregeneration.

Harnessing the potential beneficial effects of kinase signalling through the generation of direct kinase activators remains an underexplored area of drug development1-5. This also applies to the PI3K signalling pathway, which has been extensively targeted by inhibitors for conditions with PI3K overactivation, such as cancer and immune dysregulation. Here we report the discovery of UCL-TRO-1938 (referred to as 1938 hereon), a small-molecule activator of the PI3Kα isoform, a crucial effector of growth factor signalling. 1938 allosterically activates PI3Kα through a distinct mechanism by enhancing multiple steps of the PI3Kα catalytic cycle and causes both local and global conformational changes in the PI3Kα structure. This compound is selective for PI3Kα over other PI3K isoforms and multiple protein and lipid kinases. It transiently activates PI3K signalling in all rodent and human cells tested, resulting in cellular responses such as proliferation and neurite outgrowth. In rodent models, acute treatment with 1938 provides cardioprotection from ischaemia-reperfusion injury and, after local administration, enhances nerve regeneration following nerve crush. This study identifies a chemical tool to directly probe the PI3Kα signalling pathway and a new approach to modulate PI3K activity, widening the therapeutic potential of targeting these enzymes through short-term activation for tissue protection and regeneration. Our findings illustrate the potential of activating kinases for therapeutic benefit, a currently largely untapped area of drug development.

Cardiomyocyte BRAF and type 1 RAF inhibitors promote cardiomyocyte and cardiac hypertrophy in mice in vivo.

The extracellular signal-regulated kinase 1/2 (ERK1/2) cascade promotes cardiomyocyte hypertrophy and is cardioprotective, with the three RAF kinases forming a node for signal integration. Our aims were to determine if BRAF is relevant for human heart failure, whether BRAF promotes cardiomyocyte hypertrophy, and if Type 1 RAF inhibitors developed for cancer (that paradoxically activate ERK1/2 at low concentrations: the 'RAF paradox') may have the same effect. BRAF was up-regulated in heart samples from patients with heart failure compared with normal controls. We assessed the effects of activated BRAF in the heart using mice with tamoxifen-activated Cre for cardiomyocyte-specific knock-in of the activating V600E mutation into the endogenous gene. We used echocardiography to measure cardiac dimensions/function. Cardiomyocyte BRAFV600E induced cardiac hypertrophy within 10 d, resulting in increased ejection fraction and fractional shortening over 6 weeks. This was associated with increased cardiomyocyte size without significant fibrosis, consistent with compensated hypertrophy. The experimental Type 1 RAF inhibitor, SB590885, and/or encorafenib (a RAF inhibitor used clinically) increased ERK1/2 phosphorylation in cardiomyocytes, and promoted hypertrophy, consistent with a 'RAF paradox' effect. Both promoted cardiac hypertrophy in mouse hearts in vivo, with increased cardiomyocyte size and no overt fibrosis. In conclusion, BRAF potentially plays an important role in human failing hearts, activation of BRAF is sufficient to induce hypertrophy, and Type 1 RAF inhibitors promote hypertrophy via the 'RAF paradox'. Cardiac hypertrophy resulting from these interventions was not associated with pathological features, suggesting that Type 1 RAF inhibitors may be useful to boost cardiomyocyte function.

Structural Insight and Development of EGFR Tyrosine Kinase Inhibitors.

Lung cancer has a high prevalence, with a growing number of new cases and mortality every year. Furthermore, the survival rate of patients with non-small-cell lung carcinoma (NSCLC) is still quite low in the majority of cases. Despite the use of conventional therapy such as tyrosine kinase inhibitor for Epidermal Growth Factor Receptor (EGFR), which is highly expressed in most NSCLC cases, there was still no substantial improvement in patient survival. This is due to the drug's ineffectiveness and high rate of resistance among individuals with mutant EGFR. Therefore, the development of new inhibitors is urgently needed. Understanding the EGFR structure, including its kinase domain and other parts of the protein, and its activation mechanism can accelerate the discovery of novel compounds targeting this protein. This study described the structure of the extracellular, transmembrane, and intracellular domains of EGFR. This was carried out along with identifying the binding pose of commercially available inhibitors in the ATP-binding and allosteric sites, thereby clarifying the research gaps that can be filled. The binding mechanism of inhibitors that have been used clinically was also explained, thereby aiding the structure-based development of new drugs.

A new porphyrin as selective substrate-based inhibitor of breast cancer resistance protein (BCRP/ABCG2).

The ABCG2 transporter plays a pivotal role in multidrug resistance, however, no clinical trial using specific ABCG2 inhibitors have been successful. Although ABC transporters actively extrude a wide variety of substrates, photodynamic therapeutic agents with porphyrinic scaffolds are exclusively transported by ABCG2. In this work, we describe for the first time a porphyrin derivative (4B) inhibitor of ABCG2 and capable to overcome multidrug resistance in vitro. The inhibition was time-dependent and 4B was not itself transported by ABCG2. Independently of the substrate, the porphyrin 4B showed an IC50 value of 1.6 µM and a mixed type of inhibition. This compound inhibited the ATPase activity and increased the binding of the conformational-sensitive antibody 5D3. A thermostability assay confirmed allosteric protein changes triggered by the porphyrin. Long-timescale molecular dynamics simulations revealed a different behavior between the ABCG2 porphyrinic substrate pheophorbide a and the porphyrin 4B. Pheophorbide a was able to bind in three different protein sites but 4B showed one binding conformation with a strong ionic interaction with GLU446. The inhibition was selective toward ABCG2, since no inhibition was observed for P-glycoprotein and MRP1. Finally, this compound successfully chemosensitized cells that overexpress ABCG2. These findings reinforce that substrates may be a privileged source of chemical scaffolds for identification of new inhibitors of multidrug resistance-linked ABC transporters.

Study on the cytotoxic, antimetastatic and albumin binding properties of the oxidovanadium(IV) chrysin complex. Structural elucidation by computational methodologies.

We have previously synthesized and characterized the chrysin coordination complex with the oxidovanadium(IV) cation (VIVO(chrys)2) and characterized in ethanolic solution and in solid state. Because suitable single crystals for X-ray diffraction determinations could not be obtained, in the present work, we elucidate the geometrical parameters of this complex by computational methodologies. The optimization and vibrational investigation were carried out both in ethanolic solution and in gas phase. The computational results support the experimentally proposed geometries of the VIVO(chrys)2 complex, thus leading to the conclusion that the complex exists as conformers with trans-octahedral geometry in ethanolic solution and as conformers with cis-octahedral geometry in the solid state. The complex also exists as conformers with trans-octahedral geometry in aqueous media. The active species formed after dissolution in DMSO showed anticancer and antimetastatic behavior in human lung cell line A549 with moderate binding (Kaca. 105 M-1) to bovine serum albumin (BSA). The interaction through hydrogen bonding and van der Waals forces resulted in a spontaneous process. Site marker competitive experiments showed binding sites for chrysin mainly located in site II (subdomain IIIA) and in site I (subdomain IIIA) for the complex. FT-IR spectral measurements showed evidences of the alterations of protein secondary structure in the presence of chrysin and VIVO(chrys)2.

Drug Repositioning For Allosteric Modulation of VIP and PACAP Receptors.

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two neuropeptides that contribute to the regulation of intestinal motility and secretion, exocrine and endocrine secretions, and homeostasis of the immune system. Their biological effects are mediated by three receptors named VPAC1, VPAC2 and PAC1 that belong to class B GPCRs. VIP and PACAP receptors have been identified as potential therapeutic targets for the treatment of chronic inflammation, neurodegenerative diseases and cancer. However, pharmacological use of endogenous ligands for these receptors is limited by their lack of specificity (PACAP binds with high affinity to VPAC1, VPAC2 and PAC1 receptors while VIP recognizes both VPAC1 and VPAC2 receptors), their poor oral bioavailability (VIP and PACAP are 27- to 38-amino acid peptides) and their short half-life. Therefore, the development of non-peptidic small molecules or specific stabilized peptidic ligands is of high interest. Structural similarities between VIP and PACAP receptors are major causes of difficulties in the design of efficient and selective compounds that could be used as therapeutics. In this study we performed structure-based virtual screening against the subset of the ZINC15 drug library. This drug repositioning screen provided new applications for a known drug: ticagrelor, a P2Y12 purinergic receptor antagonist. Ticagrelor inhibits both VPAC1 and VPAC2 receptors which was confirmed in VIP-binding and calcium mobilization assays. A following analysis of detailed ticagrelor binding modes to all three VIP and PACAP receptors with molecular dynamics revealed its allosteric mechanism of action. Using a validated homology model of inactive VPAC1 and a recently released cryo-EM structure of active VPAC1 we described how ticagrelor could block conformational changes in the region of 'tyrosine toggle switch' required for the receptor activation. We also discuss possible modifications of ticagrelor comparing other P2Y12 antagonist - cangrelor, closely related to ticagrelor but not active for VPAC1/VPAC2. This comparison with inactive cangrelor could lead to further improvement of the ticagrelor activity and selectivity for VIP and PACAP receptor sub-types.

Barnase-Barstar Pair: Contemporary Application in Cancer Research and Nanotechnology.

Barnase is an extracellular ribonuclease secreted by Bacillus amyloliquefaciens that was originally studied as a small stable enzyme with robust folding. The identification of barnase intracellular inhibitor barstar led to the discovery of an incredibly strong protein-protein interaction. Together, barnase and barstar provide a fully genetically encoded toxin-antitoxin pair having an extremely low dissociation constant. Moreover, compared to other dimerization systems, the barnase-barstar module provides the exact one-to-one ratio of the complex components and possesses high stability of each component in a complex and high solubility in aqueous solutions without self-aggregation. The unique properties of barnase and barstar allow the application of this pair for the engineering of different variants of targeted anticancer compounds and cytotoxic supramolecular complexes. Using barnase in suicide gene therapy has also found its niche in anticancer therapy. The application of barnase and barstar in contemporary experimental cancer therapy is reflected in the review.

Deciphering conformational selectivity in the A2A adenosine G protein-coupled receptor by free energy simulations.

Transmembranal G Protein-Coupled Receptors (GPCRs) transduce extracellular chemical signals to the cell, via conformational change from a resting (inactive) to an active (canonically bound to a G-protein) conformation. Receptor activation is normally modulated by extracellular ligand binding, but mutations in the receptor can also shift this equilibrium by stabilizing different conformational states. In this work, we built structure-energetic relationships of receptor activation based on original thermodynamic cycles that represent the conformational equilibrium of the prototypical A2A adenosine receptor (AR). These cycles were solved with efficient free energy perturbation (FEP) protocols, allowing to distinguish the pharmacological profile of different series of A2AAR agonists with different efficacies. The modulatory effects of point mutations on the basal activity of the receptor or on ligand efficacies could also be detected. This methodology can guide GPCR ligand design with tailored pharmacological properties, or allow the identification of mutations that modulate receptor activation with potential clinical implications.

An In Vitro Comparative Study of the Effects of Tetrabromobisphenol A and Tetrabromobisphenol S on Human Erythrocyte Membranes-Changes in ATP Level, Perturbations in Membrane Fluidity, Alterations in Conformational State and Damage to Proteins.

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)-a compound with a very similar structure-is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

Flavonoids in Ampelopsis grossedentata as covalent inhibitors of SARS-CoV-2 3CLpro: Inhibition potentials, covalent binding sites and inhibitory mechanisms.

Coronavirus 3C-like protease (3CLpro) is a crucial target for treating coronavirus diseases including COVID-19. Our preliminary screening showed that Ampelopsis grossedentata extract (AGE) displayed potent SARS-CoV-2-3CLpro inhibitory activity, but the key constituents with SARS-CoV-2-3CLpro inhibitory effect and their mechanisms were unrevealed. Herein, a practical strategy via integrating bioactivity-guided fractionation and purification, mass spectrometry-based peptide profiling and time-dependent biochemical assay, was applied to identify the crucial constituents in AGE and to uncover their inhibitory mechanisms. The results demonstrated that the flavonoid-rich fractions (10-17.5 min) displayed strong SARS-CoV-2-3CLpro inhibitory activities, while the constituents in these fractions were isolated and their SARS-CoV-2-3CLpro inhibitory activities were investigated. Among all isolated flavonoids, dihydromyricetin, isodihydromyricetin and myricetin strongly inhibited SARS-CoV-2 3CLpro in a time-dependent manner. Further investigations demonstrated that myricetin could covalently bind on SARS-CoV-2 3CLpro at Cys300 and Cys44, while dihydromyricetin and isodihydromyricetin covalently bound at Cys300. Covalent docking coupling with molecular dynamics simulations showed the detailed interactions between the orthoquinone form of myricetin and two covalent binding sites (surrounding Cys300 and Cys44) of SARS-CoV-2 3CLpro. Collectively, the flavonoids in AGE strongly and time-dependently inhibit SARS-CoV-2 3CLpro, while the newly identified SARS-CoV-2 3CLpro inhibitors in AGE offer promising lead compounds for developing novel antiviral agents.

Structural Basis of Inhibition of DCLK1 by Ruxolitinib.

Given the functional attributes of Doublecortin-like kinase 1 (DCLK1) in tumor growth, invasion, metastasis, cell motility, and tumor stemness, it is emerging as a therapeutic target in gastrointestinal cancers. Although a series of specific or nonspecific ATP-competitive inhibitors were identified against DCLK1, different types of scaffolds that can be utilized for the development of highly selective inhibitors or structural understanding of binding specificities of the compounds remain limited. Here, we present our work to repurpose a Janus kinase 1 inhibitor, ruxolitinib as a DCLK1 inhibitor, showing micromolar binding affinity and inhibitory activity. Furthermore, to gain an insight into its interaction mode with DCLK1, a crystal structure of the ruxolitinib-complexed DCLK1 has been determined and analyzed. Ruxolitinib as a nonspecific DCLK1 inhibitor characterized in this work is anticipated to provide a starting point for the structure-guided discovery of selective DCLK1 inhibitors.

Identification of new GLUT2-selective inhibitors through in silico ligand screening and validation in eukaryotic expression systems.

Glucose is an essential energy source for cells. In humans, its passive diffusion through the cell membrane is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT2 transports both glucose and fructose with low affinity and plays a critical role in glucose sensing mechanisms. Alterations in the function or expression of GLUT2 are involved in the Fanconi-Bickel syndrome, diabetes, and cancer. Distinguishing GLUT2 transport in tissues where other GLUTs coexist is challenging due to the low affinity of GLUT2 for glucose and fructose and the scarcity of GLUT-specific modulators. By combining in silico ligand screening of an inward-facing conformation model of GLUT2 and glucose uptake assays in a hexose transporter-deficient yeast strain, in which the GLUT1-5 can be expressed individually, we identified eleven new GLUT2 inhibitors (IC50 ranging from 0.61 to 19.3 µM). Among them, nine were GLUT2-selective, one inhibited GLUT1-4 (pan-Class I GLUT inhibitor), and another inhibited GLUT5 only. All these inhibitors dock to the substrate cavity periphery, close to the large cytosolic loop connecting the two transporter halves, outside the substrate-binding site. The GLUT2 inhibitors described here have various applications; GLUT2-specific inhibitors can serve as tools to examine the pathophysiological role of GLUT2 relative to other GLUTs, the pan-Class I GLUT inhibitor can block glucose entry in cancer cells, and the GLUT2/GLUT5 inhibitor can reduce the intestinal absorption of fructose to combat the harmful effects of a high-fructose diet.

Prospecting the therapeutic edge of a novel compound (B12) over berberine in the selective targeting of Retinoid X Receptor in colon cancer.

The Retinoid X Receptor (RXR) is an attractive target in the treatment of colon cancer. Different therapeutic binders with high potency have been used to specifically target RXR. Among these compounds is a novel analogue of berberine, B12. We provided structural and molecular insights into the therapeutic activity properties of B12 relative to its parent compound, berberine, using force field estimations and thermodynamic calculations. Upon binding of B12 to RXR, the high instability elicited by RXR was markedly reduced; similar observation was seen in the berberine-bound RXR. However, our analysis revealed that B12 could have a more stabilizing effect on RXR when compared to berberine. Interestingly, the mechanistic behaviour of B12 in the active site of RXR opposed its impact on RXR protein. This disparity could be due to the bond formation and breaking elicited between B12/berberine and the active site residues. We observed that B12 and berberine could induce a disparate conformational change in regions Gly250-Asp258 located on the His-RXRα/LBD domain. Comparatively, the high agonistic and activation potential reported for B12 compared to berberine might be due to its superior binding affinity as evidenced in the thermodynamic estimations. The total affinity for B12 (-25.76 kcal/mol) was contributed by electrostatic interactions from Glu243 and Glu239. Also, Arg371, which plays a crucial role in the activity of RXR, formed a strong hydrogen bond with B12; however, a weak interaction was elicited between Arg371 and berberine. Taken together, our study has shown the RXRα activating potential of B12, and findings from this study could provide a framework in the future design of RXRα binders specifically tailored in the selective treatment of colon cancer.

The Structural Effect of FLT3 Mutations at 835th Position and Their Interaction with Acute Myeloid Leukemia Inhibitors: In Silico Approach.

FMS-like tyrosine kinase 3 (FLT3) gene mutations have been found in more than one-third of Acute Myeloid Leukemia (AML) cases. The most common point mutation in FLT3 occurs at the 835th residue (D835A/E/F/G/H/I/N/V/Y), in the activation loop region. The D835 residue is critical in maintaining FLT3 inactive conformation; these mutations might influence the interaction with clinically approved AML inhibitors used to treat the AML. The molecular mechanism of each of these mutations and their interactions with AML inhibitors at the atomic level is still unknown. In this manuscript, we have investigated the structural consequence of native and mutant FLT-3 proteins and their molecular mechanisms at the atomic level, using molecular dynamics simulations (MDS). In addition, we use the molecular docking method to investigate the binding pattern between the FLT-3 protein and AML inhibitors upon mutations. This study apparently elucidates that, due to mutations in the D835, the FLT-3 structure loses its conformation and becomes more flexible compared to the native FLT3 protein. These structural changes are suggested to contribute to the relapse and resistance responses to AML inhibitors. Identifying the effects of FLT3 at the molecular level will aid in developing a personalized therapeutic strategy for treating patients with FLT-3-associated AML.

Dynamics of aqueous peptide solutions in folded and disordered states examined by dynamic light scattering and dielectric spectroscopy.

Characterizing the segmental dynamics of proteins, and intrinsically disordered proteins in particular, is a challenge in biophysics. In this study, by combining data from broadband dielectric spectroscopy (BDS) and both depolarized (DDLS) and polarized (PDLS) dynamic light scattering, we were able to determine the dynamics of a small peptide [ε-poly(lysine)] in water solutions in two different conformations (pure ß-sheet at pH = 10 and a more disordered conformation at pH = 7). We found that the segmental (α-) relaxation, as probed by DDLS, is faster in the disordered state than in the folded conformation. The water dynamics, as detected by BDS, is also faster in the disordered state. In addition, the combination of BDS and DDLS results allows us to confirm the molecular origin of water-related processes observed by BDS. Finally, we discuss the origin of two slow processes (A and B processes) detected by DDLS and PDLS in both conformations and usually observed in other types of water solutions. For fully homogeneous ε-PLL solutions at pH = 10, the A-DLS process is assigned to the diffusion of individual ß-sheets. The combination of both techniques opens a route for understanding the dynamics of peptides and other biological solutions.

Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2.

Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.

Conserved binding site in the N-lobe of prokaryotic MATE transporters suggests a role for Na+ in ion-coupled drug efflux.

In both prokaryotes and eukaryotes, multidrug and toxic-compound extrusion (MATE) transporters catalyze the efflux of a broad range of cytotoxic compounds, including human-made antibiotics and anticancer drugs. MATEs are secondary-active antiporters, i.e., their drug-efflux activity is coupled to, and powered by, the uptake of ions down a preexisting transmembrane electrochemical gradient. Key aspects of this mechanism, however, remain to be delineated, such as its ion specificity and stoichiometry. We previously revealed the existence of a Na+-binding site in a MATE transporter from Pyroccocus furiosus (PfMATE) and hypothesized that this site might be broadly conserved among prokaryotic MATEs. Here, we evaluate this hypothesis by analyzing VcmN and ClbM, which along with PfMATE are the only three prokaryotic MATEs whose molecular structures have been determined at atomic resolution, i.e. better than 3 Å. Reinterpretation of existing crystallographic data and molecular dynamics simulations indeed reveal an occupied Na+-binding site in the N-terminal lobe of both structures, analogous to that identified in PfMATE. We likewise find this site to be strongly selective against K+, suggesting it is mechanistically significant. Consistent with these computational results, DEER spectroscopy measurements for multiple doubly-spin-labeled VcmN constructs demonstrate Na+-dependent changes in protein conformation. The existence of this binding site in three MATE orthologs implicates Na+ in the ion-coupled drug-efflux mechanisms of this class of transporters. These results also imply that observations of H+-dependent activity likely stem either from a site elsewhere in the structure, or from H+ displacing Na+ under certain laboratory conditions, as has been noted for other Na+-driven transport systems.

A chemical genetics approach to examine the functions of AAA proteins.

The structural conservation across the AAA (ATPases associated with diverse cellular activities) protein family makes designing selective chemical inhibitors challenging. Here, we identify a triazolopyridine-based fragment that binds the AAA domain of human katanin, a microtubule-severing protein. We have developed a model for compound binding and designed ASPIR-1 (allele-specific, proximity-induced reactivity-based inhibitor-1), a cell-permeable compound that selectively inhibits katanin with an engineered cysteine mutation. Only in cells expressing mutant katanin does ASPIR-1 treatment increase the accumulation of CAMSAP2 at microtubule minus ends, confirming specific on-target cellular activity. Importantly, ASPIR-1 also selectively inhibits engineered cysteine mutants of human VPS4B and FIGL1-AAA proteins, involved in organelle dynamics and genome stability, respectively. Structural studies confirm our model for compound binding at the AAA ATPase site and the proximity-induced reactivity-based inhibition. Together, our findings suggest a chemical genetics approach to decipher AAA protein functions across essential cellular processes and to test hypotheses for developing therapeutics.

Proteases and Their Modulators in Cancer Therapy: Challenges and Opportunities.

Proteostasis is the process of regulating intracellular proteins to maintain the balance of the cell proteome, which is crucial for cancer cell survival. Several proteases located in the cytoplasm, mitochondria, lysosome, and extracellular environment have been identified as potential antitumor targets because of their involvement in proteostasis. Although the discovery of small-molecule inhibitors targeting proteases faces particular challenges, rapid advances in chemical biology and structural biology, and the new technology of drug discovery have facilitated the development of promising protease modulators. In this review, the protein structure and function of important tumor-related proteases and their inhibitors are presented. We also provide a prospective on advances and the outlook of new drug strategies that target these proteases.