A new ALK inhibitor overcomes resistance to first- and second-generation inhibitors in NSCLC.

More than 60% of nonsmall cell lung cancer (NSCLC) patients show a positive response to the first ALK inhibitor, crizotinib, which has been used as the standard treatment for newly diagnosed patients with ALK rearrangement. However, most patients inevitably develop crizotinib resistance due to acquired secondary mutations in the ALK kinase domain, such as the gatekeeper mutation L1196M and the most refractory mutation, G1202R. Here, we develop XMU-MP-5 as a new-generation ALK inhibitor to overcome crizotinib resistance mutations, including L1196M and G1202R. XMU-MP-5 blocks ALK signaling pathways and inhibits the proliferation of cells harboring either wild-type or mutant EML4-ALK in vitro and suppresses tumor growth in xenograft mouse models in vivo. Structural analysis provides insights into the mode of action of XMU-MP-5. In addition, XMU-MP-5 induces significant regression of lung tumors in two genetically engineered mouse (GEM) models, further demonstrating its pharmacological efficacy and potential for clinical application. These preclinical data support XMU-MP-5 as a novel selective ALK inhibitor with high potency and selectivity. XMU-MP-5 holds great promise as a new therapeutic against clinically relevant secondary ALK mutations.

Structural Basis of Specificity for Carboxyl-Terminated Acyl Donors in a Bacterial Acyltransferase.

Macrolactins (MLNs) are a class of important antimacular degeneration and antitumor agents. Malonylated/succinylated MLNs are even more important due to their efficacy in overcoming multi-drug-resistant bacteria. However, which enzyme catalyzes this reaction remains enigmatic. Herein, we deciphered a ß-lactamase homologue BmmI to be responsible for this step. BmmI could specifically attach C3-C5 alkyl acid thioesters onto 7-OH of MLN A and also exhibits substrate promiscuity toward acyl acceptors with different scaffolds. The crystal structure of BmmI covalently linked to the succinyl group and systematic mutagenesis highlighted the role of oxyanion holelike geometry in the recognition of carboxyl-terminated acyl donors. The engineering of this geometry expanded its substrate scope, with the R166A/G/Q variants recognizing up to C12 alkyl acid thioester. The structure of BmmI with acyl acceptor MLN A revealed the importance of Arg292 in the recognition of macrolide substrates. Moreover, the mechanism of the BmmI-catalyzed acyltransfer reaction was established, unmasking the deft role of Lys76 in governing acyl donors as well as catalysis. Our studies uncover the delicate mechanism underlying the substrate selectivity of acyltransferases, which would guide rational enzyme engineering for drug development.

Structural Basis of AZD9291 Selectivity for EGFR T790M.

AZD9291 (Osimertinib) is highly effective in treating EGFR-mutated non-small-cell lung cancers (NSCLCs) with T790M-mediated drug resistance. Despite the remarkable success of AZD9291, its binding pose with EGFR T790M remains unclear. Here, we report unbiased, atomic-level molecular dynamics (MD) simulations in which spontaneous association of AZD9291 with EGFR kinases having WT and T790M mutant gatekeepers was observed. Simulation-generated structural models suggest that the binding pose of AZD9291 with T790M differs from its binding pose with the WT, and that AZD9291 interacts extensively with the gatekeeper residue (Met 790) in T790M but not with Thr 790 in the WT, which explains why AZD9291 binds T790M with higher affinity. The MD simulation-generated models were confirmed by experimentally determined EGFR/T790M complex crystal structures. This work may facilitate the rational design of drugs that can overcome resistance mutations to AZD9291, and more generally it suggests the potential of using unbiased MD simulation to elucidate small-molecule binding poses.

Discovery and Optimization of Dibenzodiazepinones as Allosteric Mutant-Selective EGFR Inhibitors.

Allosteric kinase inhibitors represent a promising new therapeutic strategy for targeting kinases harboring oncogenic driver mutations in cancers. Here, we report the discovery, optimization, and structural characterization of allosteric mutant-selective EGFR inhibitors comprising a 5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one scaffold. Our structure-based medicinal chemistry effort yielded an inhibitor (3) of the EGFR(L858R/T790M) and EGFR(L858R/T790M/C797S) mutants with an IC50 of ∼10 nM and high selectivity, as assessed by kinome profiling. Further efforts to develop allosteric dibenzodiazepinone inhibitors may serve as the basis for new therapeutic options for targeting drug-resistant EGFR mutations.

A non-covalent inhibitor XMU-MP-3 overrides ibrutinib-resistant BtkC481S mutation in B-cell malignancies.

BACKGROUND AND PURPOSE: Bruton's tyrosine kinase (BTK) plays a key role in B-cell receptor signalling by regulating cell proliferation and survival in various B-cell malignancies. Covalent low-MW BTK kinase inhibitors have shown impressive clinical efficacy in B-cell malignancies. However, the mutant BtkC481S poses a major challenge in the management of B-cell malignancies by disrupting the formation of the covalent bond between BTK and irreversible inhibitors, such as ibrutinib. The present studies were designed to develop novel BTK inhibitors targeting ibrutinib-resistant BtkC481S mutation. EXPERIMENTAL APPROACH: BTK-Ba/F3, BTK(C481S)-Ba/F3 cells, and human malignant B-cells JeKo-1, Ramos, and NALM-6 were used to evaluate cellular potency of BTK inhibitors. The in vitro pharmacological efficacy and compound selectivity were assayed via cell viability, colony formation, and BTK-mediated signalling. A tumour xenograft model with BTK-Ba/F3, Ramos and BTK(C481S)-Ba/F3 cells in Nu/nu BALB/c mice was used to assess in vivo efficacy of XMU-MP-3. KEY RESULTS: XMU-MP-3 is one of a group of low MW compounds that are potent non-covalent BTK inhibitors. XMU-MP-3 inhibited both BTK and the acquired mutant BTKC481S, in vitro and in vivo. Further computational modelling, site-directed mutagenesis analysis, and structure-activity relationships studies indicated that XMU-MP-3 displayed a typical Type-II inhibitor binding mode. CONCLUSION AND IMPLICATIONS: XMU-MP-3 directly targets the BTK signalling pathway in B-cell lymphoma. These findings establish XMU-MP-3 as a novel inhibitor of BTK, which could serve as both a tool compound and a lead for further drug development in BTK relevant B-cell malignancies, especially those with the acquired ibrutinib-resistant C481S mutation.

Structure-Based Design of 5-Methylpyrimidopyridone Derivatives as New Wild-Type Sparing Inhibitors of the Epidermal Growth Factor Receptor Triple Mutant (EGFRL858R/T790M/C797S).

Tertiary EGFRC797S mutation induced resistance against osimertinib (1) is an emerging "unmet clinical need" for non-small-cell lung cancer (NSCLC) patients. A series of 5-methylpyrimidopyridone derivatives were designed and synthesized as new selective EGFRL858R/T790M/C797S inhibitors. A representative compound, 8r-B, exhibited an IC50 of 27.5 nM against the EGFRL858R/T790M/C797S mutant, while being a significantly less potent for EGFRWT (IC50 > 1.0 µM). Cocrystallographic structure determination and computational investigation were conducted to elucidate its target selectivity.

Discovery of JND3229 as a New EGFRC797S Mutant Inhibitor with In Vivo Monodrug Efficacy.

EGFRC797S mutation inducing resistance against third generation EGFR inhibitor drugs is an emerging "unmet clinical need" for nonsmall cell lung cancer patients. The pyrimidopyrimidinone derivative JND3229 was identified as a new highly potent EGFRC797S inhibitor with single digit nM potency. It also exhibited good in vitro and in vivo monodrug anticancer efficacy in a xenograft mouse model of BaF3/EGFR19D/T790M/C797S cells. A high-resolution X-ray crystallographic structure was also determined to elucidate the interactions between JND3229 and EGFRT790M/C797S. Our study provides an important structural and chemical basis for future development of new generation EGFRC797S inhibitors as anticancer drugs.

Crystal structures of pan-IDH inhibitor AG-881 in complex with mutant human IDH1 and IDH2.

Some mutations of isocitrate dehydrogenase 1 and 2 observed in multiple kinds of malignant tumors can lead to a neomorphic enzyme activity that converts alpha-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). As an oncometabolite, 2-HG can cause epigenetic changes and impair cell differentiation. Inhibiting the activity of isocitrate dehydrogenase mutants (mIDH) is considered to be an effective therapy for the treatment of mIDH positive cancers, including glioma and acute myeloid leukemia (AML). The presently disclosed allosteric inhibitors work only on one of the mIDH1 and mIDH2, and it is shown that mIDH1 and mIDH2 have different allosteric inhibition pockets. However, AG-881 from Agios Pharmaceuticals was found to be a pan-IDH inhibitor against both mIDH1 and mIDH2, and is undergoing Phase I clinical trials for tumors with an IDH1 and/or IDH2 mutation. To understand the binding mode of AG-881 to mIDHs, we solved the crystal structures of IDH1-R132H/NADPH/AG-881 and IDH2-R140Q/NADPH/AG-881 complexes, and acquired the IC50 values of AG-881 for IDH1-R132H and IDH2-R140Q homodimers after different pre-incubation times. Our data show that AG-881 binds IDH1-R132H and IDH2-R140Q in the same allosteric pockets and that the subtle difference in the pockets of these two proteins may contribute to their remarkably different inhibitory kinetics by AG-881. The structural pharmacological data provided in this report may benefit the future development of pan-IDH inhibitors targeting mIDH1 and mIDH2.

A Novel Microtubule Inhibitor Overcomes Multidrug Resistance in Tumors.

Microtubule inhibitors as chemotherapeutic drugs are widely used for cancer treatment. However, the development of multidrug resistance (MDR) in cancer is a major challenge for microtubule inhibitors in their clinical implementation. From a high-throughput drug screen using cells transformed by oncogenic RAS, we identify a lead heteroaryl amide compound that blocks cell proliferation. Analysis of the structure-activity relationship indicated that this series of scaffolds (exemplified by MP-HJ-1b) represents a potent inhibitor of tumor cell growth. MP-HJ-1b showed activities against a panel of more than 1,000 human cancer cell lines with a wide variety of tissue origins. This compound depolymerized microtubules and affected spindle formation. It also induced the spike-like conformation of microtubules in vitro and in vivo, which is different from typical microtubule modulators. Structural analysis revealed that this series of compounds bound the colchicine pocket at the intra-dimer interface, although mostly not overlapping with colchicine binding. MP-HJ-1b displayed favorable pharmacological properties for overcoming tumor MDR, both in vitro and in vivo Taken together, our data reveal a novel scaffold represented by MP-HJ-1b that can be developed as a cancer therapeutic against tumors with MDR.Significance: Paclitaxel is a widely used chemotherapeutic drug in patients with multiple types of cancer. However, resistance to paclitaxel is a challenge. This study describes a novel class of microtubule inhibitors with the ability to circumvent multidrug resistance across multiple tumor cell lines. Cancer Res; 78(20); 5949-57. ©2018 AACR.

Crystal structure of EGFR T790M/C797S/V948R in complex with EAI045.

Lung cancer is the leading cause of cancer deaths. Epidermal growth factor receptor (EGFR) kinase domain mutations are a common cause of non-small cell lung cancers (NSCLCs), a major subtype of lung cancers. Patients harboring most of these mutations respond well to the anti-EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib initially, but soon develop resistance to them in about half of the cases due to the emergence of the gatekeeper mutation T790M. The third-generation TKIs such as AZD9291, HM61713, CO-1686 and WZ4002 can overcome T790M through covalent binding to the EGFR kinase through Cys 797, but ultimately lose their efficacy upon emergence of the C797S mutation that abolishes the covalent bonding. Therefore to develop new TKIs to overcome EGFR drug-resistant mutants harboring T790M/C797S is urgently demanded. EAI001 and EAI045 are a new type of EGFR TKIs that bind to EGFR reversibly and not relying on Cys 797. EAI045 in combination with cetuximab is effective in mouse models of lung cancer driven by EGFR L858R/T790M and L858R/T790M/C797S. Here we report the crystal structure of EGFR T790M/C797S/V948R in complex with EAI045, and compare it to EGFR T790M/V948R in complex with EAI001. The complex structure reveals why EAI045 binds tighter to EGFR than does EAI001, and why EAI001 and EAI045 prefer binding to EGFR T790M. The knowledge may facilitate future drug development studies targeting this very important cancer target.

Structural insights into drug development strategy targeting EGFR T790M/C797S.

Treatment of non-small-cell lung cancers (NSCLCs) harboring primary EGFR oncogenic mutations such as L858R and exon 19 deletion delE746_A750 (Del-19) using gefitinib/erlotinib ultimately fails due to the emergence of T790M mutation. Though WZ4002/CO-1686/AZD9291 are effective in overcoming EGFR T790M by targeting Cys797 via covalent bonding, their efficacy is again limited due to the emergence of C797S mutation. New agents effectively inhibiting EGFR T790M without covalent linkage through Cys 797 may solve this problem. We presented here crystal structures of EGFR activating/drug-resistant mutants in complex with a panel of reversible inhibitors along with mutagenesis and enzyme kinetic data. These data revealed a previously un-described hydrophobic clamp structure in the EGFR kinase which may be exploited to facilitate development of next generation drugs targeting EGFR T790M with or without concomitant C797S. Interestingly, mutations in the hydrophobic clamp that hinder drug binding often also weaken ATP binding and/or abolish kinase activity, thus do not readily result in resistance to the drugs.

Discovery of 4-((N-(2-(dimethylamino)ethyl)acrylamido)methyl)-N-(4-methyl-3-((4-(pyridin-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide (CHMFL-PDGFR-159) as a highly selective type II PDGFRα kinase inhibitor for PDGFRα driving chronic eosinophilic leukemia.

Through exploration of the non-highly conserved allosteric hydrophobic pocket generated by DFG-out shifting in the inactive conformation, we discovered a highly selective type II PDGFRα kinase inhibitor 15i (CHMFL-PDGFRα-159), which exhibited strong potency against purified PDGFRα (IC50: 132 nM) but not structurally similar PDGFRß, ABL, c-KIT and VEGFR2 kinases. In addition, it displayed a high selectivity profile (S score (10) = 0.02) at the concentration of 1 µM among 468 kinases/mutants in the KINOMEscan profiling. X-ray crystal structure of 15i in complex with PDGFRα revealed a distinct binding feature in the allosteric hydrophobic pocket which might help to expand the diversity of type II kinase inhibitors. Compound 15i potently inhibited the proliferation of PDGFRα driving Chronic Eosinophilic Leukemia (CEL) cell line EOL-1 through strong blockage of PDGFRα mediated signaling pathways, arresting cell cycle progression, and induction of apoptosis. Furthermore, compound 15i effectively suppressed the EOL-1 tumor progression in the xenograft model and increased the survival rate in the engraftment tumor model.

Acetylation of 53BP1 dictates the DNA double strand break repair pathway.

P53-binding protein 1 (53BP1) plays critical roles in DNA double strand break (DSB) repair by promoting non-homologous end joining (NHEJ), and loss of 53BP1 abolishes PARPi sensitivity in BRCA1-deficient cells by restoring homologous recombination (HR). 53BP1 is one of the proteins initially recruited to sites of DSBs via recognition of H4K20me2 through the Tudor-UDR domain and H2AK15ub through the UDR motif. Although extensive studies have been conducted, it remains unclear how the post-translational modification of 53BP1 affects DSB repair pathway choice. Here, we identified 53BP1 as an acetylated protein and determined that acetylation of 53BP1 inhibit NHEJ and promote HR by negatively regulating 53BP1 recruitment to DSBs. Mechanistically, CBP-mediated acetylation of K1626/1628 in the UDR motif disrupted the interaction between 53BP1 and nucleosomes, subsequently blocking the recruitment of 53BP1 and its downstream factors PTIP and RIF1 to DSBs. Hyperacetylation of 53BP1, similar to depletion of 53BP1, restored PARPi resistance in BRCA1-deficient cells. Interestingly, 53BP1 acetylation was tightly regulated by HDAC2 to maintain balance between the HR and NHEJ pathways. Together, our results demonstrate that the acetylation status of 53BP1 plays a key role in its recruitment to DSBs and reveal how specific 53BP1 modification modulates the choice of DNA repair pathway.

Structural basis of mutant-selectivity and drug-resistance related to CO-1686.

Non-small-cell lung cancers (NSCLCs) caused by activating mutations in the kinase domain of epidermal growth factor receptor (EGFR) initially respond to first-generation reversible drugs gefitinib and erlotinib. However, clinical efficacy is limited due to the development of drug-resistance that in more than half of the cases are driven by the secondary T790M mutation. CO-1686 is one of the third generation irreversible inhibitors that inhibits EGFR activating mutants, including those with concurrent T790M, while avoiding the off-target toxicity owing to inhibition of wild-type EGFR in treating EGFR mutation-positive NSCLCs. Despite the remarkable success, the experimentally determined structure of this agent in complex with EGFR T790M remains unknown. In this study, we determined crystal structures of EGFR T790M or L858R mutants covalently bound by CO-1686. Based on these structural data, we can explain why CO-1686 irreversibly inhibits EGFR and selectively prefers T790M, which may help improving this or similar compounds, and explain why EGFR L718Q and L844V mutations incur resistance to this agent.

Structural pharmacological studies on EGFR T790M/C797S.

Drug-resistance is a major challenge in targeted therapy of EGFR mutated non-small cell lung cancers (NSCLCs). The third-generation irreversible inhibitors such as AZD9291, CO-1686 and WZ4002 can overcome EGFR T790M drug-resistance mutant through covalent binding through Cys 797, but ultimately lose their efficacy upon emergence of the new mutation C797S. To develop new reversible inhibitors not relying on covalent binding through Cys 797 is therefore urgently demanded. Gö6976 is a staurosporine-like reversible inhibitor targeting T790M while sparing the wild-type EGFR. In the present work, we reported the complex crystal structures of EGFR T790M/C797S + Gö6976 and T790M + Gö6976, along with enzyme kinetic data of EGFR wild-type, T790M and T790M/C797S. These data showed that the C797S mutation does not significantly alter the structure and function of the EGFR kinase, but increases the local hydrophilicity around residue 797. The complex crystal structures also elucidated the detailed binding mode of Gö6976 to EGFR and explained why this compound prefers binding to T790M mutant. These structural pharmacological data would facilitate future drug development studies.

Discovery and characterization of a novel irreversible EGFR mutants selective and potent kinase inhibitor CHMFL-EGFR-26 with a distinct binding mode.

EGFR T790M mutation accounts for about 40-55% drug resistance for the first generation EGFR kinase inhibitors in the NSCLC. Starting from ibrutinib, a highly potent irreversible BTK kinase inhibitor, which was also found to be moderately active to EGFR T790M mutant, we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26, which is selectively potent against EGFR mutants including L858R, del19, and L858R/T790M. It displayed proper selectivity window between the EGFR mutants and the wide-type. CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested (S score (1)=0.02). In addition, X-ray crystallography revealed a distinct "DFG-in" and "cHelix-out" inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein. The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway, induction of apoptosis and arresting of cell cycle progression. CHMFL-EGFR-26 bore acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975 (EGFR L858R/T790M) and PC-9 (EGFR del19) inoculated xenograft mouse models. Currently CHMFL-EGFR-26 is undergoing extensive pre-clinical evaluation for the clinical trial purpose.

Discovery of (R)-1-(3-(4-Amino-3-(3-chloro-4-(pyridin-2-ylmethoxy)phenyl)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)prop-2-en-1-one (CHMFL-EGFR-202) as a Novel Irreversible EGFR Mutant Kinase Inhibitor with a Distinct Binding Mode.

On the basis of Ibrutinib's core pharmacophore, which was moderately active to EGFR T790M mutant, we discovered novel epidermal growth factor receptor (EGFR) inhibitor compound 19 (CHMFL-EGFR-202), which potently inhibited EGFR primary mutants (L858R, del19) and drug-resistant mutant L858R/T790M. Compound 19 displayed a good selectivity profile among 468 kinases/mutants tested in the KINOMEscan assay (S score (1) = 0.02). In particular, it did not exhibit apparent activities against INSR and IGF1R kinases. The X-ray crystal structure revealed that this class of inhibitors formed a covalent bond with Cys797 in a distinct "DFG-in-C-helix-out" inactive EGFR conformation. Compound 19 displayed strong antiproliferative effects against EGFR mutant-driven nonsmall cell lung cancer (NSCLC) cell lines such as H1975, PC9, HCC827, and H3255 but not the wild-type EGFR expressing cells. In the H1975 and PC9 cell-inoculated xenograft mouse models, compound 19 exhibited dose-dependent tumor growth suppression efficacy without obvious toxicity. Compound 19 might be a potential drug candidate for EGFR mutant-driven NSCLC.

Discovery of N-(3-(5-((3-acrylamido-4-(morpholine-4-carbonyl)phenyl)amino)-1-methyl-6-oxo-1,6-dihydropyridin-3-yl)-2-methylphenyl)-4-(tert-butyl)benzamide (CHMFL-BTK-01) as a highly selective irreversible Bruton's tyrosine kinase (BTK) inhibitor.

Currently there are several irreversible BTK inhibitors targeting Cys481 residue under preclinical or clinical development. However, most of these inhibitors also targeted other kinases such as BMX, JAK3, and EGFR that bear the highly similar active cysteine residues. Through a structure-based drug design approach, we discovered a highly potent (IC50: 7 nM) irreversible BTK inhibitor compound 9 (CHMFL-BTK-01), which displayed a high selectivity profile in KINOMEscan (S score (35) = 0.00) among 468 kinases/mutants at the concentration of 1 µM. Compound 9 completely abolished BMX, JAK3 and EGFR's activity. Both X-ray crystal structure and cysteine-serine mutation mediated rescue experiment confirmed 9's irreversible binding mode. 9 also potently inhibited BTK Y223 auto-phosphorylation (EC50: <30 nM), arrested cell cycle in G0/G1 phase and induced apoptosis in U2932 and Pfeiffer cells. We believe these features would make 9 a good pharmacological tool to study the BTK related pathology.

Ibrutinib targets mutant-EGFR kinase with a distinct binding conformation.

Ibrutinib, a clinically approved irreversible BTK kinase inhibitor for Mantle Cell Lymphoma (MCL) and Chronic Lymphocytic Leukemia (CLL) etc, has been reported to be potent against EGFR mutant kinase and currently being evaluated in clinic for Non Small Cell Lung Cancer (NSCLC). Through EGFR wt/mutant engineered isogenic BaF3 cell lines we confirmed the irreversible binding mode of Ibrutinib with EGFR wt/mutant kinase via Cys797. However, comparing to typical irreversible EGFR inhibitor, such as WZ4002, the washing-out experiments revealed a much less efficient covalent binding for Ibrutinib. The biochemical binding affinity examination in the EGFR L858R/T790M kinase revealed that, comparing to more efficient irreversible inhibitor WZ4002 (Kd: 0.074 µM), Ibrutinib exhibited less efficient binding (Kd: 0.18 µM). An X-ray crystal structure of EGFR (T790M) in complex with Ibrutinib exhibited a unique DFG-in/c-Helix-out inactive binding conformation, which partially explained the less efficiency of covalent binding and provided insight for further development of highly efficient irreversible binding inhibitor for the EGFR mutant kinase. These results also imply that, unlike the canonical irreversible inhibitor, sustained effective concentration might be required for Ibrutinib in order to achieve the maximal efficacy in the clinic application against EGFR driven NSCLC.

Pharmacological targeting of kinases MST1 and MST2 augments tissue repair and regeneration.

Tissue repair and regenerative medicine address the important medical needs to replace damaged tissue with functional tissue. Most regenerative medicine strategies have focused on delivering biomaterials and cells, yet there is the untapped potential for drug-induced regeneration with good specificity and safety profiles. The Hippo pathway is a key regulator of organ size and regeneration by inhibiting cell proliferation and promoting apoptosis. Kinases MST1 and MST2 (MST1/2), the mammalian Hippo orthologs, are central components of this pathway and are, therefore, strong target candidates for pharmacologically induced tissue regeneration. We report the discovery of a reversible and selective MST1/2 inhibitor, 4-((5,10-dimethyl-6-oxo-6,10-dihydro-5H-pyrimido[5,4-b]thieno[3,2-e][1,4]diazepin-2-yl)amino)benzenesulfonamide (XMU-MP-1), using an enzyme-linked immunosorbent assay-based high-throughput biochemical assay. The cocrystal structure and the structure-activity relationship confirmed that XMU-MP-1 is on-target to MST1/2. XMU-MP-1 blocked MST1/2 kinase activities, thereby activating the downstream effector Yes-associated protein and promoting cell growth. XMU-MP-1 displayed excellent in vivo pharmacokinetics and was able to augment mouse intestinal repair, as well as liver repair and regeneration, in both acute and chronic liver injury mouse models at a dose of 1 to 3 mg/kg via intraperitoneal injection. XMU-MP-1 treatment exhibited substantially greater repopulation rate of human hepatocytes in the Fah-deficient mouse model than in the vehicle-treated control, indicating that XMU-MP-1 treatment might facilitate human liver regeneration. Thus, the pharmacological modulation of MST1/2 kinase activities provides a novel approach to potentiate tissue repair and regeneration, with XMU-MP-1 as the first lead for the development of targeted regenerative therapeutics.