Strategies for the drug discovery and development of taxane anticancer therapeutics.

INTRODUCTION: Paclitaxel and docetaxel have been extensively used in the clinic over the past three decades. Although the patents of these first-generation taxanes have expired, their clinical applications, particularly new formulations and combination therapies, are under active investigations. Inspired by the notable success of Abraxane and Lipusu, new formulations have been extensively developed. In parallel, to overcome multidrug resistance (MDR) and to eradicate cancer stem cells, immense efforts have been made on the discovery and development of new-generation taxanes with improved potency and superior pharmacological properties. AREAS COVERED: This review covers (a) natural sources of advanced intermediates used for semi-synthesis of taxane API, (b) new formulations, (c) the major issues of FDA approved taxanes, (d) the design and development of next-generation taxanes, (e) new mechanisms of action, and (f) a variety of taxane-based drug delivery systems. EXPERT OPINION: As the highly potent next-generation taxanes can eradicate cancer stem cells and overcome MDR, the priority is to develop these compounds as an integral part of cancer therapy, especially for pancreatic, colon and prostate cancers which hardly respond to checkpoint inhibitors. In order to mitigate undesirable side effects, the exploration of effective nanoformulations and tumor-targeted drug delivery systems are essential.

Therapeutic Strategies to Activate p53.

The p53 protein has appropriately been named the "guardian of the genome". In almost all human cancers, the powerful tumor suppressor function of p53 is compromised by a variety of mechanisms, including mutations with either loss of function or gain of function and inhibition by its negative regulators MDM2 and/or MDMX. We review herein the progress made on different therapeutic strategies for targeting p53.

Discovery of M-1121 as an Orally Active Covalent Inhibitor of Menin-MLL Interaction Capable of Achieving Complete and Long-Lasting Tumor Regression.

Targeting the menin-MLL protein-protein interaction is being pursued as a new therapeutic strategy for the treatment of acute leukemia carrying MLL-rearrangements (MLLr leukemia). Herein, we report M-1121, a covalent and orally active inhibitor of the menin-MLL interaction capable of achieving complete and persistent tumor regression. M-1121 establishes covalent interactions with Cysteine 329 located in the MLL binding pocket of menin and potently inhibits growth of acute leukemia cell lines carrying MLL translocations with no activity in cell lines with wild-type MLL. Consistent with the mechanism of action, M-1121 drives dose-dependent down-regulation of HOXA9 and MEIS1 gene expression in the MLL-rearranged MV4;11 leukemia cell line. M-1121 is orally bioavailable and shows potent antitumor activity in vivo with tumor regressions observed at tolerated doses in the MV4;11 subcutaneous and disseminated models of MLL-rearranged leukemia. Together, our findings support development of an orally active covalent menin inhibitor as a new therapy for MLLr leukemia.

The ubiquitin ligase MDM2 sustains STAT5 stability to control T cell-mediated antitumor immunity.

Targeting the p53-MDM2 pathway to reactivate tumor p53 is a chemotherapeutic approach. However, the involvement of this pathway in CD8+ T cell-mediated antitumor immunity is unknown. Here, we report that mice with MDM2 deficiency in T cells exhibit accelerated tumor progression and a decrease in tumor-infiltrating CD8+ T cell survival and function. Mechanistically, MDM2 competes with c-Cbl for STAT5 binding, reduces c-Cbl-mediated STAT5 degradation and enhances STAT5 stability in tumor-infiltrating CD8+ T cells. Targeting the p53-MDM2 interaction with a pharmacological agent, APG-115, augmented MDM2 in T cells, thereby stabilizing STAT5, boosting T cell immunity and synergizing with cancer immunotherapy. Unexpectedly, these effects of APG-115 were dependent on p53 and MDM2 in T cells. Clinically, MDM2 abundance correlated with T cell function and interferon-γ signature in patients with cancer. Thus, the p53-MDM2 pathway controls T cell immunity, and targeting this pathway may treat patients with cancer regardless of tumor p53 status.

Discovery of M-808 as a Highly Potent, Covalent, Small-Molecule Inhibitor of the Menin-MLL Interaction with Strong In Vivo Antitumor Activity.

Targeting the menin-MLL protein-protein interaction is a new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein the structure-based optimization of a class of covalent menin inhibitors, which led to the discovery of M-808 (16) as a highly potent and efficacious covalent menin inhibitor. M-808 effectively inhibits leukemia cell growth at low nanomolar concentrations and is capable of achieving partial tumor regression in an MV4;11 xenograft tumor model in mice at a well-tolerated dose schedule. Determination of the co-crystal structure of M-808 in complex with menin provides a structural basis for their high-affinity, covalent interactions. M-808 represents a promising, covalent menin inhibitor for further optimization and evaluation toward developing a new therapy for the treatment of MLL leukemia.

Simple Structural Modifications Converting a Bona fide MDM2 PROTAC Degrader into a Molecular Glue Molecule: A Cautionary Tale in the Design of PROTAC Degraders.

Inducing protein degradation by proteolysis targeting chimeras (PROTACs) has gained tremendous momentum for its promise to discover and develop new therapies. Based upon our previously reported PROTAC MDM2 degraders, we have designed and synthesized additional analogues. Surprisingly, we found that simple structural modifications of MD-222, a bona fide MDM2 PROTAC degrader, converts it into a "molecular glue", as exemplified by MG-277. MG-277 induces only moderate MDM2 degradation and fails to activate wild-type p53 but is highly potent in inhibition of tumor cell growth in a p53-independent manner. Our mechanistic investigation established that MG-277 is not a PROTAC MDM2 degrader but instead works as a molecular glue, inducing degradation of a translation termination factor, GSPT1 to achieve its potent anticancer activity. Our study provides the first example that simple structural modifications can convert a bona fide PROTAC degrader into a molecular glue compound, which has a completely different mechanism of action.

Structure-Based Discovery of M-89 as a Highly Potent Inhibitor of the Menin-Mixed Lineage Leukemia (Menin-MLL) Protein-Protein Interaction.

Inhibition of the menin-mixed lineage leukemia (MLL) protein-protein interaction is a promising new therapeutic strategy for the treatment of acute leukemia carrying MLL fusion (MLL leukemia). We describe herein our structure-based design, synthesis, and evaluation of a new class of small-molecule inhibitors of the menin-MLL interaction (hereafter called menin inhibitors). Our efforts have resulted in the discovery of highly potent menin inhibitors, as exemplified by compound 42 (M-89). M-89 binds to menin with a Kd value of 1.4 nM and effectively engages cellular menin protein at low nanomolar concentrations. M-89 inhibits cell growth in the MV4;11 and MOLM-13 leukemia cell lines carrying MLL fusion with IC50 values of 25 and 55 nM, respectively, and demonstrates >100-fold selectivity over the HL-60 leukemia cell line lacking MLL fusion. The determination of a co-crystal structure of M-89 in a complex with menin provides the structural basis for their high-affinity interaction. Further optimization of M-89 may lead to a new class of therapy for the treatment of MLL leukemia.

Discovery of MD-224 as a First-in-Class, Highly Potent, and Efficacious Proteolysis Targeting Chimera Murine Double Minute 2 Degrader Capable of Achieving Complete and Durable Tumor Regression.

Human murine double minute 2 (MDM2) protein is a primary endogenous cellular inhibitor of the tumor suppressor p53 and has been pursued as an attractive cancer therapeutic target. Several potent, nonpeptide, small-molecule inhibitors of MDM2 are currently in clinical development. In this paper, we report our design, synthesis, and evaluation of small-molecule MDM2 degraders based on the proteolysis targeting chimera (PROTAC) concept. The most promising compound (MD-224) effectively induces rapid degradation of MDM2 at concentrations <1 nM in human leukemia cells. It achieves an IC50 value of 1.5 nM in inhibition of growth of RS4;11 cells and also low nanomolar IC50 values in a panel of leukemia cell lines. MD-224 achieves complete and durable tumor regression in vivo in the RS4;11 xenograft tumor model in mice at well-tolerated dose schedules. MD-224 is thus a highly potent and efficacious MDM2 degrader and warrants extensive evaluations as a new class of anticancer agent.

Structure-Based Discovery of CF53 as a Potent and Orally Bioavailable Bromodomain and Extra-Terminal (BET) Bromodomain Inhibitor.

We report the structure-based discovery of CF53 (28) as a highly potent and orally active inhibitor of bromodomain and extra-terminal (BET) proteins. By the incorporation of a NH-pyrazole group into the 9H-pyrimido[4,5- b]indole core, we identified a series of compounds that bind to BRD4 BD1 protein with Ki values of <1 nM and achieve low nanomolar potencies in the cell growth inhibition of leukemia and breast cancer cells. The most-promising compound, CF53, possesses excellent oral pharmacokinetic properties and achieves significant antitumor activity in both triple-negative breast cancer and acute leukemia xenograft models in mice. Determination of the co-crystal structure of CF53 with the BRD4 BD1 protein provides a structural basis for its high binding affinity to BET proteins. CF53 is very selective over non-BET bromodomain-containing proteins. These data establish CF53 as a potent, selective, and orally active BET inhibitor, which warrants further evaluation for advanced preclinical development.

Design of the First-in-Class, Highly Potent Irreversible Inhibitor Targeting the Menin-MLL Protein-Protein Interaction.

The structure-based design of M-525 as the first-in-class, highly potent, irreversible small-molecule inhibitor of the menin-MLL interaction is presented. M-525 targets cellular menin protein at sub-nanomolar concentrations and achieves low nanomolar potencies in cell growth inhibition and in the suppression of MLL-regulated gene expression in MLL leukemia cells. M-525 demonstrates high cellular specificity over non-MLL leukemia cells and is more than 30 times more potent than its corresponding reversible inhibitors. Mass spectrometric analysis and co-crystal structure of M-525 in complex with menin firmly establish its mode of action. A single administration of M-525 effectively suppresses MLL-regulated gene expression in tumor tissue. An efficient procedure was developed to synthesize M-525. This study demonstrates that irreversible inhibition of menin may be a promising therapeutic strategy for MLL leukemia.

Targeting the MDM2-p53 Protein-Protein Interaction for New Cancer Therapy: Progress and Challenges.

MDM2 is a primary cellular inhibitor of p53. It inhibits p53 function by multiple mechanisms, each of which, however, is mediated by their direct interaction. It has been proposed that small-molecule inhibitors designed to block the MDM2-p53 interaction may be effective in the treatment of human cancer retaining wild-type p53 by reactivating the p53 tumor suppressor function. Through nearly two decades of intense efforts, a number of structurally distinct, highly potent, nonpeptide, small-molecule inhibitors of the MDM2-p53 interaction (MDM2 inhibitors) have been successfully designed and developed, and at least seven such compounds have now been advanced into human clinical trials as new anticancer drugs. This review offers a perspective on the design and development of MDM2 small-molecule inhibitors and discusses early clinical data for some of the MDM2 small-molecule inhibitors and future challenges for the successful clinical development of MDM2 inhibitors for cancer treatment.

Discovery of 4-((3'R,4'S,5'R)-6″-Chloro-4'-(3-chloro-2-fluorophenyl)-1'-ethyl-2″-oxodispiro[cyclohexane-1,2'-pyrrolidine-3',3″-indoline]-5'-carboxamido)bicyclo[2.2.2]octane-1-carboxylic Acid (AA-115/APG-115): A Potent and Orally Active Murine Double Minute 2 (MDM2) Inhibitor in Clinical Development.

We previously reported the design of spirooxindoles with two identical substituents at the carbon-2 of the pyrrolidine core as potent MDM2 inhibitors. In this paper we describe an extensive structure-activity relationship study of this class of MDM2 inhibitors, which led to the discovery of 60 (AA-115/APG-115). Compound 60 has a very high affinity to MDM2 (Ki < 1 nM), potent cellular activity, and an excellent oral pharmacokinetic profile. Compound 60 is capable of achieving complete and long-lasting tumor regression in vivo and is currently in phase I clinical trials for cancer treatment.

Chemical synthesis of tetracyclic terpenes and evaluation of antagonistic activity on endothelin-A receptors and voltage-gated calcium channels.

A class of tetracyclic terpenes was synthesized and evaluated for antagonistic activity of endothelin-1 (ET-1) induced vasoconstriction and inhibitory activity of voltage-activated Ca(2+) channels. Three repeated Robinson annulation reactions were utilized to construct the tetracyclic molecules. A stereoselective reductive Robinson annulation was discovered for the formation of optically pure tricyclic terpenes. Stereoselective addition of cyanide to the hindered α-face of tetracyclic enone (-)-18 was found and subsequent transformation into the aldehyde function was affected by the formation of bicyclic hemiiminal (-)-4. Six selected synthetic tetracyclic terpenes show inhibitory activities in ET-1 induced vasoconstriction in the gerbil spiral modiolar artery with putative affinity constants ranging between 93 and 319 nM. Moreover, one compound, (-)-3, was evaluated further and found to inhibit voltage-activated Ca(2+) currents but not to affect Na(+) or K(+) currents in dorsal root ganglion cells under similar concentrations. These observations imply a dual mechanism of action. In conclusion, tetracyclic terpenes represent a new class of hit molecules for the discovery of new drugs for the treatment of pulmonary hypertension and vascular related diseases.

Small-molecule inhibitors of the MDM2-p53 protein-protein interaction (MDM2 Inhibitors) in clinical trials for cancer treatment.

Design of small-molecule inhibitors (MDM2 inhibitors) to block the MDM2-p53 protein-protein interaction has been pursued as a new cancer therapeutic strategy. In recent years, potent, selective, and efficacious MDM2 inhibitors have been successfully obtained and seven such compounds have been advanced into early phase clinical trials for the treatment of human cancers. Here, we review the design, synthesis, properties, preclinical, and clinical studies of these clinical-stage MDM2 inhibitors.

Design of chemically stable, potent, and efficacious MDM2 inhibitors that exploit the retro-mannich ring-opening-cyclization reaction mechanism in spiro-oxindoles.

Inhibition of the MDM2-p53 protein-protein interaction is being actively pursued as a new anticancer therapeutic strategy, and spiro-oxindoles have been designed as a class of potent and efficacious small-molecule inhibitors of this interaction (MDM2 inhibitors). Our previous study showed that some of our first-generation spiro-oxindoles undergo a reversible ring-opening-cyclization reaction that, from a single compound in protic solution, results in an equilibrium mixture of four diastereoisomers. By exploiting the ring-opening-cyclization reaction mechanism, we have designed and synthesized a series of second-generation spiro-oxindoles with symmetrical pyrrolidine C2 substitution. These compounds undergo a rapid and irreversible conversion to a single, stable diastereoisomer. Our study has yielded compound 31 (MI-1061), which binds to MDM2 with Ki = 0.16 nM, shows excellent chemical stability, and achieves tumor regression in the SJSA-1 xenograft tumor model in mice.

SAR405838: an optimized inhibitor of MDM2-p53 interaction that induces complete and durable tumor regression.

Blocking the oncoprotein murine double minute 2 (MDM2)-p53 protein-protein interaction has long been considered to offer a broad cancer therapeutic strategy, despite the potential risks of selecting tumors harboring p53 mutations that escape MDM2 control. In this study, we report a novel small-molecule inhibitor of the MDM2-p53 interaction, SAR405838 (MI-77301), that has been advanced into phase I clinical trials. SAR405838 binds to MDM2 with K(i) = 0.88 nmol/L and has high specificity over other proteins. A cocrystal structure of the SAR405838:MDM2 complex shows that, in addition to mimicking three key p53 amino acid residues, the inhibitor captures additional interactions not observed in the p53-MDM2 complex and induces refolding of the short, unstructured MDM2 N-terminal region to achieve its high affinity. SAR405838 effectively activates wild-type p53 in vitro and in xenograft tumor tissue of leukemia and solid tumors, leading to p53-dependent cell-cycle arrest and/or apoptosis. At well-tolerated dose schedules, SAR405838 achieves either durable tumor regression or complete tumor growth inhibition in mouse xenograft models of SJSA-1 osteosarcoma, RS4;11 acute leukemia, LNCaP prostate cancer, and HCT-116 colon cancer. Remarkably, a single oral dose of SAR405838 is sufficient to achieve complete tumor regression in the SJSA-1 model. Mechanistically, robust transcriptional upregulation of PUMA induced by SAR405838 results in strong apoptosis in tumor tissue, leading to complete tumor regression. Our findings provide a preclinical basis upon which to evaluate SAR405838 as a therapeutic agent in patients whose tumors retain wild-type p53.

BM-1197: a novel and specific Bcl-2/Bcl-xL inhibitor inducing complete and long-lasting tumor regression in vivo.

Bcl-2 and Bcl-xL are critical regulators of apoptosis that are overexpressed in a variety of human cancers and pharmacological inhibition of Bcl-2 and Bcl-xL represents a promising strategy for cancer treatment. Using a structure-based design approach, we have designed BM-1197 as a potent and efficacious dual inhibitor of Bcl-2 and Bcl-xL. BM-1197 binds to Bcl-2 and Bcl-xL proteins with Ki values less than 1 nM and shows >1,000-fold selectivity over Mcl-1. Mechanistic studies performed in the Mcl-1 knockout mouse embryonic fibroblast (MEF) cells revealed that BM-1197 potently disassociates the heterodimeric interactions between anti-apoptotic and pro-apoptotic Bcl-2 family proteins, concomitant with conformational changes in Bax protein, loss of mitochondrial membrane potential and subsequent cytochrome c release to the cytosol, leading to activation of the caspase cascade and apoptosis. BM-1197 exerts potent growth-inhibitory activity in 7 of 12 small cell lung cancer cell lines tested and induces mechanism-based apoptotic cell death. When intravenously administered at daily or weekly in H146 and H1963 small-cell lung cancer xenograft models, it achieves complete and long-term tumor regression. Consistent with its targeting of Bcl-xL, BM-1197 causes transit platelet reduction in mice. Collectively, our data indicate that BM-1197 is a promising dual Bcl-2/Bcl-xL inhibitor which warrants further investigation as a new anticancer drug.

A potent and highly efficacious Bcl-2/Bcl-xL inhibitor.

Our previously reported Bcl-2/Bcl-xL inhibitor, 4, effectively inhibited tumor growth but failed to achieve complete regression in vivo. We have now performed extensive modifications on its pyrrole core structure, which has culminated in the discovery of 32 (BM-1074). Compound 32 binds to Bcl-2 and Bcl-xL proteins with K(i) values of <1 nM and inhibits cancer cell growth with IC50 values of 1-2 nM in four small-cell lung cancer cell lines sensitive to potent and specific Bcl-2/Bcl-xL inhibitors. Compound 32 is capable of achieving rapid, complete, and durable tumor regression in vivo at a well-tolerated dose schedule. Compound 32 is the most potent and efficacious Bcl-2/Bcl-xL inhibitor reported to date.

Structure-based discovery of BM-957 as a potent small-molecule inhibitor of Bcl-2 and Bcl-xL capable of achieving complete tumor regression.

Bcl-2 and Bcl-xL antiapoptotic proteins are attractive cancer therapeutic targets. We have previously reported the design of 4,5-diphenyl-1H-pyrrole-3-carboxylic acids as a class of potent Bcl-2/Bcl-xL inhibitors. In the present study, we report our structure-based optimization for this class of compounds based upon the crystal structure of Bcl-xL complexed with a potent lead compound. Our efforts accumulated into the design of compound 30 (BM-957), which binds to Bcl-2 and Bcl-xL with K(i) < 1 nM and has low nanomolar IC(50) values in cell growth inhibition in cancer cell lines. Significantly, compound 30 achieves rapid, complete, and durable tumor regression in the H146 small-cell lung cancer xenograft model at a well-tolerated dose schedule.

Structure-based design of potent Bcl-2/Bcl-xL inhibitors with strong in vivo antitumor activity.

Bcl-2 and Bcl-xL are key apoptosis regulators and attractive cancer therapeutic targets. We have designed and optimized a class of small-molecule inhibitors of Bcl-2 and Bcl-xL containing a 4,5-diphenyl-1H-pyrrole-3-carboxylic acid core structure. A 1.4 Å resolution crystal structure of a lead compound, 12, complexed with Bcl-xL has provided a basis for our optimization. The most potent compounds, 14 and 15, bind to Bcl-2 and Bcl-xL with subnanomolar K(i) values and are potent antagonists of Bcl-2 and Bcl-xL in functional assays. Compounds 14 and 15 inhibit cell growth with low nanomolar IC(50) values in multiple small-cell lung cancer cell lines and induce robust apoptosis in cancer cells at concentrations as low as 10 nM. Compound 14 also achieves strong antitumor activity in an animal model of human cancer.