Ribonucleotide Reductases: Structure, Chemistry, and Metabolism Suggest New Therapeutic Targets.

Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.

Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy.

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Targeting KRAS Mutant Cancers with a Covalent G12C-Specific Inhibitor.

KRASG12C was recently identified to be potentially druggable by allele-specific covalent targeting of Cys-12 in vicinity to an inducible allosteric switch II pocket (S-IIP). Success of this approach requires active cycling of KRASG12C between its active-GTP and inactive-GDP conformations as accessibility of the S-IIP is restricted only to the GDP-bound state. This strategy proved feasible for inhibiting mutant KRAS in vitro; however, it is uncertain whether this approach would translate to in vivo. Here, we describe structure-based design and identification of ARS-1620, a covalent compound with high potency and selectivity for KRASG12C. ARS-1620 achieves rapid and sustained in vivo target occupancy to induce tumor regression. We use ARS-1620 to dissect oncogenic KRAS dependency and demonstrate that monolayer culture formats significantly underestimate KRAS dependency in vivo. This study provides in vivo evidence that mutant KRAS can be selectively targeted and reveals ARS-1620 as representing a new generation of KRASG12C-specific inhibitors with promising therapeutic potential.

At the Intersection of Chemistry, Biology, and Medicine.

After an undergraduate degree in biology at Harvard, I started graduate school at The Rockefeller Institute for Medical Research in New York City in July 1965. I was attracted to the chemical side of biochemistry and joined Fritz Lipmann's large, hierarchical laboratory to study enzyme mechanisms. That work led to postdoctoral research with Robert Abeles at Brandeis, then a center of what, 30 years later, would be called chemical biology. I spent 15 years on the Massachusetts Institute of Technology faculty, in both the Chemistry and Biology Departments, and then 26 years on the Harvard Medical School Faculty. My research interests have been at the intersection of chemistry, biology, and medicine. One unanticipated major focus has been investigating the chemical logic and enzymatic machinery of natural product biosynthesis, including antibiotics and antitumor agents. In this postgenomic era it is now recognized that there may be from 105 to 106 biosynthetic gene clusters as yet uncharacterized for potential new therapeutic agents.

Where is the Future of Drug Discovery for Cancer?

With both small molecules and biologics succeeding in trials and in the clinic, the scope of drug discovery in cancer is changing. We asked a group of researchers to share their visions for how to identify new targets and how to approach taming them.

First Approved Kinase Inhibitor for AML.

Activating mutations of FLT3 occur in about 30% of acute myeloid leukemia (AML) cases and are associated with relapse and poor prognosis. Midostaurin is the first drug approved for AML since 2000, and the first multi-kinase inhibitor approved for the FLT3-mutant subtype. To view this Bench to Bedside, open or download the PDF.

Multivalent Small-Molecule Pan-RAS Inhibitors.

Design of small molecules that disrupt protein-protein interactions, including the interaction of RAS proteins and their effectors, may provide chemical probes and therapeutic agents. We describe here the synthesis and testing of potential small-molecule pan-RAS ligands, which were designed to interact with adjacent sites on the surface of oncogenic KRAS. One compound, termed 3144, was found to bind to RAS proteins using microscale thermophoresis, nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry and to exhibit lethality in cells partially dependent on expression of RAS proteins. This compound was metabolically stable in liver microsomes and displayed anti-tumor activity in xenograft mouse cancer models. These findings suggest that pan-RAS inhibition may be an effective therapeutic strategy for some cancers and that structure-based design of small molecules targeting multiple adjacent sites to create multivalent inhibitors may be effective for some proteins.

Inhibition of Dihydroorotate Dehydrogenase Overcomes Differentiation Blockade in Acute Myeloid Leukemia.

While acute myeloid leukemia (AML) comprises many disparate genetic subtypes, one shared hallmark is the arrest of leukemic myeloblasts at an immature and self-renewing stage of development. Therapies that overcome differentiation arrest represent a powerful treatment strategy. We leveraged the observation that the majority of AML, despite their genetically heterogeneity, share in the expression of HoxA9, a gene normally downregulated during myeloid differentiation. Using a conditional HoxA9 model system, we performed a high-throughput phenotypic screen and defined compounds that overcame differentiation blockade. Target identification led to the unanticipated discovery that inhibition of the enzyme dihydroorotate dehydrogenase (DHODH) enables myeloid differentiation in human and mouse AML models. In vivo, DHODH inhibitors reduced leukemic cell burden, decreased levels of leukemia-initiating cells, and improved survival. These data demonstrate the role of DHODH as a metabolic regulator of differentiation and point to its inhibition as a strategy for overcoming differentiation blockade in AML.

Elucidating Compound Mechanism of Action by Network Perturbation Analysis.

Genome-wide identification of the mechanism of action (MoA) of small-molecule compounds characterizing their targets, effectors, and activity modulators represents a highly relevant yet elusive goal, with critical implications for assessment of compound efficacy and toxicity. Current approaches are labor intensive and mostly limited to elucidating high-affinity binding target proteins. We introduce a regulatory network-based approach that elucidates genome-wide MoA proteins based on the assessment of the global dysregulation of their molecular interactions following compound perturbation. Analysis of cellular perturbation profiles identified established MoA proteins for 70% of the tested compounds and elucidated novel proteins that were experimentally validated. Finally, unknown-MoA compound analysis revealed altretamine, an anticancer drug, as an inhibitor of glutathione peroxidase 4 lipid repair activity, which was experimentally confirmed, thus revealing unexpected similarity to the activity of sulfasalazine. This suggests that regulatory network analysis can provide valuable mechanistic insight into the elucidation of small-molecule MoA and compound similarity.

Discovery of WRN inhibitor HRO761 with synthetic lethality in MSI cancers.

The Werner syndrome RecQ helicase WRN was identified as a synthetic lethal target in cancer cells with microsatellite instability (MSI) by several genetic screens1-6. Despite advances in treatment with immune checkpoint inhibitors7-10, there is an unmet need in the treatment of MSI cancers11-14. Here we report the structural, biochemical, cellular and pharmacological characterization of the clinical-stage WRN helicase inhibitor HRO761, which was identified through an innovative hit-finding and lead-optimization strategy. HRO761 is a potent, selective, allosteric WRN inhibitor that binds at the interface of the D1 and D2 helicase domains, locking WRN in an inactive conformation. Pharmacological inhibition by HRO761 recapitulated the phenotype observed by WRN genetic suppression, leading to DNA damage and inhibition of tumour cell growth selectively in MSI cells in a p53-independent manner. Moreover, HRO761 led to WRN degradation in MSI cells but not in microsatellite-stable cells. Oral treatment with HRO761 resulted in dose-dependent in vivo DNA damage induction and tumour growth inhibition in MSI cell- and patient-derived xenograft models. These findings represent preclinical pharmacological validation of WRN as a therapeutic target in MSI cancers. A clinical trial with HRO761 (NCT05838768) is ongoing to assess the safety, tolerability and preliminary anti-tumour activity in patients with MSI colorectal cancer and other MSI solid tumours.

A glimpse of structural biology through X-ray crystallography.

Since determination of the myoglobin structure in 1957, X-ray crystallography, as the anchoring tool of structural biology, has played an instrumental role in deciphering the secrets of life. Knowledge gained through X-ray crystallography has fundamentally advanced our views on cellular processes and greatly facilitated development of modern medicine. In this brief narrative, I describe my personal understanding of the evolution of structural biology through X-ray crystallography-using as examples mechanistic understanding of protein kinases and integral membrane proteins-and comment on the impact of technological development and outlook of X-ray crystallography.

MEN1 mutations mediate clinical resistance to menin inhibition.

Chromatin-binding proteins are critical regulators of cell state in haematopoiesis1,2. Acute leukaemias driven by rearrangement of the mixed lineage leukaemia 1 gene (KMT2Ar) or mutation of the nucleophosmin gene (NPM1) require the chromatin adapter protein menin, encoded by the MEN1 gene, to sustain aberrant leukaemogenic gene expression programs3-5. In a phase 1 first-in-human clinical trial, the menin inhibitor revumenib, which is designed to disrupt the menin-MLL1 interaction, induced clinical responses in patients with leukaemia with KMT2Ar or mutated NPM1 (ref. 6). Here we identified somatic mutations in MEN1 at the revumenib-menin interface in patients with acquired resistance to menin inhibition. Consistent with the genetic data in patients, inhibitor-menin interface mutations represent a conserved mechanism of therapeutic resistance in xenograft models and in an unbiased base-editor screen. These mutants attenuate drug-target binding by generating structural perturbations that impact small-molecule binding but not the interaction with the natural ligand MLL1, and prevent inhibitor-induced eviction of menin and MLL1 from chromatin. To our knowledge, this study is the first to demonstrate that a chromatin-targeting therapeutic drug exerts sufficient selection pressure in patients to drive the evolution of escape mutants that lead to sustained chromatin occupancy, suggesting a common mechanism of therapeutic resistance.

Synthesis of portimines reveals the basis of their anti-cancer activity.

Marine-derived cyclic imine toxins, portimine A and portimine B, have attracted attention because of their chemical structure and notable anti-cancer therapeutic potential1-4. However, access to large quantities of these toxins is currently not feasible, and the molecular mechanism underlying their potent activity remains unknown until now. To address this, a scalable and concise synthesis of portimines is presented, which benefits from the logic used in the two-phase terpenoid synthesis5,6 along with other tactics such as exploiting ring-chain tautomerization and skeletal reorganization to minimize protecting group chemistry through self-protection. Notably, this total synthesis enabled a structural reassignment of portimine B and an in-depth functional evaluation of portimine A, revealing that it induces apoptosis selectively in human cancer cell lines with high potency and is efficacious in vivo in tumour-clearance models. Finally, practical access to the portimines and their analogues simplified the development of photoaffinity analogues, which were used in chemical proteomic experiments to identify a primary target of portimine A as the 60S ribosomal export protein NMD3.

An interactive resource to identify cancer genetic and lineage dependencies targeted by small molecules.

The high rate of clinical response to protein-kinase-targeting drugs matched to cancer patients with specific genomic alterations has prompted efforts to use cancer cell line (CCL) profiling to identify additional biomarkers of small-molecule sensitivities. We have quantitatively measured the sensitivity of 242 genomically characterized CCLs to an Informer Set of 354 small molecules that target many nodes in cell circuitry, uncovering protein dependencies that: (1) associate with specific cancer-genomic alterations and (2) can be targeted by small molecules. We have created the Cancer Therapeutics Response Portal (http://www.broadinstitute.org/ctrp) to enable users to correlate genetic features to sensitivity in individual lineages and control for confounding factors of CCL profiling. We report a candidate dependency, associating activating mutations in the oncogene ß-catenin with sensitivity to the Bcl-2 family antagonist, navitoclax. The resource can be used to develop novel therapeutic hypotheses and to accelerate discovery of drugs matched to patients by their cancer genotype and lineage.

Pharmacological inactivation of Skp2 SCF ubiquitin ligase restricts cancer stem cell traits and cancer progression.

Skp2 E3 ligase is overexpressed in numerous human cancers and plays a critical role in cell-cycle progression, senescence, metabolism, cancer progression, and metastasis. In the present study, we identified a specific Skp2 inhibitor using high-throughput in silico screening of large and diverse chemical libraries. This Skp2 inhibitor selectively suppresses Skp2 E3 ligase activity, but not activity of other SCF complexes. It also phenocopies the effects observed upon genetic Skp2 deficiency, such as suppressing survival and Akt-mediated glycolysis and triggering p53-independent cellular senescence. Strikingly, we discovered a critical function of Skp2 in positively regulating cancer stem cell populations and self-renewal ability through genetic and pharmacological approaches. Notably, Skp2 inhibitor exhibits potent antitumor activities in multiple animal models and cooperates with chemotherapeutic agents to reduce cancer cell survival. Our study thus provides pharmacological evidence that Skp2 is a promising target for restricting cancer stem cell and cancer progression.

A microbial supply chain for production of the anti-cancer drug vinblastine.

Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.

Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

Interactome Rewiring Following Pharmacological Targeting of BET Bromodomains.

Targeting bromodomains (BRDs) of the bromo-and-extra-terminal (BET) family offers opportunities for therapeutic intervention in cancer and other diseases. Here, we profile the interactomes of BRD2, BRD3, BRD4, and BRDT following treatment with the pan-BET BRD inhibitor JQ1, revealing broad rewiring of the interaction landscape, with three distinct classes of behavior for the 603 unique interactors identified. A group of proteins associate in a JQ1-sensitive manner with BET BRDs through canonical and new binding modes, while two classes of extra-terminal (ET)-domain binding motifs mediate acetylation-independent interactions. Last, we identify an unexpected increase in several interactions following JQ1 treatment that define negative functions for BRD3 in the regulation of rRNA synthesis and potentially RNAPII-dependent gene expression that result in decreased cell proliferation. Together, our data highlight the contributions of BET protein modules to their interactomes allowing for a better understanding of pharmacological rewiring in response to JQ1.

Curvature-mediated rapid extravasation and penetration of nanoparticles against interstitial fluid pressure for improved drug delivery.

Elevated interstitial fluid pressure (IFP) within pathological tissues (e.g., tumors, obstructed kidneys, and cirrhotic livers) creates a significant hindrance to the transport of nanomedicine, ultimately impairing the therapeutic efficiency. Among these tissues, solid tumors present the most challenging scenario. While several strategies through reducing tumor IFP have been devised to enhance nanoparticle delivery, few approaches focus on modulating the intrinsic properties of nanoparticles to effectively counteract IFP during extravasation and penetration, which are precisely the stages obstructed by elevated IFP. Herein, we propose an innovative solution by engineering nanoparticles with a fusiform shape of high curvature, enabling efficient surmounting of IFP barriers during extravasation and penetration within tumor tissues. Through experimental and theoretical analyses, we demonstrate that the elongated nanoparticles with the highest mean curvature outperform spherical and rod-shaped counterparts against elevated IFP, leading to superior intratumoral accumulation and antitumor efficacy. Super-resolution microscopy and molecular dynamics simulations uncover the underlying mechanisms in which the high curvature contributes to diminished drag force in surmounting high-pressure differentials during extravasation. Simultaneously, the facilitated rotational movement augments the hopping frequency during penetration. This study effectively addresses the limitations posed by high-pressure impediments, uncovers the mutual interactions between the physical properties of NPs and their environment, and presents a promising avenue for advancing cancer treatment through nanomedicine.

Iridium(III) carbene complexes as potent girdin inhibitors against metastatic cancers.

Many cancer-driving protein targets remain undruggable due to a lack of binding molecular scaffolds. In this regard, octahedral metal complexes with unique and versatile three-dimensional structures have rarely been explored as inhibitors of undruggable protein targets. Here, we describe antitumor iridium(III) pyridinium-N-heterocyclic carbene complex 1a, which profoundly reduces the viability of lung and breast cancer cells as well as cancer patient-derived organoids at low micromolar concentrations. Compound 1a effectively inhibits the growth of non-small-cell lung cancer and triple-negative breast cancer xenograft tumors, impedes the metastatic spread of breast cancer cells, and can be modified into an antibody-drug conjugate payload to achieve precise tumor delivery in mice. Identified by thermal proteome profiling, an important molecular target of 1a in cellulo is Girdin, a multifunctional adaptor protein that is overexpressed in cancer cells and unequivocally serves as a signaling hub for multiple pivotal oncogenic pathways. However, specific small-molecule inhibitors of Girdin have not yet been developed. Notably, 1a exhibits high binding affinity to Girdin with a Kd of 1.3 µM and targets the Girdin-linked EGFR/AKT/mTOR/STAT3 cancer-driving pathway, inhibiting cancer cell proliferation and metastatic activity. Our study reveals a potent Girdin-targeting anticancer compound and demonstrates that octahedral metal complexes constitute an untapped library of small-molecule inhibitors that can fit into the ligand-binding pockets of key oncoproteins.