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Knudson's "two-hit" paradigm posits that carcinogenesis requires inactivation of both copies of an autosomal tumor suppressor gene. Here, we report that the glycolytic metabolite methylglyoxal (MGO) transiently bypasses Knudson's paradigm by inactivating the breast cancer suppressor protein BRCA2 to elicit a cancer-associated, mutational single-base substitution (SBS) signature in nonmalignant mammary cells or patient-derived organoids. Germline monoallelic BRCA2 mutations predispose to these changes. An analogous SBS signature, again without biallelic BRCA2 inactivation, accompanies MGO accumulation and DNA damage in Kras-driven, Brca2-mutant murine pancreatic cancers and human breast cancers. MGO triggers BRCA2 proteolysis, temporarily disabling BRCA2's tumor suppressive functions in DNA repair and replication, causing functional haploinsufficiency. Intermittent MGO exposure incites episodic SBS mutations without permanent BRCA2 inactivation. Thus, a metabolic mechanism wherein MGO-induced BRCA2 haploinsufficiency transiently bypasses Knudson's two-hit requirement could link glycolysis activation by oncogenes, metabolic disorders, or dietary challenges to mutational signatures implicated in cancer evolution.
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Proteína BRCA2 , Neoplasias de la Mama , Glucólisis , Piruvaldehído , Animales , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Ratones , Humanos , Femenino , Piruvaldehído/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Haploinsuficiencia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Mutación , Daño del ADN , Reparación del ADN , Línea Celular TumoralRESUMEN
Post-pregnancy breast cancer often carries a poor prognosis, posing a major clinical challenge. The increasing trend of later-life pregnancies exacerbates this risk, highlighting the need for effective chemoprevention strategies. Current options, limited to selective estrogen receptor modulators, aromatase inhibitors, or surgical procedures, offer limited efficacy and considerable side effects. Here, we report that cabergoline, a dopaminergic agonist, reduces the risk of breast cancer post-pregnancy in a Brca1/P53-deficient mouse model, with implications for human breast cancer prevention. We show that a single dose of cabergoline administered post-pregnancy significantly delayed the onset and reduced the incidence of breast cancer in Brca1/P53-deficient mice. Histological analysis revealed a notable acceleration in post-lactational involution over the short term, characterized by increased apoptosis and altered gene expression related to ion transport. Over the long term, histological changes in the mammary gland included a reduction in the ductal component, decreased epithelial proliferation, and a lower presence of recombinant Brca1/P53 target cells, which are precursors of tumors. These changes serve as indicators of reduced breast cancer susceptibility. Additionally, RNA sequencing identified gene expression alterations associated with decreased proliferation and mammary gland branching. Our findings highlight a mechanism wherein cabergoline enhances the protective effect of pregnancy against breast cancer by potentiating postlactational involution. Notably, a retrospective cohort study in women demonstrated a markedly lower incidence of post-pregnancy breast cancer in those treated with cabergoline compared to a control group. Our work underscores the importance of enhancing postlactational involution as a strategy for breast cancer prevention, and identifies cabergoline as a promising, low-risk option in breast cancer chemoprevention. This strategy has the potential to revolutionize breast cancer prevention approaches, particularly for women at increased risk due to genetic factors or delayed childbirth, and has wider implications beyond hereditary breast cancer cases.
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Germline BRCA2 mutation carriers frequently develop luminal-like breast cancers, but it remains unclear how BRCA2 mutations affect mammary epithelial subpopulations. Here, we report that monoallelic Brca2mut/WT mammary organoids subjected to replication stress activate a transcriptional response that selectively expands Brca2mut/WT luminal cells lacking hormone receptor expression (HR-). While CyTOF analyses reveal comparable epithelial compositions among wildtype and Brca2mut/WT mammary glands, Brca2mut/WT HR- luminal cells exhibit greater organoid formation and preferentially survive and expand under replication stress. ScRNA-seq analysis corroborates the expansion of HR- luminal cells which express elevated transcript levels of Tetraspanin-8 (Tspan8) and Thrsp, plus pathways implicated in replication stress survival including Type I interferon responses. Notably, CRISPR/Cas9-mediated deletion of Tspan8 or Thrsp prevents Brca2mut/WT HR- luminal cell expansion. Our findings indicate that Brca2mut/WT cells activate a transcriptional response after replication stress that preferentially favours outgrowth of HR- luminal cells through the expression of interferon-responsive and mammary alveolar genes.
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Células Epiteliales , Interferón Tipo I , Proliferación Celular , Ciclo Celular , Expresión GénicaRESUMEN
Glucose dependency of cancer cells can be targeted with a high-fat, low-carbohydrate ketogenic diet (KD). However, in IL-6-producing cancers, suppression of the hepatic ketogenic potential hinders the utilization of KD as energy for the organism. In IL-6-associated murine models of cancer cachexia, we describe delayed tumor growth but accelerated cachexia onset and shortened survival in mice fed KD. Mechanistically, this uncoupling is a consequence of the biochemical interaction of two NADPH-dependent pathways. Within the tumor, increased lipid peroxidation and, consequently, saturation of the glutathione (GSH) system lead to the ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impair corticosterone biosynthesis. Administration of dexamethasone, a potent glucocorticoid, increases food intake, normalizes glucose levels and utilization of nutritional substrates, delays cachexia onset, and extends the survival of tumor-bearing mice fed KD while preserving reduced tumor growth. Our study emphasizes the need to investigate the effects of systemic interventions on both the tumor and the host to accurately assess therapeutic potential. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.
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Dieta Cetogénica , Ferroptosis , Neoplasias , Ratones , Animales , Caquexia , Corticosterona , Interleucina-6 , NADP , Cuerpos Cetónicos , Glucosa , Neoplasias/complicacionesRESUMEN
Mis-sense mutations affecting TP53 promote carcinogenesis both by inactivating tumor suppression, and by conferring pro-carcinogenic activities. We report here that p53 DNA-binding domain (DBD) and transactivation domain (TAD) mis-sense mutants unexpectedly activate pro-carcinogenic epidermal growth factor receptor (EGFR) signaling via distinct, previously unrecognized molecular mechanisms. DBD- and TAD-specific TP53 mutants exhibited different cellular localization and induced distinct gene expression profiles. In multiple tissues, EGFR is stabilized by TAD and DBD mutants in the cytosolic and nuclear compartments respectively. TAD mutants promote EGFR-mediated signaling by enhancing EGFR interaction with AKT via DDX31 in the cytosol. Conversely, DBD mutants maintain EGFR activity in the nucleus, by blocking EGFR interaction with the phosphatase SHP1, triggering c-Myc and Cyclin D1 upregulation. Our findings suggest that p53 mutants carrying gain-of-function, mis-sense mutations affecting two different domains form new protein complexes that promote carcinogenesis by enhancing EGFR signaling via distinctive mechanisms, exposing clinically relevant therapeutic vulnerabilities.
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Receptores ErbB , Proteína p53 Supresora de Tumor , Proteína p53 Supresora de Tumor/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transducción de Señal , Activación Transcripcional , FosforilaciónRESUMEN
The dependency of cancer cells on glucose can be targeted with high-fat low-carbohydrate ketogenic diet (KD). However, hepatic ketogenesis is suppressed in IL-6 producing cancers, which prevents the utilization of this nutrient source as energy for the organism. In two IL-6 associated murine models of cancer cachexia we describe delayed tumor growth but accelerated onset of cancer cachexia and shortened survival when mice are fed KD. Mechanistically, we find this uncoupling is a consequence of the biochemical interaction of two simultaneously occurring NADPH-dependent pathways. Within the tumor, increased production of lipid peroxidation products (LPPs) and, consequently, saturation of the glutathione (GSH) system leads to ferroptotic death of cancer cells. Systemically, redox imbalance and NADPH depletion impairs the biosynthesis of corticosterone, the main regulator of metabolic stress, in the adrenal glands. Administration of dexamethasone, a potent glucocorticoid, improves food intake, normalizes glucose homeostasis and utilization of nutritional substrates, delays onset of cancer cachexia and extends survival of tumor-bearing mice fed KD, while preserving reduced tumor growth. Our study highlights that the outcome of systemic interventions cannot necessarily be extrapolated from the effect on the tumor alone, but that they have to be investigated for anti-cancer and host effects. These findings may be relevant to clinical research efforts that investigate nutritional interventions such as KD in patients with cancer.
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CK2 is a ubiquitous protein kinase with an anti-apoptotic role and is found to be overexpressed in multiple cancer types. To this end, the inhibition of CK2 is of great interest with regard to the development of novel anti-cancer therapeutics. ATP-site inhibition of CK2 is possible; however, this typically results in poor selectivity due to the highly conserved nature of the catalytic site amongst kinases. An alternative methodology for the modulation of CK2 activity is through allosteric inhibition. The recently identified αD site represents a promising binding site for allosteric inhibition of CK2α. The work presented herein describes the development of a series of CK2α allosteric inhibitors through iterative cycles of X-ray crystallography and enzymatic assays, in addition to both fragment growing and fragment merging design strategies. The lead fragment developed, fragment 8, exhibits a high ligand efficiency, displays no drop off in activity between enzymatic and cellular assays, and successfully engages CK2α in cells. Furthermore, X-ray crystallographic analysis provided indications towards a novel mechanism of allosteric inhibition through αD site binding. Fragments described in this paper therefore represent promising starting points for the development of highly selective allosteric CK2 inhibitors.
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SignificanceOur work focuses on the critical longstanding question of the nontranscriptional role of p53 in tumor suppression. We demonstrate here that poly(ADP-ribose) polymerase (PARP)-dependent modification of p53 enables rapid recruitment of p53 to damage sites, where it in turn directs early repair pathway selection. Specifically, p53-mediated recruitment of 53BP1 at early time points promotes nonhomologous end joining over the more error-prone microhomology end-joining. Similarly, p53 directs nucleotide excision repair by mediating DDB1 recruitment. This property of p53 also correlates with tumor suppression in vivo. Our study provides mechanistic insight into how certain transcriptionally deficient p53 mutants may retain tumor-suppressive functions through regulating the DNA damage response.
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Daño del ADN , Reparación del ADN por Unión de Extremidades , Proteína p53 Supresora de Tumor/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Proteínas de Unión al ADN , Humanos , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos , Proteína p53 Supresora de Tumor/genética , Proteína 1 de Unión al Supresor Tumoral P53/genéticaRESUMEN
An elevated neutrophil-lymphocyte ratio negatively predicts the outcome of patients with cancer and is associated with cachexia, the terminal wasting syndrome. Here, using murine model systems of colorectal and pancreatic cancer we show that neutrophilia in the circulation and multiple organs, accompanied by extramedullary hematopoiesis, is an early event during cancer progression. Transcriptomic and metabolic assessment reveals that neutrophils in tumor-bearing animals utilize aerobic glycolysis, similar to cancer cells. Although pharmacological inhibition of aerobic glycolysis slows down tumor growth in C26 tumor-bearing mice, it precipitates cachexia, thereby shortening the overall survival. This negative effect may be explained by our observation that acute depletion of neutrophils in pre-cachectic mice impairs systemic glucose homeostasis secondary to altered hepatic lipid processing. Thus, changes in neutrophil number, distribution, and metabolism play an adaptive role in host metabolic homeostasis during cancer progression. Our findings provide insight into early events during cancer progression to cachexia, with implications for therapy.
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Oxidative stress is a ubiquitous cellular challenge implicated in aging, neurodegeneration, and cancer. By studying pathogenic mutations in the tumor suppressor BRCA2, we identify a general mechanism by which oxidative stress restricts mitochondrial (mt)DNA replication. BRCA2 inactivation induces R-loop accumulation in the mtDNA regulatory region and diminishes mtDNA replication initiation. In BRCA2-deficient cells, intracellular reactive oxygen species (ROS) are elevated, and ROS scavengers suppress the mtDNA defects. Conversely, wild-type cells exposed to oxidative stress by pharmacologic or genetic manipulation phenocopy these defects. Mechanistically, we find that 8-oxoguanine accumulation in mtDNA caused by oxidative stress suffices to impair recruitment of the mitochondrial enzyme RNaseH1 to sites of R-loop accrual, restricting mtDNA replication initiation. Thus, oxidative stress impairs RNaseH1 function to cripple mtDNA maintenance. Our findings highlight a molecular mechanism that links oxidative stress to mitochondrial dysfunction and is elicited by the inactivation of genes implicated in neurodegeneration and cancer.
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Proteína BRCA2/deficiencia , ADN Mitocondrial/genética , Estrés Oxidativo , Ribonucleasa H/metabolismo , Proteína BRCA2/metabolismo , ADN Glicosilasas/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , ADN Mitocondrial/química , Femenino , Guanina/análogos & derivados , Guanina/metabolismo , Células HeLa , Humanos , Mitocondrias/metabolismo , Mitocondrias/patología , Enzimas Multifuncionales/metabolismo , Estructuras R-Loop , ARN Helicasas/metabolismo , Proteínas de Unión al ARN/metabolismoRESUMEN
Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify "protein interference," an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.
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Descubrimiento de Drogas , Proteína Forkhead Box O3/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Células Cultivadas , Femenino , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Genes Supresores de Tumor/efectos de los fármacos , Humanos , Biblioteca de Péptidos , Fosforilación , Bibliotecas de Moléculas Pequeñas/químicaRESUMEN
Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (â¼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.
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Proteína BRCA2 , Neoplasias de la Mama/genética , Reparación del ADN , Neoplasias Ováricas/genética , Complejo de la Endopetidasa Proteasomal/fisiología , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Femenino , Células HEK293 , Células HeLa , Recombinación Homóloga , Humanos , Mutación Missense , Unión ProteicaRESUMEN
BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
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Proteína BRCA2/antagonistas & inhibidores , Recombinasa Rad51/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Muerte Celular/efectos de los fármacos , Cristalografía por Rayos X , Daño del ADN , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica/efectos de los fármacos , Recombinasa Rad51/química , Recombinasa Rad51/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Células Tumorales CultivadasRESUMEN
How oxidative stress promotes aging-related human diseases like cancer and neurodegeneration remains unclear. Here, we discuss the origins and implications of an oxidative-stress response recently reported to destabilize the mitochondrial (mt) genome via unscheduled RNA/DNA hybrid (R-loop) accumulation, by impairing the recruitment of RNAseH1 to the regulatory regions of mtDNA.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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The catalytic activity of human AURORA-A kinase (AURKA) regulates mitotic progression, and its frequent overexpression in major forms of epithelial cancer is associated with aneuploidy and carcinogenesis. Here, we report an unexpected, kinase-independent function for AURKA in DNA replication initiation whose inhibition through a class of allosteric inhibitors opens avenues for cancer therapy. We show that genetic depletion of AURKA, or its inhibition by allosteric but not catalytic inhibitors, blocks the G1-S cell cycle transition. A catalytically inactive AURKA mutant suffices to overcome this block. We identify a multiprotein complex between AURKA and the replisome components MCM7, WDHD1 and POLD1 formed during G1, and demonstrate that allosteric but not catalytic inhibitors prevent the chromatin assembly of functional replisomes. Indeed, allosteric but not catalytic AURKA inhibitors sensitize cancer cells to inhibition of the CDC7 kinase subunit of the replication-initiating factor DDK. Thus, our findings define a mechanism essential for replisome assembly during DNA replication initiation that is vulnerable to inhibition as combination therapy in cancer.
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Aurora Quinasa A/fisiología , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Complejos Multienzimáticos/metabolismo , Regulación Alostérica , Aurora Quinasa A/antagonistas & inhibidores , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/antagonistas & inhibidores , Línea Celular , Puntos de Control de la Fase G1 del Ciclo Celular , Células HeLa , Humanos , Interfase/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Origen de RéplicaRESUMEN
Gain of function (GOF) DNA binding domain (DBD) mutations of TP53 upregulate chromatin regulatory genes that promote genome-wide histone methylation and acetylation. Here, we therapeutically exploit the oncogenic GOF mechanisms of p53 codon 158 (Arg158) mutation, a DBD mutant found to be prevalent in lung carcinomas. Using high throughput compound screening and combination analyses, we uncover that acetylating mutp53R158G could render cancers susceptible to cisplatin-induced DNA stress. Acetylation of mutp53R158G alters DNA binding motifs and upregulates TRAIP, a RING domain-containing E3 ubiquitin ligase which dephosphorylates IĸB and impedes nuclear translocation of RelA (p65), thus repressing oncogenic nuclear factor kappa-B (NF-ĸB) signaling and inducing apoptosis. Given that this mechanism of cytotoxic vulnerability appears inapt in p53 wild-type (WT) or other hotspot GOF mutp53 cells, our work provides a therapeutic opportunity specific to Arg158-mutp53 tumors utilizing a regimen consisting of DNA-damaging agents and mutp53 acetylators, which is currently being pursued clinically.
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Codón/genética , Mutación/genética , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Acetilación/efectos de los fármacos , Animales , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cisplatino/farmacología , Epigénesis Genética/efectos de los fármacos , Mutación con Ganancia de Función/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ácidos Hidroxámicos/farmacología , Ratones SCID , Modelos Biológicos , Proteínas Mutantes/metabolismo , FN-kappa B/metabolismo , Neoplasias/tratamiento farmacológico , Motivos de Nucleótidos/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Unión Proteica/efectos de los fármacos , Isoformas de Proteínas/genética , Sulfonamidas/farmacología , Topotecan/farmacología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cell-autonomous changes in p53 expression govern the duration and outcome of cell-cycle arrest at the G2 checkpoint for DNA damage. Here, we report that mitogen-activated protein kinase (MAPK) signaling integrates extracellular cues with p53 dynamics to determine cell fate at the G2 checkpoint. Optogenetic tools and quantitative cell biochemistry reveal transient oscillations in MAPK activity dependent on ataxia-telangiectasia-mutated kinase after DNA damage. MAPK inhibition alters p53 dynamics and p53-dependent gene expression after checkpoint enforcement, prolonging G2 arrest. In contrast, sustained MAPK signaling induces the phosphorylation of CDC25C, and consequently, the accumulation of pro-mitotic kinases, thereby relaxing checkpoint stringency and permitting cells to evade prolonged G2 arrest and senescence induction. We propose a model in which this MAPK-mediated mechanism integrates extracellular cues with cell-autonomous p53-mediated signals, to safeguard genomic integrity during tissue proliferation. Early steps in oncogene-driven carcinogenesis may imbalance this tumor-suppressive mechanism to trigger genome instability.
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Daño del ADN , Sistema de Señalización de MAP Quinasas , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/fisiología , Expresión Génica , Humanos , Células MCF-7 , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Polo-like kinase 1 (PLK1) is a key regulator of mitosis and a recognized drug target for cancer therapy. Inhibiting the polo-box domain of PLK1 offers potential advantages of increased selectivity and subsequently reduced toxicity compared with targeting the kinase domain. However, many if not all existing polo-box domain inhibitors have been shown to be unsuitable for further development. In this paper, we describe a novel compound series, which inhibits the protein-protein interactions of PLK1 via the polo-box domain. We combine high throughput screening with molecular modeling and computer-aided design, synthetic chemistry, and cell biology to address some of the common problems with protein-protein interaction inhibitors, such as solubility and potency. We use molecular modeling to improve the solubility of a hit series with initially poor physicochemical properties, enabling biophysical and biochemical characterization. We isolate and characterize enantiomers to improve potency and demonstrate on-target activity in both cell-free and cell-based assays, entirely consistent with the proposed binding model. The resulting compound series represents a promising starting point for further progression along the drug discovery pipeline and a new tool compound to study kinase-independent PLK functions.