Mouse models to investigate in situ cell fate decisions induced by p53.

Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo.

Clustered de novo start-loss variants in GLUL result in a developmental and epileptic encephalopathy via stabilization of glutamine synthetase.

Glutamine synthetase (GS), encoded by GLUL, catalyzes the conversion of glutamate to glutamine. GS is pivotal for the generation of the neurotransmitters glutamate and gamma-aminobutyric acid and is the primary mechanism of ammonia detoxification in the brain. GS levels are regulated post-translationally by an N-terminal degron that enables the ubiquitin-mediated degradation of GS in a glutamine-induced manner. GS deficiency in humans is known to lead to neurological defects and death in infancy, yet how dysregulation of the degron-mediated control of GS levels might affect neurodevelopment is unknown. We ascertained nine individuals with severe developmental delay, seizures, and white matter abnormalities but normal plasma and cerebrospinal fluid biochemistry with de novo variants in GLUL. Seven out of nine were start-loss variants and two out of nine disrupted 5' UTR splicing resulting in splice exclusion of the initiation codon. Using transfection-based expression systems and mass spectrometry, these variants were shown to lead to translation initiation of GS from methionine 18, downstream of the N-terminal degron motif, resulting in a protein that is stable and enzymatically competent but insensitive to negative feedback by glutamine. Analysis of human single-cell transcriptomes demonstrated that GLUL is widely expressed in neuro- and glial-progenitor cells and mature astrocytes but not in post-mitotic neurons. One individual with a start-loss GLUL variant demonstrated periventricular nodular heterotopia, a neuronal migration disorder, yet overexpression of stabilized GS in mice using in utero electroporation demonstrated no migratory deficits. These findings underline the importance of tight regulation of glutamine metabolism during neurodevelopment in humans.

Direct Observation of Enhanced Iodine Binding within a Series of Functionalized Metal-Organic Frameworks with Exceptional Irradiation Stability.

Optimization of active sites and stability under irradiation are important targets for sorbent materials that might be used for iodine (I2) storage. Herein, we report the direct observation of I2 binding in a series of Cu(II)-based isostructural metal-organic frameworks, MFM-170, MFM-172, MFM-174, NJU-Bai20, and NJU-Bai21, incorporating various functional groups (-H, -CH3, - NH2, -C≡C-, and -CONH-, respectively). MFM-170 shows a reversible uptake of 3.37 g g-1 and a high packing density of 4.41 g cm-3 for physiosorbed I2. The incorporation of -NH2 and -C≡C- moieties in MFM-174 and NJU-Bai20, respectively, enhances the binding of I2, affording uptakes of up to 3.91 g g-1. In addition, an exceptional I2 packing density of 4.83 g cm-3 is achieved in MFM-174, comparable to that of solid iodine (4.93 g cm-3). In situ crystallographic studies show the formation of a range of supramolecular and chemical interactions [I···N, I···H2N] and [I···C≡C, I-C═C-I] between -NH2, -C≡C- sites, respectively, and adsorbed I2 molecules. These observations have been confirmed via a combination of solid-state nuclear magnetic resonance, X-ray photoelectron, and Raman spectroscopies. Importantly, γ-irradiation confirmed the ultraresistance of MFM-170, MFM-174, and NJU-Bai20 suggesting their potential as efficient sorbents for cleanup of radioactive waste.

Integrating multiple lines of evidence to assess the effects of maternal BMI on pregnancy and perinatal outcomes.

BACKGROUND: Higher maternal pre-pregnancy body mass index (BMI) is associated with adverse pregnancy and perinatal outcomes. However, whether these associations are causal remains unclear. METHODS: We explored the relation of maternal pre-/early-pregnancy BMI with 20 pregnancy and perinatal outcomes by integrating evidence from three different approaches (i.e. multivariable regression, Mendelian randomisation, and paternal negative control analyses), including data from over 400,000 women. RESULTS: All three analytical approaches supported associations of higher maternal BMI with lower odds of maternal anaemia, delivering a small-for-gestational-age baby and initiating breastfeeding, but higher odds of hypertensive disorders of pregnancy, gestational hypertension, preeclampsia, gestational diabetes, pre-labour membrane rupture, induction of labour, caesarean section, large-for-gestational age, high birthweight, low Apgar score at 1 min, and neonatal intensive care unit admission. For example, higher maternal BMI was associated with higher risk of gestational hypertension in multivariable regression (OR = 1.67; 95% CI = 1.63, 1.70 per standard unit in BMI) and Mendelian randomisation (OR = 1.59; 95% CI = 1.38, 1.83), which was not seen for paternal BMI (OR = 1.01; 95% CI = 0.98, 1.04). Findings did not support a relation between maternal BMI and perinatal depression. For other outcomes, evidence was inconclusive due to inconsistencies across the applied approaches or substantial imprecision in effect estimates from Mendelian randomisation. CONCLUSIONS: Our findings support a causal role for maternal pre-/early-pregnancy BMI on 14 out of 20 adverse pregnancy and perinatal outcomes. Pre-conception interventions to support women maintaining a healthy BMI may reduce the burden of obstetric and neonatal complications. FUNDING: Medical Research Council, British Heart Foundation, European Research Council, National Institutes of Health, National Institute for Health Research, Research Council of Norway, Wellcome Trust.

Social inequalities in pregnancy metabolic profile: findings from the multi-ethnic Born in Bradford cohort study.

BACKGROUND: Lower socioeconomic position (SEP) associates with adverse pregnancy and perinatal outcomes and with less favourable metabolic profile in nonpregnant adults. Socioeconomic differences in pregnancy metabolic profile are unknown. We investigated association between a composite measure of SEP and pregnancy metabolic profile in White European (WE) and South Asian (SA) women. METHODS: We included 3,905 WE and 4,404 SA pregnant women from a population-based UK cohort. Latent class analysis was applied to nineteen individual, household, and area-based SEP indicators (collected by questionnaires or linkage to residential address) to derive a composite SEP latent variable. Targeted nuclear magnetic resonance spectroscopy was used to determine 148 metabolic traits from mid-pregnancy serum samples. Associations between SEP and metabolic traits were examined using linear regressions adjusted for gestational age and weighted by latent class probabilities. RESULTS: Five SEP sub-groups were identified and labelled 'Highest SEP' (48% WE and 52% SA), 'High-Medium SEP' (77% and 23%), 'Medium SEP' (56% and 44%) 'Low-Medium SEP' (21% and 79%), and 'Lowest SEP' (52% and 48%). Lower SEP was associated with more adverse levels of 113 metabolic traits, including lower high-density lipoprotein (HDL) and higher triglycerides and very low-density lipoprotein (VLDL) traits. For example, mean standardized difference (95%CI) in concentration of small VLDL particles (vs. Highest SEP) was 0.12 standard deviation (SD) units (0.05 to 0.20) for 'Medium SEP' and 0.25SD (0.18 to 0.32) for 'Lowest SEP'. There was statistical evidence of ethnic differences in associations of SEP with 31 traits, primarily characterised by stronger associations in WE women e.g., mean difference in HDL cholesterol in WE and SA women respectively (vs. Highest-SEP) was -0.30SD (-0.41 to -0.20) and -0.16SD (-0.27 to -0.05) for 'Medium SEP', and -0.62SD (-0.72 to -0.52) and -0.29SD (-0.40 to -0.20) for 'Lowest SEP'. CONCLUSIONS: We found widespread socioeconomic differences in metabolic traits in pregnant WE and SA women residing in the UK. Further research is needed to understand whether the socioeconomic differences we observe here reflect pre-conception differences or differences in the metabolic pregnancy response. If replicated, it would be important to explore if these differences contribute to socioeconomic differences in pregnancy outcomes.

The Discovery of New Inhibitors of Insulin-Regulated Aminopeptidase by a High-Throughput Screening of 400,000 Drug-like Compounds.

With the ambition to identify novel chemical starting points that can be further optimized into small drug-like inhibitors of insulin-regulated aminopeptidase (IRAP) and serve as potential future cognitive enhancers in the clinic, we conducted an ultra-high-throughput screening campaign of a chemically diverse compound library of approximately 400,000 drug-like small molecules. Three biochemical and one biophysical assays were developed to enable large-scale screening and hit triaging. The screening funnel, designed to be compatible with high-density microplates, was established with two enzyme inhibition assays employing either fluorescent or absorbance readouts. As IRAP is a zinc-dependent enzyme, the remaining active compounds were further evaluated in the primary assay, albeit with the addition of zinc ions. Rescreening with zinc confirmed the inhibitory activity for most compounds, emphasizing a zinc-independent mechanism of action. Additionally, target engagement was confirmed using a complementary biophysical thermal shift assay where compounds causing positive/negative thermal shifts were considered genuine binders. Triaging based on biochemical activity, target engagement, and drug-likeness resulted in the selection of 50 qualified hits, of which the IC50 of 32 compounds was below 3.5 µM. Despite hydroxamic acid dominance, diverse chemotypes with biochemical activity and target engagement were discovered, including non-hydroxamic acid compounds. The most potent compound (QHL1) was resynthesized with a confirmed inhibitory IC50 of 320 nM. Amongst these compounds, 20 new compound structure classes were identified, providing many new starting points for the development of unique IRAP inhibitors. Detailed characterization and optimization of lead compounds, considering both hydroxamic acids and other diverse structures, are in progress for further exploration.

The impact of reproductive factors on the metabolic profile of females from menarche to menopause.

We explore the relation between age at menarche, parity and age at natural menopause with 249 metabolic traits in over 65,000 UK Biobank women using multivariable regression, Mendelian randomization and negative control (parity only). Older age of menarche is related to a less atherogenic metabolic profile in multivariable regression and Mendelian randomization, which is largely attenuated when accounting for adult body mass index. In multivariable regression, higher parity relates to more particles and lipids in VLDL, which are not observed in male negative controls. In multivariable regression and Mendelian randomization, older age at natural menopause is related to lower concentrations of inflammation markers, but we observe inconsistent results for LDL-related traits due to chronological age-specific effects. For example, older age at menopause is related to lower LDL-cholesterol in younger women but slightly higher in older women. Our findings support a role of reproductive traits on later life metabolic profile and provide insights into identifying novel markers for the prevention of adverse cardiometabolic outcomes in women.

Differences in menopausal quality of life, body appreciation, and body dissatisfaction between women at high and low risk of an eating disorder.

OBJECTIVE: Experiences of menopause and quality of life during menopause can vary extensively among women. While menopause has been associated with negative impacts on eating and body image, it is unclear to what extent quality of life differs by eating disorder risk status. The aim of this study was to explore how menopause symptoms and quality of life differ between those women at high- or low-risk of an eating disorder and the potential protective role of body appreciation. METHOD: This cross-sectional survey study explored differences in menopausal quality of life, body appreciation, and body dissatisfaction among women classified as high- or low-risk of an eating disorder as part of a wider survey on aging, health, and psychological complaints during midlife. Participants were 255 females aged between 40 and 60 years. Participants were classified as high-risk and low-risk of an eating disorder based on Eating Attitudes Test-26 (EAT-26) scores. Differences between groups on the Menopause-Specific Quality of Life Questionnaire (MENQOL), Body Shape Questionnaire (BSQ-16), and Body Appreciation Scale-2 were analyzed. The predictive relationship between menopausal quality of life and body appreciation was also explored. RESULTS: Participants in the high-risk group (n = 111) reported significantly poorer menopausal quality of life compared to the low-risk group (n = 144), scoring significantly higher on the sexual, physical, and psychosocial subscales of the MENQOL. The high-risk group also had significantly greater body dissatisfaction and less body appreciation than the low-risk group. Overall, menopausal quality of life was a significant predictor of body appreciation. DISCUSSION: Women with greater eating disorder risk may be faring less well with menopause. Treating and preventing menopause-related eating disorders will benefit from interventions aimed at not only reducing body dissatisfaction, but actively bolstering body appreciation and supporting the sexual, physical, and psychosocial aspects of the menopausal transition.

Multimodal framework to resolve variants of uncertain significance in TSC2.

Efforts to resolve the functional impact of variants of uncertain significance (VUS) have lagged behind the identification of new VUS; as such, there is a critical need for scalable VUS resolution technologies. Computational variant effect predictors (VEPs), once trained, can predict pathogenicity for all missense variants in a gene, set of genes, or the exome. Existing tools have employed information on known pathogenic and benign variants throughout the genome to predict pathogenicity of VUS. We hypothesize that taking a gene-specific approach will improve pathogenicity prediction over globally-trained VEPs. We tested this hypothesis using the gene TSC2, whose loss of function results in tuberous sclerosis, a multisystem mTORopathy affecting about 1 in 6,000 individuals born in the United States. TSC2 has been identified as a high-priority target for VUS resolution, with (1) well-characterized molecular and patient phenotypes associated with loss-of-function variants, and (2) more than 2,700 VUS already documented in ClinVar. We developed Tuberous sclerosis classifier to Resolve variants of Uncertain Significance in T SC2 (TRUST), a machine learning model to predict pathogenicity of TSC2 missense VUS. To test whether these predictions are accurate, we further introduce curated loci prime editing (cliPE) as an accessible strategy for performing scalable multiplexed assays of variant effect (MAVEs). Using cliPE, we tested the effects of more than 200 TSC2 variants, including 106 VUS. It is highly likely this functional data alone would be sufficient to reclassify 92 VUS with most being reclassified as likely benign. We found that TRUST's classifications were correlated with the functional data, providing additional validation for the in silico predictions. We provide our pathogenicity predictions and MAVE data to aid with VUS resolution. In the near future, we plan to host these data on a public website and deposit into relevant databases such as MAVEdb as a community resource. Ultimately, this study provides a framework to complete variant effect maps of TSC1 and TSC2 and adapt this approach to other mTORopathy genes.

Prospects for the computational humanization of antibodies and nanobodies.

To be viable therapeutics, antibodies must be tolerated by the human immune system. Rational approaches to reduce the risk of unwanted immunogenicity involve maximizing the 'humanness' of the candidate drug. However, despite the emergence of new discovery technologies, many of which start from entirely human gene fragments, most antibody therapeutics continue to be derived from non-human sources with concomitant humanization to increase their human compatibility. Early experimental humanization strategies that focus on CDR loop grafting onto human frameworks have been critical to the dominance of this discovery route but do not consider the context of each antibody sequence, impacting their success rate. Other challenges include the simultaneous optimization of other drug-like properties alongside humanness and the humanization of fundamentally non-human modalities such as nanobodies. Significant efforts have been made to develop in silico methodologies able to address these issues, most recently incorporating machine learning techniques. Here, we outline these recent advancements in antibody and nanobody humanization, focusing on computational strategies that make use of the increasing volume of sequence and structural data available and the validation of these tools. We highlight that structural distinctions between antibodies and nanobodies make the application of antibody-focused in silico tools to nanobody humanization non-trivial. Furthermore, we discuss the effects of humanizing mutations on other essential drug-like properties such as binding affinity and developability, and methods that aim to tackle this multi-parameter optimization problem.

'Building our own house' as an insider-only Community-Partnered Participatory Research Council: Co-creating a safe space for Autistic knowledge production.

LAY ABSTRACT: In recent years, there has been a growing call for participatory Autism research (i.e. research that meaningfully involves Autistic people in its design and delivery). Community Partnered Participatory Research is a research methodology that aims to share power between researchers and members of the researched community. There is some precedent for Community Partnered Participatory Research in Autism research, but it is still quite uncommon. At the start of our new research study (called Autism: From Menstruation to Menopause), we created a community council. For the first six meetings, our council was made up of four Autistic community members who were experienced in Autism advocacy and activism and three Autistic researchers. We seven are the authors of this article. In these first six meetings, we made plans for recruiting a larger number of lay community members who would join us later for the rest of the project (8 years in total). In this article, we describe and reflect what it felt like during these first six meetings to be part of a community research council where everybody is Autistic. We discuss how we co-created a safe space, how we helped each other feel valued and how we worked together to support each other's sometimes-differing access needs so that everyone could fully participate. We provide recommendations for how to support Autistic people to lead research on their own terms with their unique insights.

A metabolic redox relay supports ER proinsulin export in pancreatic islet ß cells.

ER stress and proinsulin misfolding are heralded as contributing factors to ß cell dysfunction in type 2 diabetes, yet how ER function becomes compromised is not well understood. Recent data identify altered ER redox homeostasis as a critical mechanism that contributes to insulin granule loss in diabetes. Hyperoxidation of the ER delays proinsulin export and limits the proinsulin supply available for insulin granule formation. In this report, we identified glucose metabolism as a critical determinant in the redox homeostasis of the ER. Using multiple ß cell models, we showed that loss of mitochondrial function or inhibition of cellular metabolism elicited ER hyperoxidation and delayed ER proinsulin export. Our data further demonstrated that ß cell ER redox homeostasis was supported by the metabolic supply of reductive redox donors. We showed that limiting NADPH and thioredoxin flux delayed ER proinsulin export, whereas thioredoxin-interacting protein suppression restored ER redox and proinsulin trafficking. Taken together, we propose that ß cell ER redox homeostasis is buffered by cellular redox donor cycles, which are maintained through active glucose metabolism.

Development and proof-of-concept demonstration of a clinical metagenomics method for the rapid detection of bloodstream infection.

BACKGROUND: The timely and accurate diagnosis of bloodstream infection (BSI) is critical for patient management. With longstanding challenges for routine blood culture, metagenomics is a promising approach to rapidly provide sequence-based detection and characterisation of bloodborne bacteria. Long-read sequencing technologies have successfully supported the use of clinical metagenomics for syndromes such as respiratory illness, and modified approaches may address two requisite factors for metagenomics to be used as a BSI diagnostic: depletion of the high level of host DNA to then detect the low abundance of microbes in blood. METHODS: Blood samples from healthy donors were spiked with different concentrations of four prevalent causative species of BSI. All samples were then subjected to a modified saponin-based host DNA depletion protocol and optimised DNA extraction, whole genome amplification and debranching steps in preparation for sequencing, followed by bioinformatical analyses. Two related variants of the protocol are presented: 1mL of blood processed without bacterial enrichment, and 5mL of blood processed following a rapid bacterial enrichment protocol-SepsiPURE. RESULTS: After first identifying that a large proportion of host mitochondrial DNA remained, the host depletion process was optimised by increasing saponin concentration to 3% and scaling the reaction to allow more sample volume. Compared to non-depleted controls, the 3% saponin-based depletion protocol reduced the presence of host chromosomal and mitochondrial DNA < 106 and < 103 fold respectively. When the modified depletion method was further combined with a rapid bacterial enrichment method (SepsiPURE; with 5mL blood samples) the depletion of mitochondrial DNA improved by a further > 10X while also increasing detectable bacteria by > 10X. Parameters during DNA extraction, whole genome amplification and long-read sequencing were also adjusted, and subsequently amplicons were detected for each input bacterial species at each of the spiked concentrations, ranging from 50-100 colony forming units (CFU)/mL to 1-5 CFU/mL. CONCLUSION: In this proof-of-concept study, four prevalent BSI causative species were detected in under 12 h to species level (with antimicrobial resistance determinants) at concentrations relevant to clinical blood samples. The use of a rapid and precise metagenomic protocols has the potential to advance the diagnosis of BSI.

Risk Factors and Clinical Prognosis Associated With RSV-ALRI Intensive Care Unit Admission in Children <2 Years of Age: A Multicenter Study.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infections (ALRIs) in children <2 years of age. Currently, there are limited data on risk factors for very severe RSV-ALRI requiring intensive care unit (ICU) admission. METHODS: We conducted a case-control study of children <2 years old admitted with RSV-ALRI to the Sydney Children's Hospital Network, comprising 2 large tertiary pediatric hospitals. Cases were children with laboratory-confirmed RSV-ALRI admitted to ICU, and controls were (1:2, matched on date of admission) children hospitalized with RSV-ALRI but not requiring ICU transfer. Data on risk factors were retrieved from the electronic medical record system. Adjusted odds ratios (aORs) with 95% confidence intervals (95% CI) associated with risk factors for ICU admission and the association with clinical and treatment factors were determined from logistic regression models. RESULTS: A total of 44 (44%) of 100 cases and 90 (48.1%) of 187 controls were male. Age <6 months and preterm births were associated with a 2.10-fold (95% CI: 1.14-3.79) and 2.35-fold (95% CI: 1.26-4.41) increased risk in ICU admissions, respectively. The presence of any chronic health condition was a significant risk factor for ICU admission. The clinical presentations on admission more commonly seen in cases were apnea (aOR: 5.01, 95% CI: 1.50-17.13) and respiratory distress (aOR: 15.91, 95% CI: 4.52-55.97). Cases were more likely to be hospitalized for longer duration and require respiratory support. CONCLUSIONS: Our results can be translated into a clinical risk algorithm to identify children at risk of very severe RSV disease.

Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses.

Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.

TOLLIP inhibits lipid accumulation and the integrated stress response in alveolar macrophages to control Mycobacterium tuberculosis infection.

A polymorphism causing deficiencies in Toll-interacting protein (TOLLIP), an inhibitory adaptor protein affecting endosomal trafficking, is associated with increased tuberculosis (TB) risk. It is, however, unclear how TOLLIP affects TB pathogenesis. Here we show that TB severity is increased in Tollip-/- mice, characterized by macrophage- and T cell-driven inflammation, foam cell formation and lipid accumulation. Tollip-/- alveolar macrophages (AM) specifically accumulated lipid and underwent necrosis. Transcriptional and protein analyses of Mycobacterium tuberculosis (Mtb)-infected, Tollip-/- AM revealed increased EIF2 signalling and downstream upregulation of the integrated stress response (ISR). These phenotypes were linked, as incubation of the Mtb lipid mycolic acid with Mtb-infected Tollip-/- AM activated the ISR and increased Mtb replication. Correspondingly, the ISR inhibitor, ISRIB, reduced Mtb numbers in AM and improved Mtb control, overcoming the inflammatory phenotype. In conclusion, targeting the ISR offers a promising target for host-directed anti-TB therapy towards improved Mtb control and reduced immunopathology.

Design, synthesis and modelling of photoreactive chemical probes for investigating target engagement of plasmepsin IX and X in Plasmodium falciparum.

The emergence of Plasmodium parasite resistance to current front-line antimalarial treatments poses a serious threat to global malaria control and highlights the necessity for the development of therapeutics with novel targets and mechanisms of action. Plasmepsins IX and X (PMIX/PMX) have been recognised as highly promising targets in Plasmodium due to their contribution to parasite's pathogenicity. Recent research has demonstrated that dual PMIX/PMX inhibition results in the impairment of multiple parasite's life cycle stages, which is an important feature in drug resistance prevention. Herein we report novel hydroxyethylamine photoaffinity labelling (PAL) probes, designed for PMIX/PMX target engagement and proteomics experiments in Plasmodium parasites. The prepared probes have both a photoreactive group (diazirine or benzophenone) for covalent attachment to target proteins, and a terminal alkyne handle allowing their use in bioorthogonal ligation. One of the synthesised benzophenone probes was shown to be highly promising as demonstrated by its outstanding antimalarial potency (IC50 = 15 nM versus D10 P. falciparum) and its inhibitory effect against PfPMX in an enzymatic assay. Molecular docking and molecular dynamics studies show that the inclusion of the benzophenone and alkyne handle does not alter the binding mode compared to the parent compound. The photoaffinity probe can be used in future chemical proteomics studies to allow hydroxyethylamine drug scaffold target identification and validation in Plasmodium. We expect our findings to act as a tool for future investigations on PMIX/PMX inhibition in antimalarial drug discovery.