Prognostic value of metabolic parameters measured by first responders attending patients with severe trauma: associations with the New Injury Severity Score and mortality. / Valor pronóstico de los parámetros metabólicos medidos en la asistencia inicial a pacientes con trauma grave: asociación con la puntuación de la escala NISS y la mortalidad.

OBJECTIVES: Patients with severe or potentially severe trauma must be identified early, a challenge in prehospital settings. This study aimed to analyze the possible diagnostic and prognostic usefulness of analytical markers recorded in the early moments of care. MATERIAL AND METHODS: Observational study of information extracted from the prospective multicenter Code Trauma database for 2016-2019, excluding data for isolated head injuries. Using the New Injury Severity Score (NISS), we classified cases into 4 levels of severity. NISS and mortality were considered the dependent variables in inferential analyses. We calculated the areas under receiver operating characteristic curves, identified optimal cutoff points (Youden index), and calculated positive (PPV) and negative predictive values.. RESULTS: Of the 1039 trauma patients in the registry, 709 were included in the study. Their mean (SD) age was 40.4 (17.3) years, and 77.3% were men. Motorcycle accidents were the most common causes of trauma (in 21%), and mortality was 12.1%. Lactate concentration, pH, PCO2, hemoglobin concentration, hematocrit, and blood sugar were significantly associated with severity and mortality. The PPVs corresponding to pH for the 4 NISS score groups (34-41, 42-49, 50-59, and $ 60) and mortality, respectively, were 61.2, 64.1, 70.7, 62.2, and 66.6. The PPVs of traditionally used clinical variables were lower. CONCLUSION: Patients with more severe trauma had lower pH values and higher PCO2, lactate, and base excess values. PCO2, pH, and blood sugar findings were the best predictors of severity. Metabolic variables are better predictors than traditionally recorded hemodynamic variables.

OBJETIVO: En entornos de emergencia prehospitalarios, la detección temprana de un paciente con trauma grave o potencialmente crítico es un desafío. El objetivo es analizar las posibilidades diagnósticas y pronóstico de los parámetros analíticos obtenidos en los primeros momentos de la asistencia inicial. METODO: Estudio observacional multicéntrico de la base de datos prospectiva "Código Trauma" de 2016-2019 excluyendo el trauma craneoencefálico aislado. La evaluación de las lesiones se realizó utilizando el New Injury Severity Score (NISS). Los pacientes fueron clasificados en 4 grupos según nivel de gravedad. Para el análisis inferencial, las puntuaciones NISS y el resultado de mortalidad se consideraron variables dependientes. Se realizó el análisis de la curva ROC, puntos de corte óptimos mediante el índice de Youden y se calcularon los valores predictivos positivo (VPP) y negativo. RESULTADOS: De los 1.039 pacientes traumatizados del registro, 709 fueron incluidos en el estudio, con una edad media de 40,4 años (DE 17,3), 77,3% eran varones, el mecanismo lesional principal accidentes de moto (21%) y la mortalidad del 12,1%. El pH, lactato, pCO2, hemoglobina, hematocrito y glucemia influyeron significativamente en gravedad y mortalidad. El VPP de mortalidad para pH fue 61,2, 64,1, 70,7, 62,2 y 66,6 para los grupos de NISS 34- 41, 42-49, 50-59 y $ 60 puntos la mortalidad, respectivamente. Las variables clínicas clásicas obtuvieron valores más bajos. CONCLUSIONES: Los pacientes con mayor gravedad presentaron menor pH y concentraciones más altas de pCO2, lactato y exceso de bases. El pH, la pCO2 y la glucemia tuvieron la mayor capacidad predictiva de gravedad. La capacidad predictiva de los valores metabólicos es superior a la de los valores hemodinámicos clásicos.

LRRK2 Inhibition by BIIB122 in Healthy Participants and Patients with Parkinson's Disease.

BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) inhibition is a promising therapeutic approach for the treatment of Parkinson's disease (PD). OBJECTIVE: The aim of this study was to evaluate the safety, tolerability, pharmacokinetics, and pharmacodynamics of the potent, selective, CNS-penetrant LRRK2 inhibitor BIIB122 (DNL151) in healthy participants and patients with PD. METHODS: Two randomized, double-blind, placebo-controlled studies were completed. The phase 1 study (DNLI-C-0001) evaluated single and multiple doses of BIIB122 for up to 28 days in healthy participants. The phase 1b study (DNLI-C-0003) evaluated BIIB122 for 28 days in patients with mild to moderate PD. The primary objectives were to investigate the safety, tolerability, and plasma pharmacokinetics of BIIB122. Pharmacodynamic outcomes included peripheral and central target inhibition and lysosomal pathway engagement biomarkers. RESULTS: A total of 186/184 healthy participants (146/145 BIIB122, 40/39 placebo) and 36/36 patients (26/26 BIIB122, 10/10 placebo) were randomized/treated in the phase 1 and phase 1b studies, respectively. In both studies, BIIB122 was generally well tolerated; no serious adverse events were reported, and the majority of treatment-emergent adverse events were mild. BIIB122 cerebrospinal fluid/unbound plasma concentration ratio was ~1 (range, 0.7-1.8). Dose-dependent median reductions from baseline were observed in whole-blood phosphorylated serine 935 LRRK2 (≤98%), peripheral blood mononuclear cell phosphorylated threonine 73 pRab10 (≤93%), cerebrospinal fluid total LRRK2 (≤50%), and urine bis (monoacylglycerol) phosphate (≤74%). CONCLUSIONS: At generally safe and well-tolerated doses, BIIB122 achieved substantial peripheral LRRK2 kinase inhibition and modulation of lysosomal pathways downstream of LRRK2, with evidence of CNS distribution and target inhibition. These studies support continued investigation of LRRK2 inhibition with BIIB122 for the treatment of PD. © 2023 Denali Therapeutics Inc and The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Preclinical and clinical evaluation of the LRRK2 inhibitor DNL201 for Parkinson's disease.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic risk factors for Parkinson's disease (PD). Increased LRRK2 kinase activity is thought to impair lysosomal function and may contribute to the pathogenesis of PD. Thus, inhibition of LRRK2 is a potential disease-modifying therapeutic strategy for PD. DNL201 is an investigational, first-in-class, CNS-penetrant, selective, ATP-competitive, small-molecule LRRK2 kinase inhibitor. In preclinical models, DNL201 inhibited LRRK2 kinase activity as evidenced by reduced phosphorylation of both LRRK2 at serine-935 (pS935) and Rab10 at threonine-73 (pT73), a direct substrate of LRRK2. Inhibition of LRRK2 by DNL201 demonstrated improved lysosomal function in cellular models of disease, including primary mouse astrocytes and fibroblasts from patients with Gaucher disease. Chronic administration of DNL201 to cynomolgus macaques at pharmacologically relevant doses was not associated with adverse findings. In phase 1 and phase 1b clinical trials in 122 healthy volunteers and in 28 patients with PD, respectively, DNL201 at single and multiple doses inhibited LRRK2 and was well tolerated at doses demonstrating LRRK2 pathway engagement and alteration of downstream lysosomal biomarkers. Robust cerebrospinal fluid penetration of DNL201 was observed in both healthy volunteers and patients with PD. These data support the hypothesis that LRRK2 inhibition has the potential to correct lysosomal dysfunction in patients with PD at doses that are generally safe and well tolerated, warranting further clinical development of LRRK2 inhibitors as a therapeutic modality for PD.

Molecular architecture determines brain delivery of a transferrin receptor-targeted lysosomal enzyme.

Delivery of biotherapeutics across the blood-brain barrier (BBB) is a challenge. Many approaches fuse biotherapeutics to platforms that bind the transferrin receptor (TfR), a brain endothelial cell target, to facilitate receptor-mediated transcytosis across the BBB. Here, we characterized the pharmacological behavior of two distinct TfR-targeted platforms fused to iduronate 2-sulfatase (IDS), a lysosomal enzyme deficient in mucopolysaccharidosis type II (MPS II), and compared the relative brain exposures and functional activities of both approaches in mouse models. IDS fused to a moderate-affinity, monovalent TfR-binding enzyme transport vehicle (ETV:IDS) resulted in widespread brain exposure, internalization by parenchymal cells, and significant substrate reduction in the CNS of an MPS II mouse model. In contrast, IDS fused to a standard high-affinity bivalent antibody (IgG:IDS) resulted in lower brain uptake, limited biodistribution beyond brain endothelial cells, and reduced brain substrate reduction. These results highlight important features likely to impact the clinical development of TfR-targeting platforms in MPS II and potentially other CNS diseases.

Iduronate-2-sulfatase transport vehicle rescues behavioral and skeletal phenotypes in a mouse model of Hunter syndrome.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the iduronate-2-sulfatase (IDS) enzyme, resulting in cellular accumulation of glycosaminoglycans (GAGs) throughout the body. Treatment of MPS II remains a considerable challenge as current enzyme replacement therapies do not adequately control many aspects of the disease, including skeletal and neurological manifestations. We developed an IDS transport vehicle (ETV:IDS) that is engineered to bind to the transferrin receptor; this design facilitates receptor-mediated transcytosis of IDS across the blood-brain barrier and improves its distribution into the brain while maintaining distribution to peripheral tissues. Here we show that chronic systemic administration of ETV:IDS in a mouse model of MPS II reduced levels of peripheral and central nervous system GAGs, microgliosis, and neurofilament light chain, a biomarker of neuronal injury. Additionally, ETV:IDS rescued auricular and skeletal abnormalities when introduced in adult MPS II mice. These effects were accompanied by improvements in several neurobehavioral domains, including motor skills, sensorimotor gating, and learning and memory. Together, these results highlight the therapeutic potential of ETV:IDS for treating peripheral and central abnormalities in MPS II. DNL310, an investigational ETV:IDS molecule, is currently in clinical trials as a potential treatment for patients with MPS II.

Rescue of a lysosomal storage disorder caused by Grn loss of function with a brain penetrant progranulin biologic.

GRN mutations cause frontotemporal dementia (GRN-FTD) due to deficiency in progranulin (PGRN), a lysosomal and secreted protein with unclear function. Here, we found that Grn-/- mice exhibit a global deficiency in bis(monoacylglycero)phosphate (BMP), an endolysosomal phospholipid we identified as a pH-dependent PGRN interactor as well as a redox-sensitive enhancer of lysosomal proteolysis and lipolysis. Grn-/- brains also showed an age-dependent, secondary storage of glucocerebrosidase substrate glucosylsphingosine. We investigated a protein replacement strategy by engineering protein transport vehicle (PTV):PGRN-a recombinant protein linking PGRN to a modified Fc domain that binds human transferrin receptor for enhanced CNS biodistribution. PTV:PGRN rescued various Grn-/- phenotypes in primary murine macrophages and human iPSC-derived microglia, including oxidative stress, lysosomal dysfunction, and endomembrane damage. Peripherally delivered PTV:PGRN corrected levels of BMP, glucosylsphingosine, and disease pathology in Grn-/- CNS, including microgliosis, lipofuscinosis, and neuronal damage. PTV:PGRN thus represents a potential biotherapeutic for GRN-FTD.

Discovery of A-1331852, a First-in-Class, Potent, and Orally-Bioavailable BCL-XL Inhibitor.

Herein we describe the discovery of A-1331852, a first-in-class orally active BCL-XL inhibitor that selectively and potently induces apoptosis in BCL-XL-dependent tumor cells. This molecule was generated by re-engineering our previously reported BCL-XL inhibitor A-1155463 using structure-based drug design. Key design elements included rigidification of the A-1155463 pharmacophore and introduction of sp3-rich moieties capable of generating highly productive interactions within the key P4 pocket of BCL-XL. A-1331852 has since been used as a critical tool molecule for further exploring BCL-2 family protein biology, while also representing an attractive entry into a drug discovery program.

Characterization of Fluid Biomarkers Reveals Lysosome Dysfunction and Neurodegeneration in Neuronopathic MPS II Patients.

Mucopolysaccharidosis type II is a lysosomal storage disorder caused by a deficiency of iduronate-2-sulfatase (IDS) and characterized by the accumulation of the primary storage substrate, glycosaminoglycans (GAGs). Understanding central nervous system (CNS) pathophysiology in neuronopathic MPS II (nMPS II) has been hindered by the lack of CNS biomarkers. Characterization of fluid biomarkers has been largely focused on evaluating GAGs in cerebrospinal fluid (CSF) and the periphery; however, GAG levels alone do not accurately reflect the broad cellular dysfunction in the brains of MPS II patients. We utilized a preclinical mouse model of MPS II, treated with a brain penetrant form of IDS (ETV:IDS) to establish the relationship between markers of primary storage and downstream pathway biomarkers in the brain and CSF. We extended the characterization of pathway and neurodegeneration biomarkers to nMPS II patient samples. In addition to the accumulation of CSF GAGs, nMPS II patients show elevated levels of lysosomal lipids, neurofilament light chain, and other biomarkers of neuronal damage and degeneration. Furthermore, we find that these biomarkers of downstream pathology are tightly correlated with heparan sulfate. Exploration of the responsiveness of not only CSF GAGs but also pathway and disease-relevant biomarkers during drug development will be crucial for monitoring disease progression, and the development of effective therapies for nMPS II.

Brain delivery and activity of a lysosomal enzyme using a blood-brain barrier transport vehicle in mice.

Most lysosomal storage diseases (LSDs) involve progressive central nervous system (CNS) impairment, resulting from deficiency of a lysosomal enzyme. Treatment of neuronopathic LSDs remains a considerable challenge, as approved intravenously administered enzyme therapies are ineffective in modifying CNS disease because they do not effectively cross the blood-brain barrier (BBB). We describe a therapeutic platform for increasing the brain exposure of enzyme replacement therapies. The enzyme transport vehicle (ETV) is a lysosomal enzyme fused to an Fc domain that has been engineered to bind to the transferrin receptor, which facilitates receptor-mediated transcytosis across the BBB. We demonstrate that ETV fusions containing iduronate 2-sulfatase (ETV:IDS), the lysosomal enzyme deficient in mucopolysaccharidosis type II, exhibited high intrinsic activity and degraded accumulated substrates in both IDS-deficient cell and in vivo models. ETV substantially improved brain delivery of IDS in a preclinical model of disease, enabling enhanced cellular distribution to neurons, astrocytes, and microglia throughout the brain. Improved brain exposure for ETV:IDS translated to a reduction in accumulated substrates in these CNS cell types and peripheral tissues and resulted in a complete correction of downstream disease-relevant pathologies in the brain, including secondary accumulation of lysosomal lipids, perturbed gene expression, neuroinflammation, and neuroaxonal damage. These data highlight the therapeutic potential of the ETV platform for LSDs and provide preclinical proof of concept for TV-enabled therapeutics to treat CNS diseases more broadly.

Cell-penetrating peptides containing 2,5-diketopiperazine (DKP) scaffolds as shuttles for anti-cancer drugs: conformational studies and biological activity.

A series of linear and cyclic peptidomimetics composed of a cell-penetrating peptide and a non-natural, bifunctional 2,5-diketopiperazine scaffold is reported. Conformational studies revealed well-defined helical structures in micellar medium for linear structures, while cyclic peptidomimetics were more flexible. Biological investigations showed higher membrane-activity of cyclic derivatives allowing their use as shuttles for anti-cancer drugs.

Molecular determinants of the mechanism and substrate specificity of Clostridium difficile proline-proline endopeptidase-1.

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

Clinical manifestations of homozygote allele carriers in Huntington disease.

OBJECTIVE: Because patients homozygous for Huntington disease (HD) receive the gain-of-function mutation in a double dose, one would expect a more toxic effect in homozygotes than in heterozygotes. Our aim was to investigate the phenotypic differences between homozygotes with both alleles ≥36 CAG repeats and heterozygotes with 1 allele ≥36 CAG repeats. METHODS: This was an international, longitudinal, case-control study (European Huntington's Disease Network Registry database). Baseline and longitudinal total functional capacity, motor, cognitive, and behavioral scores of the Unified Huntington's Disease Rating Scale (UHDRS) were compared between homozygotes and heterozygotes. Four-year follow-up data were analyzed using longitudinal mixed-effects models. To estimate the association of age at onset with the length of the shorter and larger allele in homozygotes and heterozygotes, regression analysis was applied. RESULTS: Of 10,921 participants with HD (5,777 female [52.9%] and 5,138 male [47.0%]) with a mean age of 55.1 ± 14.1 years, 28 homozygotes (0.3%) and 10,893 (99.7%) heterozygotes were identified. After correcting for multiple comparisons, homozygotes and heterozygotes had similar age at onset and UHDRS scores and disease progression. In the multivariate linear regression analysis, the longer allele was the most contributing factor to decreased age at HD onset in the homozygotes (p < 0.0001) and heterozygotes (p < 0.0001). CONCLUSIONS: CAG repeat expansion on both alleles of the HTT gene is infrequent. Age at onset, HD phenotype, and disease progression do not significantly differ between homozygotes and heterozygotes, indicating similar effect on the mutant protein. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that age at onset, the motor phenotype and rate of motor decline, and symptoms and signs progression is similar in homozygotes compared to heterozygotes.

Teaching courses in the management of visceral trauma. / Docencia en el manejo del traumatismo visceral.

Several changes introduced in the management of trauma during the last two decades have considerably decreased the practical exposure to bleeding trauma patients by residents and young surgeons. Hemorrhage still represents the second cause of death from trauma worldwide, and the surgical maneuvers required for its control must be learned and practised in specific courses. These courses address the "second hour" of trauma, beyond ATLS©, and also emphasize the decision-making process, communication among team members, and discussion of clinical scenarios. The significant progress made in simulation technologies and virtual reality systems have yet to replace living tissue models to train surgeons in the rapid control of active bleeding, although that replacement is probably not far away.

Rhizoctonia solani fucomannogalactan: Chemical characterization and antiproliferative activity.

A fucomannogalactan from Rhizoctonia solani biomass was obtained after hot aqueous extraction and purified by freeze-thaw cycles and gel filtration chromatography on Sepharose CL-6B. The polysaccharide was homogeneous after HPSEC/RID analysis (Mw/Mn~1.1), displaying an average molecular weight of 15.4×103Da. Its chemical structure was determined by methylation analysis (GC/MS) and spectroscopy (FTIR, 1D and 2D NMR). The polysaccharide had a branched α-1,6-linked Galp backbone with 66% linear residues, a number of which were at O-3 methylated. Side chains (34%) were always linked at O-2 positions of the main chain and consisted of single, non-reducing ends of α-d-Manp (6%) and α-l-Fucp (28%). Analysis of its biological activity showed that the highly purified fucomannogalactan from R. solani inhibited the proliferation of colon cancer cells in vitro, but that it did not have the same activity against lung cancer cells.

Exploring the Role of Solvent on Carbohydrate-Aryl Interactions by Diffusion NMR-Based Studies.

Carbohydrate-protein interactions play an important role in many molecular recognition processes. An exquisite combination of multiple factors favors the interaction of the receptor with one specific type of sugar, whereas others are excluded. Stacking CH-aromatic interactions within the binding site provide a relevant contribution to the stabilization of the resulting sugar-protein complex. Being experimentally difficult to detect and analyze, the key CH-π interaction features have been very often dissected using a variety of techniques and simple model systems. In the present work, diffusion NMR spectroscopy has been employed to separate the components of sugar mixtures in different solvents on the basis of their differential ability to interact through CH-π interactions with one particular aromatic cosolute in solution. The experimental data show that the properties of the solvent did also influence the diffusion behavior of the sugars present in the mixture, inhibiting or improving their separation. Overall, the results showed that, for the considered monosaccharide derivatives, their diffusion coefficient values and, consequently, their apparent molecular sizes and/or shapes depend on the balance between solute/cosolute as well as solute/solvent interactions. Thus, in certain media and in the presence of the aromatic cosolute, the studied saccharides that are more suited to display CH-π interactions exhibited a lower diffusion coefficient than the noncomplexing sugars in the mixture. However, when dissolved in another medium, the interaction with the solvent strongly competes with that of the aromatic cosolute.

An Industry Perspective on the 2017 EMA Guideline on First-in-Human and Early Clinical Trials.

The European Medicines Agency (EMA) in 2017 issued a revised guideline on nonclinical and clinical aspects of first-in-human (FIH) and early clinical trials (CTs). External input was solicited during a draft comment phase, and although some industry suggestions were adopted, others were not. We agree that subject safety is of utmost priority, and believe that minimizing risk must be balanced with efficient and informative study designs to bring new medicines to patients.

Current nonclinical testing paradigms in support of safe clinical trials: An IQ Consortium DruSafe perspective.

The transition from nonclinical to First-in-Human (FIH) testing is one of the most challenging steps in drug development. In response to serious outcomes in a recent Phase 1 trial (sponsored by Bial), IQ Consortium/DruSafe member companies reviewed their nonclinical approach to progress small molecules safely to FIH trials. As a common practice, safety evaluation begins with target selection and continues through iterative in silico and in vitro screening to identify molecules with increased probability of acceptable in vivo safety profiles. High attrition routinely occurs during this phase. In vivo exploratory and pivotal FIH-enabling toxicity studies are then conducted to identify molecules with a favorable benefit-risk profile for humans. The recent serious incident has reemphasized the importance of nonclinical testing plans that are customized to the target, the molecule, and the intended clinical plan. Despite the challenges and inherent risks of transitioning from nonclinical to clinical testing, Phase 1 studies have a remarkably good safety record. Given the rapid scientific evolution of safety evaluation, testing paradigms and regulatory guidance must evolve with emerging science. The authors posit that the practices described herein, together with science-based risk assessment and management, support safe FIH trials while advancing development of important new medicines.

MEK and ERK Kinase Inhibitors Increase Circulating Ceruloplasmin and Cause Green Serum in Rats.

Inhibition of the mitogen-activated protein kinase/extracellular signal-regulated (MAPK/ERK) pathway is an attractive therapeutic approach for human cancer therapy. In the course of evaluating structurally distinct small molecule inhibitors that target mitogen-activated protein kinase kinase (MEK) and ERK kinases in this pathway, we observed an unusual, dose-related increase in the incidence of green serum in preclinical safety studies in rats. Having ruled out changes in bilirubin metabolism, we demonstrated a 2- to 3-fold increase in serum ceruloplasmin levels, likely accounting for the observed green color. This was not associated with an increase in α-2-macroglobulin, the major acute phase protein in rats, indicating that ceruloplasmin levels increased independently of an inflammatory response. Elevated serum ceruloplasmin was also not correlated with changes in total hepatic copper, adverse clinical signs, or pathology findings indicative of copper toxicity, therefore discounting copper overload as the etiology. Both ERK and MEK inhibitors led to increased ceruloplasmin secretion in rat primary hepatocyte cultures in vitro, and this increase was associated with activation of the Forkhead box, class O1 (FOXO1) transcription factor. In conclusion, increased serum ceruloplasmin induced by MEK and ERK inhibition is due to increased synthesis by hepatocytes from FOXO1 activation and results in the nonadverse development of green serum in rats.

Chemically Diverse Group I p21-Activated Kinase (PAK) Inhibitors Impart Acute Cardiovascular Toxicity with a Narrow Therapeutic Window.

p21-activated kinase 1 (PAK1) has an important role in transducing signals in several oncogenic pathways. The concept of inhibiting this kinase has garnered significant interest over the past decade, particularly for targeting cancers associated with PAK1 amplification. Animal studies with the selective group I PAK (pan-PAK1, 2, 3) inhibitor G-5555 from the pyrido[2,3-d]pyrimidin-7-one class uncovered acute toxicity with a narrow therapeutic window. To attempt mitigating the toxicity, we introduced significant structural changes, culminating in the discovery of the potent pyridone side chain analogue G-9791. Mouse tolerability studies with this compound, other members of this series, and compounds from two structurally distinct classes revealed persistent toxicity and a correlation of minimum toxic concentrations and PAK1/2 mediated cellular potencies. Broad screening of selected PAK inhibitors revealed PAK1, 2, and 3 as the only overlapping targets. Our data suggest acute cardiovascular toxicity resulting from the inhibition of PAK2, which may be enhanced by PAK1 inhibition, and cautions against continued pursuit of pan-group I PAK inhibitors in drug discovery.