A PAK1 Mutational Hotspot Within the Regulatory CRIPaK Domain is Associated With Severe Neurodevelopmental Disorders in Children.

BACKGROUND: P-21-activated kinases (PAKs) are protein serine/threonine kinases, part of the RAS/mitogen-activated protein kinase pathway. PAK1 is highly expressed in the central nervous system and crucially involved in neuronal migration and brain developmental processes. Recently, de novo heterozygous missense variants in PAK1 have been identified as an ultrarare cause of pediatric neurodevelopmental disorders. METHODS: We report a series of children affected with postnatal macrocephaly, neurodevelopmental impairment, and drug-resistant epilepsy. Repeated electroencephalographic (EEG) and video-EEG evaluations were performed over a two- to 10-year period during follow-up to delineate electroclinical histories. Genetic sequencing studies and computational evaluation of the identified variants were performed in our patient cohort. RESULTS: We identified by whole-exome sequencing three novel de novo variants in PAK1 (NM_001128620: c.427A>G, p.Met143Val; c.428T>C, p.Met143Thr; c.428T>A, p.Met143Lys) as the underlying cause of the disease in our families. The three variants affected the same highly conserved Met143 residue within the cysteine-rich inhibitor of PAK1 (CRIPaK) domain, which was identified before as a PAK1 inhibitor target. Computational studies suggested a defective autoinhibition presumably due to impaired PAK1 autoregulation as a result of the recurrent substitution. CONCLUSIONS: We delineated the electroclinical phenotypes of PAK1-related neurological disorders and highlight a novel mutational hotspot that may involve defective autoinhibition of the PAK1 protein. The three novel variants affecting the same hotspot residue within the CRIPaK domain highlight potentially impaired PAK1-CRIPaK interaction as a novel disease mechanism. These findings shed light on possible future treatments targeted at the CRIPaK domain, to modulate PAK1 activity and function.

PAK1 c.1409 T > a (p. Leu470Gln) de novo variant affects the protein kinase domain, leading to epilepsy, macrocephaly, spastic quadriplegia, and hydrocephalus: Case report and review of the literature.

The p-21-activated kinase 1 (PAK1) protein, encoded by the PAK1 gene, is an evolutionarily conserved serine/threonine-protein kinase that regulates key cellular developmental processes. To date, seven de novo PAK1 variants have been reported to cause the Intellectual Developmental Disorder with Macrocephaly, Seizures, and Speech Delay (IDDMSSD). In addition to the namesake features, other common characteristics include structural brain anomalies, delayed development, hypotonia, and dysmorphic features. Here, we report a de novo PAK1 NM_002576.5: c.1409 T > A variant (p.Leu470Gln) identified by trio genome sequencing (GS) in a 13-year-old boy with postnatal macrocephaly, obstructive hydrocephalus, medically refractory epilepsy, spastic quadriplegia, white matter hyperintensities, profound developmental disabilities, and a horseshoe kidney. This is the first recurrently affected residue identified in the protein kinase domain. Combined assessment of the eight pathogenic PAK1 missense variants reveal that the variants cluster in either the protein kinase or autoregulatory domains. Although interpretation of the phenotypic spectrum is limited by the sample size, neuroanatomical alterations were found more often in individuals with PAK1 variants in the autoregulatory domain. In contrast, non-neurological comorbidities were found more often in individuals with PAK1 variants in the protein kinase domain. Together, these findings expand the clinical spectrum of PAK1-associated IDDMSSD and reveal potential correlations with the affected protein domains.

Rho family GTPase signaling through type II p21-activated kinases.

Signaling from the Rho family small GTPases controls a wide range of signaling outcomes. Key among the downstream effectors for many of the Rho GTPases are the p21-activated kinases, or PAK group. The PAK family comprises two types, the type I PAKs (PAK1, 2 and 3) and the type II PAKs (PAK4, 5 and 6), which have distinct structures and mechanisms of regulation. In this review, we discuss signal transduction from Rho GTPases with a focus on the type II PAKs. We discuss the role of PAKs in signal transduction pathways and selectivity of Rho GTPases for PAK family members. We consider the less well studied of the Rho GTPases and their PAK-related signaling. We then discuss the molecular basis for kinase domain recognition of substrates and for regulation of signaling. We conclude with a discussion of the role of PAKs in cross talk between Rho family small GTPases and the roles of PAKs in disease.

Molecular modelling approaches predicted 1,2,3-triazolyl ester of ketorolac (15K) to be a novel allosteric modulator of the oncogenic kinase PAK1.

P21-activated kinases (PAKs) are serine/threonine protein kinase which have six different isoforms (PAK1-6). Of those, PAK1 is overexpressed in many cancers and considered to be a major chemotherapeutic target. Most of the developed PAK1 inhibitor drugs work as pan-PAK inhibitors and show undesirable toxicity due to having untargeted kinase inhibition activities. Selective PAK1 inhibitors are therefore highly desired and oncogenic drug hunters are trying to develop allosteric PAK1 inhibitors. We previously synthesized 1,2,3-triazolyl ester of ketorolac (15K) through click chemistry technique, which exhibits significant anti-cancer effects via inhibiting PAK1. Based on the selective anticancer effects of 15K against PAK1-dependent cancer cells, we hypothesize that it may act as an allosteric PAK1 inhibitor. In this study, computational analysis was done with 15K to explore its quantum chemical and thermodynamic properties, molecular interactions and binding stability with PAK1, physicochemical properties, ADMET, bioactivities, and druglikeness features. Molecular docking analysis demonstrates 15K as a potent allosteric ligand that strongly binds to a novel allosteric site of PAK1 (binding energy ranges - 8.6 to - 9.2 kcal/mol) and does not target other PAK isoforms; even 15K shows better interactions than another synthesized PAK1 inhibitor. Molecular dynamics simulation clearly supports the stable binding properties of 15K with PAK1 crystal. Density functional theory-based calculations reveal that it can be an active drug with high softness and moderate polarity, and ADMET predictions categorize it as a non-toxic drug as evidenced by in vitro studies with brine shrimp and fibroblast cells. Structure-activity relationship clarifies the role of ester bond and triazol moiety of 15K in establishing novel allosteric interactions. Our results summarize that 15K selectively inhibits PAK1 as an allosteric inhibitor and in turn shows anticancer effects without toxicity.

Derivation of stationary distributions of biochemical reaction networks via structure transformation.

Long-term behaviors of biochemical reaction networks (BRNs) are described by steady states in deterministic models and stationary distributions in stochastic models. Unlike deterministic steady states, stationary distributions capturing inherent fluctuations of reactions are extremely difficult to derive analytically due to the curse of dimensionality. Here, we develop a method to derive analytic stationary distributions from deterministic steady states by transforming BRNs to have a special dynamic property, called complex balancing. Specifically, we merge nodes and edges of BRNs to match in- and out-flows of each node. This allows us to derive the stationary distributions of a large class of BRNs, including autophosphorylation networks of EGFR, PAK1, and Aurora B kinase and a genetic toggle switch. This reveals the unique properties of their stochastic dynamics such as robustness, sensitivity, and multi-modality. Importantly, we provide a user-friendly computational package, CASTANET, that automatically derives symbolic expressions of the stationary distributions of BRNs to understand their long-term stochasticity.

Integrated bioinformatics analysis reveals novel key biomarkers and potential candidate small molecule drugs in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is the metabolic disorder that appears during pregnancy. The current investigation aimed to identify central differentially expressed genes (DEGs) in GDM. The transcription profiling by array data (E-MTAB-6418) was obtained from the ArrayExpress database. The DEGs between GDM samples and non-GDM samples were analyzed. Functional enrichment analysis were performed using ToppGene. Then we constructed the protein-protein interaction (PPI) network of DEGs by the Search Tool for the Retrieval of Interacting Genes database (STRING) and module analysis was performed. Subsequently, we constructed the miRNA-hub gene network and TF-hub gene regulatory network. The validation of hub genes was performed through receiver operating characteristic curve (ROC). Finally, the candidate small molecules as potential drugs to treat GDM were predicted by using molecular docking. Through transcription profiling by array data, a total of 869 DEGs were detected including 439 up-regulated and 430 down-regulated genes. Functional enrichment analysis showed these DEGs were mainly enriched in reproduction, cell adhesion, cell surface interactions at the vascular wall and extracellular matrix organization. Ten genes, HSP90AA1, EGFR, RPS13, RBX1, PAK1, FYN, ABL1, SMAD3, STAT3 and PRKCA were associated with GDM, according to ROC analysis. Finally, the most significant small molecules were predicted based on molecular docking. This investigation identified hub genes, signal pathways and therapeutic agents, which might help us, enhance our understanding of the mechanisms of GDM and find some novel therapeutic agents for GDM.

PAK4 methylation by the methyltransferase SETD6 attenuates cell adhesion.

P21-activated kinase 4 (PAK4), a member of serine/threonine kinases family is over-expressed in numerous cancer tumors and is associated with oncogenic cell proliferation, migration and invasion. Our recent work demonstrated that the SET-domain containing protein 6 (SETD6) interacts with and methylates PAK4 at chromatin in mammalian cells, leading to activation of the Wnt/ß-catenin signaling pathway. In our current work, we identified lysine 473 (K473) on PAK4 as the primary methylation site by SETD6. Methylation of PAK4 at K473 activates ß-catenin transcriptional activity and inhibits cell adhesion. Specific methylation of PAK4 at K473 also attenuates paxillin localization to focal adhesions leading to overall reduction in adhesion-related features, such as filopodia and actin structures. The altered adhesion of the PAK4 wild-type cells is accompanied with a decrease in the migrative and invasive characteristics of the cells. Taken together, our results suggest that methylation of PAK4 at K473 plays a vital role in the regulation of cell adhesion and migration.

The molecular basis for immune dysregulation by the hyperactivated E62K mutant of the GTPase RAC2.

The RAS-related C3 botulinum toxin substrate 2 (RAC2) is a member of the RHO subclass of RAS superfamily GTPases required for proper immune function. An activating mutation in a key switch II region of RAC2 (RAC2E62K) involved in recognizing modulatory factors and effectors has been identified in patients with common variable immune deficiency. To better understand how the mutation dysregulates RAC2 function, we evaluated the structure and stability, guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP) activity, and effector binding of RAC2E62K Our findings indicate the E62K mutation does not alter RAC2 structure or stability. However, it does alter GEF specificity, as RAC2E62K is activated by the DOCK GEF, DOCK2, but not by the Dbl homology GEF, TIAM1, both of which activate the parent protein. Our previous data further showed that the E62K mutation impairs GAP activity for RAC2E62K As this disease mutation is also found in RAS GTPases, we assessed GAP-stimulated GTP hydrolysis for KRAS and observed a similar impairment, suggesting that the mutation plays a conserved role in GAP activation. We also investigated whether the E62K mutation alters effector binding, as activated RAC2 binds effectors to transmit signaling through effector pathways. We find that RAC2E62K retains binding to an NADPH oxidase (NOX2) subunit, p67phox, and to the RAC-binding domain of p21-activated kinase, consistent with our earlier findings. Taken together, our findings indicate that the RAC2E62K mutation promotes immune dysfunction by promoting RAC2 hyperactivation, altering GEF specificity, and impairing GAP function yet retaining key effector interactions.

Recognition of physiological phosphorylation sites by p21-activated kinase 4.

Many serine/threonine protein kinases discriminate between serine and threonine substrates as a filter to control signaling output. Among these, the p21-activated kinase (PAK) group strongly favors phosphorylation of Ser over Thr residues. PAK4, a group II PAK, almost exclusively phosphorylates its substrates on serine residues. The only well documented exception is LIM domain kinase 1 (LIMK1), which is phosphorylated on an activation loop threonine (Thr508) to promote its catalytic activity. To understand the molecular and kinetic basis for PAK4 substrate selectivity we compared its mode of recognition of LIMK1 (Thr508) with that of a known serine substrate, ß-catenin (Ser675). We determined X-ray crystal structures of PAK4 in complex with synthetic peptides corresponding to its phosphorylation sites in LIMK1 and ß-catenin to 1.9 Å and 2.2 Å resolution, respectively. We found that the PAK4 DFG + 1 residue, a key determinant of phosphoacceptor preference, adopts a sub-optimal orientation when bound to LIMK1 compared to ß-catenin. In peptide kinase activity assays, we find that phosphoacceptor identity impacts catalytic efficiency but does not affect the Km value for both phosphorylation sites. Although catalytic efficiency of wild-type LIMK1 and ß-catenin are equivalent, T508S mutation of LIMK1 creates a highly efficient substrate. These results suggest suboptimal phosphorylation of LIMK1 as a mechanism for controlling the dynamics of substrate phosphorylation by PAK4.

Serine phosphorylation of the small phosphoprotein ICAP1 inhibits its nuclear accumulation.

Nuclear accumulation of the small phosphoprotein integrin cytoplasmic domain-associated protein-1 (ICAP1) results in recruitment of its binding partner, Krev/Rap1 interaction trapped-1 (KRIT1), to the nucleus. KRIT1 loss is the most common cause of cerebral cavernous malformation, a neurovascular dysplasia resulting in dilated, thin-walled vessels that tend to rupture, increasing the risk for hemorrhagic stroke. KRIT1's nuclear roles are unknown, but it is known to function as a scaffolding or adaptor protein at cell-cell junctions and in the cytosol, supporting normal blood vessel integrity and development. As ICAP1 controls KRIT1 subcellular localization, presumably influencing KRIT1 function, in this work, we investigated the signals that regulate ICAP1 and, hence, KRIT1 nuclear localization. ICAP1 contains a nuclear localization signal within an unstructured, N-terminal region that is rich in serine and threonine residues, several of which are reportedly phosphorylated. Using quantitative microscopy, we revealed that phosphorylation-mimicking substitutions at Ser-10, or to a lesser extent at Ser-25, within this N-terminal region inhibit ICAP1 nuclear accumulation. Conversely, phosphorylation-blocking substitutions at these sites enhanced ICAP1 nuclear accumulation. We further demonstrate that p21-activated kinase 4 (PAK4) can phosphorylate ICAP1 at Ser-10 both in vitro and in cultured cells and that active PAK4 inhibits ICAP1 nuclear accumulation in a Ser-10-dependent manner. Finally, we show that ICAP1 phosphorylation controls nuclear localization of the ICAP1-KRIT1 complex. We conclude that serine phosphorylation within the ICAP1 N-terminal region can prevent nuclear ICAP1 accumulation, providing a mechanism that regulates KRIT1 localization and signaling, potentially influencing vascular development.

A novel PAK1 variant causative of neurodevelopmental disorder with postnatal macrocephaly.

p21-activated kinases (PAKs) are protein serine/threonine kinases stimulated by Rho-family p21 GTPases such as CDC42 and RAC. PAKs have been implicated in several human disorders, with pathogenic variants in PAK3 associated with intellectual disability and several PAK members, especially PAK1 and PAK4, overexpressed in human cancer. Recently, de novo PAK1 variants were reported to be causative of neurodevelopmental disorder (ND) with secondary macrocephaly in three patients. We herein report a fourth patient with ND, epilepsy, and macrocephaly caused by a de novo PAK1 missense variant. Two previously reported missense PAK1 variants functioned as activating alleles by reducing PAK1 homodimerization. To examine the pathogenicity of the identified novel p.Ser110Thr variant, we carried out in silico structural analysis. Our findings suggest that this variant also prevents PAK1 homodimerization, leading to constitutive PAK1 activation.

Identification of Pak1 inhibitors using water thermodynamic analysis.

p21-activated kinases (Paks) play an integral component in various cellular diverse processes. The full activation of Pak is dependent upon several serine residues present in the N-terminal region, a threonine present at the activation loop, and finally the phosphorylation of these residues ensure the complete activation of Pak1. The present study deals with the identification of novel potent candidates of Pak1 using computational methods as anti-cancer compounds. A diverse energy based pharmacophore (e-pharmacophore) was developed using four co-crystal inhibitors of Pak1 having pharmacophore features of 5 (DRDRR), 6 (DRHADR), and 7 (RRARDRP and DRRDADH) hypotheses. These models were used for rigorous screening against e-molecule database. The obtained hits were filtered using ADME/T and molecular docking to identify the high affinity binders. These hits were subjected to hierarchical clustering using dendritic fingerprint inorder to identify structurally diverse molecules. The diverse hits were scored against generated water maps to obtain WM/MM ΔG binding energy. Furthermore, molecular dynamics simulation and density functional theory calculations were performed on the final hits to understand the stability of the complexes. Five structurally diverse novel Pak1 inhibitors (4835785, 32198676, 32407813, 76038049, and 32945545) were obtained from virtual screening, water thermodynamics and WM/MM ΔG binding energy. All hits revealed similar mode of binding pattern with the hinge region residues replacing the unstable water molecules in the binding site. The obtained novel hits could be used as a platform to design potent drugs that could be experimentally tested against cancer patients having increased Pak1 expression.

PAK3 mutations responsible for severe intellectual disability and callosal agenesis inhibit cell migration.

Corpus callosum agenesis (CCA) is a brain malformation associated with a wide clinical spectrum including intellectual disability (ID) and an etiopathological complexity. We identified a novel missense G424R mutation in the X-linked p21-activated kinase 3 (PAK3) gene in a boy presenting with severe ID, microcephaly and CCA and his fetal sibling with CCA and severe hydrocephaly. PAK3 kinase is known to control synaptic plasticity and dendritic spine dynamics but its implication is less characterized in brain ontogenesis. In order to identify developmental functions of PAK3 impacted by mutations responsible for CCA, we compared the biochemical and biological effects of three PAK3 mutations localized in the catalytic domain. These mutations include two "severe" G424R and K389N variants (responsible for severe ID and CCA) and the "mild" A365E variant (responsible for nonsyndromic mild ID). Whereas they suppressed kinase activity, only the two severe variants displayed normal protein stability. Furthermore, they increased interactions between PAK3 and the guanine exchange factor αPIX/ARHGEF6, disturbed adhesion point dynamics and cell spreading, and severely impacted cell migration. Our findings highlight new molecular defects associated with mutations responsible for severe clinical phenotypes with developmental brain defects.

Human p21-activated kinase 5 (PAK5) expression and potential mechanisms in relevant cancers: Basic and clinical perspectives for molecular cancer therapeutics.

An oncogenic role, p21-activated kinase 5 (PAK5), has proven as a significant mediator for many cellular progression, which is expressed highly in human organs such as lung, liver, kidney, blood vessels endothelial cells and inflammatory cells. PAK5 was primitively detected in the cerebrum and accelerated the filopodia formation in neurocytes. It can reverse the effect of Rho and adjust its activity to mediate maintenance and development of nerve axon by binding with Cdc42-GTP. Moreover, PAK5 has been suggested to mediate protean, multitudinous and inscrutable functions in cancer. Currently, many researches indicated that PAK5 was dysregulated in ovarian cancer, cervical cancer, melanoma, osteosarcoma, renal carcinoma, breast cancer, gastric cancer and so on, which was involved in cell proliferation, apoptosis, migration and invasion. This review focuses the latest knowledge on the structure, expression, signalling pathway of PAK5, emphasizing its function in cancer.

Phosphorylation-dependent activity-based conformational changes in P21-activated kinase family members and screening of novel ATP competitive inhibitors.

P21-activated kinases (PAKs) are serine/threonine protein kinases that are subdivided into two groups on the basis of their domain architecture: group-I (PAK1-3) and group-II (PAK4-6). PAKs are considered as attractive drug targets that play vital role in cell proliferation, survival, motility, angiogenesis and cytoskeletal dynamics. In current study, molecular dynamics simulation-based comparative residual contributions and differential transitions were monitored in both active and inactive states of human PAK homologs for therapeutic intervention. Due to their involvement in cancer, infectious diseases, and neurological disorders, it is inevitable to develop novel therapeutic strategies that specifically target PAKs on the basis of their activity pattern. In order to isolate novel inhibitors that are able to bind at the active sites of PAK1 and PAK4, high throughput structure-based virtual screening was performed. Multiple lead compounds were proposed on the basis of their binding potential and targeting region either phosphorylated (active) or unphosphorylated PAK isoform (inactive). Thus, ATP-competitive inhibitors may prove ideal therapeutic choice against PAK family members. The detailed conformational readjustements occurring in the PAKs upon phosphorylation-dephosphorylation events may serve as starting point for devising novel drug molecules that are able to target on activity basis. Overall, the observations of current study may add valuable contribution in the inventory of novel inhibitors that may serve as attractive lead compounds for targeting PAK family members on the basis of activity-based conformational changes.

Molecular dynamics simulation and QM/MM calculation reveal the selectivity mechanism of type I 1/2 kinase inhibitors: the effect of intramolecular H-bonds and conformational restriction for improved selectivity.

Understanding the selectivity mechanisms of inhibitors towards highly similar proteins is extremely important work on the way to a new drug. Here, we aim to reveal the selectivity mechanisms of type I 1/2 kinase inhibitors towards p21-activated kinase (PAK4) and mitogen-activated protein kinase kinase kinase 14 (MAP3K14, NIK). PAK4, belonging to the serine/threonine protein kinases, is involved in cell signaling pathways and controls cellular functions and has received attention as an attractive drug target. The high sequence identity between PAK4 and NIK makes it challenging to design selective PAK4 inhibitors. In this work, computational methods including protein comparison, molecular docking, QM/MM, molecular dynamics simulations, and density functional theory (DFT) calculation were employed to explore the binding mechanisms of selective inhibitors against NIK and PAK4. The simulation results revealed the crucial factors accounting for selective inhibition of PAK4 over NIK, including different protein-ligand interactions, the positions and conformations of key residues, and the ligands flexibilities. This study will shed light on understanding the selectivity mechanisms of PAK4 and NIK inhibitors.

De novo variants in PAK1 lead to intellectual disability with macrocephaly and seizures.

Using trio exome sequencing, we identified de novo heterozygous missense variants in PAK1 in four unrelated individuals with intellectual disability, macrocephaly and seizures. PAK1 encodes the p21-activated kinase, a major driver of neuronal development in humans and other organisms. In normal neurons, PAK1 dimers reside in a trans-inhibited conformation, where each autoinhibitory domain covers the kinase domain of the other monomer. Upon GTPase binding via CDC42 or RAC1, the PAK1 dimers dissociate and become activated. All identified variants are located within or close to the autoinhibitory switch domain that is necessary for trans-inhibition of resting PAK1 dimers. Protein modelling supports a model of reduced ability of regular autoinhibition, suggesting a gain of function mechanism for the identified missense variants. Alleviated dissociation into monomers, autophosphorylation and activation of PAK1 influences the actin dynamics of neurite outgrowth. Based on our clinical and genetic data, as well as the role of PAK1 in brain development, we suggest that gain of function pathogenic de novo missense variants in PAK1 lead to moderate-to-severe intellectual disability, macrocephaly caused by the presence of megalencephaly and ventriculomegaly, (febrile) seizures and autism-like behaviour.

The subcellular localization of type I p21-activated kinases is controlled by the disordered variable region and polybasic sequences.

p21-activated kinases (PAKs) are serine/threonine kinase effectors of the small GTPases Rac and Cdc42 and major participants in cell adhesion, motility, and survival. Type II PAKs (PAK4, -5, and -6) are recruited to cell-cell boundaries, where they regulate adhesion dynamics and colony escape. In contrast, the type I PAK, PAK1, does not localize to cell-cell contacts. We have now found that the other type I PAKs (PAK2 and PAK3) also fail to target to cell-cell junctions. PAKs contain extensive similarities in sequence and domain organization; therefore, focusing on PAK1 and PAK6, we used chimeras and truncation mutants to investigate their differences in localization. We observed that a weakly conserved sequence region (the variable region), located between the Cdc42-binding CRIB domain and the kinase domain, inhibits PAK1 targeting to cell-cell junctions. Accordingly, substitution of the PAK1 variable region with that from PAK6 or removal of this region of PAK1 resulted in its localization to cell-cell contacts. We further show that Cdc42 binding is required, but not sufficient, to direct PAKs to cell-cell contacts and that an N-terminal polybasic sequence is necessary for PAK1 recruitment to cell-cell contacts, but only if the variable region-mediated inhibition is released. We propose that all PAKs contain cell-cell boundary-targeting motifs but that the variable region prevents type I PAK accumulation at junctions. This highlights the importance of this poorly conserved, largely disordered region in PAK regulation and raises the possibility that variable region inhibition may be released by cellular signals.

Strategy and validation of a structure-based method for the discovery of selective inhibitors of PAK isoforms and the evaluation of their anti-cancer activity.

p21 activated kinase 4 (PAK4), which belongs to the serine/threonine (Ser/Thr) protein kinase family, is a representative member of the PAK family and plays a significant role in multiple processes associated with cancer development. In this study, structure-based virtual screening was performed to discover novel and selective small molecule scaffolds, and a 6-hydroxy-2-mercapto-3-phenylpyrimidin-4(3H)-one-based compound (SPU-106, 14#) was identified as an effective PAK4 inhibitor. By combining both a molecular docking study and molecular dynamics (MD) simulation strategies, the binding mode was determined in the PAK4 site. The SPU-106 compound could efficiently and selectively bind to the PAK4 kinase domain at an IC50 of 21.36 µM according to the kinase analysis. The designed molecular probe demonstrated that SPU-106 binds to the kinase domain in the C-terminus of PAK4. Further investigation revealed that the SPU-106 had a strong inhibitory effect on the invasion of SGC7901 cells but without any cytotoxicity. The western blot analysis indicated that the compound potently inhibited the PAK4/LIMK1/cofilin and PAK4/SCG10 signaling pathways. Thus, our work shows the successful application of computational strategies for the discovery of selective hits, and SPU-106 may be an effective PAK4 inhibitor for further development as an antitumor agent.

Solution structures and biophysical analysis of full-length group A PAKs reveal they are monomeric and auto-inhibited in cis.

The group A p21-activated kinases (PAKs) exist in an auto-inhibited form until activated by GTPase binding and auto-phosphorylation. In the auto-inhibited form, a regulatory domain binds to the kinase domain (KD) blocking the binding of substrates, and CDC42 or Rac binding to the regulatory domain relieves this auto-inhibition allowing auto-phosphorylation on the KD activation loop. We have determined the crystal structure of the PAK3 catalytic domain and by small angle X-ray scattering, the solution-phase structures of full-length inactive PAK1 and PAK3. The structures reveal a compact but elongated molecular shape that demonstrates that, together with multiple independent biophysical measurements and in contrast with previous assumptions, group A PAKs are monomeric both before and after activation, consistent with an activation mechanism of cis-auto-inhibition and initial cis-auto-phosphorylation, followed by transient dimerisation to allow trans-auto-phosphorylation for full activation, yielding a monomeric active PAK protein.