P21-activated Kinase Inhibitor Research Articles

Antitumor effects of all-trans retinoic acid and its synergism with gemcitabine are associated with downregulation of p21-activated kinases in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal malignancies worldwide. All-trans retinoic acid (ATRA) has been used as an antistromal agent in PDA, and its antitumor effect has also been reported in various kinds of cancer, including PDA. Inhibition of p21-activated kinases (PAKs) is associated with decreased tumor growth and increased gemcitabine sensitivity. The aim of this study was to evaluate the inhibitory effects of ATRA alone and in combination with gemcitabine on cell growth and migration of wild-type and gemcitabine-resistant PDA cells and the potential mechanism(s) involved. Human (MiaPaCa-2) and murine (TB33117) PDA cell lines were incubated in increasing concentrations of gemcitabine to establish resistant clones. Cell growth, clonogenicity, and migration/invasion were determined using a sulforhodamine B assay, a colony formation assay, and a Boyden chamber assay, respectively. Protein expression was measured by Western blotting. ATRA reduced cell proliferation, colony formation, and migration/invasion in both wild-type and gemcitabine-resistant cell lines. PAK1 expression was significantly increased in resistant cells. Cells treated with ATRA showed decreased expression of PAK1, PAK2, PAK4, and α-smooth muscle actin. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant cell lines. Depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. In conclusion, the antitumor effects of ATRA and its synergism with gemcitabine are associated with downregulation of PAKs. NEW & NOTEWORTHY The inhibitory effect of all-trans retinoic acid (ATRA) on cell proliferation, colony formation, and migration/invasion was associated with downregulation of p21-activated kinases (PAKs), and depletion of PAK1 enhanced ATRA sensitivity in MiaPaCa-2 cells. The combination of ATRA and gemcitabine synergistically reduced cell growth in both wild-type and gemcitabine-resistant pancreatic ductal adenocarcinoma cells. As an important prognostic marker, α-smooth muscle actin also can be downregulated by ATRA in pancreatic ductal adenocarcinoma cells.

Inhibition of PAK1 alleviates cerulein-induced acute pancreatitis via p38 and NF-κB pathways.

Acute pancreatitis is a life-threatening disease accompanied by systemic inflammatory response. NF-κB and p38 signal pathways are activated in AP induced by cerulein. And PAKs are multifunctional effectors of Rho GTPases with kinase activity. In the present study, the function of P21-activated kinase 1 (PAK1) in AP was investigated, and found that PAK1 was up-regulated in pancreas of AP mice model, and led to NF-κB and p38 pathway activation. PAK1 inhibition by shRNA or small molecule inhibitor FRAX597 decreased NF-κB and p38 activity, also alleviated the pathological damage in the pancreas of AP mice model, including decreasing the amylase and lipase levels in serum, decreasing the levels of tumor necrosis factor-α, interleukin-6, and interleukin-1β in AP. These results suggested that PAK1 inhibition protects against AP by inhibiting NF-κB and p38 pathways, and indicated that PAK1 is a potential therapy to alleviate AP patients in clinic, and these need to be explored further.

New strategies for inhibition of non-adrenergic prostate smooth muscle contraction by pharmacologic intervention.

Inhibition of prostate smooth muscle contraction by α1 -adrenoceptor antagonists (α1 -blockers) is a first-line medical treatment of lower urinary tract symptoms suggestive of benign prostatic hyperplasia. Increased smooth muscle tone in the hyperplastic prostate may drive urethral obstruction, resulting in bladder outlet obstruction and voiding symptoms. However, efficacy of α1 -blockers is limited, as non-adrenergic mediators including endothelin-1 and thromboxane A2 (TXA2 ) increase prostate smooth muscle tension in parallel to α1 -adrenoceptors. This may maintain urethral obstruction despite therapy with α1 -blockers. Consequently, future treatment options with higher efficacy need to target α1 -adrenergic and non-adrenergic contractions simultaneouly. Recently, several compounds were reported to inhibit adrenergic or neurogenic prostate contractions, however, their effects on non-adrenergic contraction are unknown. Here, we examined effects of inhibitors for Rac-GTPase, Src family kinases (SFKs), and p21-activated kinases (PAKs) on non-adrenergic prostate contractions. Prostate tissues were obtained from radical prostatectomy. Contractions were studied in an organ bath. Viability of cultured stromal cells was assessed by CCK-8 assay. Inhibition of α1 -adrenergic contractions by Rac inhibitors EHT1864 (100 μM) and NSC23766 (100 μM), and SFK inhibitors AZM475721 (10 μM) and PP2 (10 μM) was confirmed by inhibition of methoxamine-induced contractions. No effects of the PAK inhibitors FRAX486 (30 μM) and IPA3 (300 μM) on α1 -adrenergic contraction were confirmed by absent effects on methoxamine-inuced contractions. EHT1864 caused inhibition of endothelin-1- and U46619-induced contractions. EHT1864 reduced the viability of stromal cells concentration- and time-dependently. EHT1864 attenuated KCl-induced contractions of prostate strips only slightly, so that toxic effects may not account alone for inhibition of agonist-induced contractions. NSC23766 inhibited U46619-induced contractions, but not endothelin-1-induced contractions. AZM475271 had no effects on endothelin-1- or U46619-induced contractions, while PP2 inhibited U46619- but not endothelin-1-induced contractions. FRAX486 caused inhibition of U46619-induced contractions. IPA3 inhibited U46619-, but not endothelin-1-induced contractions. Of all six inhibitors, EHT1864 seems to be most promising from a translational point of view, as it inhibited TXA2 - and endothelin-1-induced besides α1 -adrenergic prostate contractions. This reflects divergent pharmacologic profiles of EHT1864 and NSC23766, although both are Rac-GTPase inhibitors. In vivo, urodynamic effects of EHT1864 and possibly of FRAX486 may exceed those of α1 -blockers.

Combined inhibition of receptor tyrosine and p21-activated kinases as a therapeutic strategy in childhood ALL

AbstractReceptor tyrosine kinase (RTK)-dependent signaling has been implicated in the pathogenesis of acute lymphoblastic leukemia (ALL) of childhood. However, the RTK-dependent signaling state and its interpretation with regard to biological behavior are often elusive. To decipher signaling circuits that link RTK activity with biological output in vivo, we established patient-derived xenograft ALL (PDX-ALL) models with dependencies on fms-like tyrosine kinase 3 (FLT3) and platelet-derived growth factor receptor β (PDGFRB), which were interrogated by phosphoproteomics using iTRAQ mass spectrometry. Signaling circuits were determined by receptor type and cellular context with few generic features, among which we identified group I p21-activated kinases (PAKs) as potential therapeutic targets. Growth factor stimulation markedly increased catalytic activities of PAK1 and PAK2. RNA interference (RNAi)-mediated or pharmacological inhibition of PAKs using allosteric or adenosine triphosphate (ATP)-competitive compounds attenuated cell growth and increased apoptosis in vitro. Notably, PAK1- or PAK2-directed RNAi enhanced the antiproliferative effects of the type III RTK and protein kinase C inhibitor midostaurin. Treatment of FLT3- or PDGFRB-dependent ALLs with ATP-competitive PAK inhibitors markedly decreased catalytic activities of both PAK isoforms. In FLT3-driven ALL, this effect was augmented by coadministration of midostaurin resulting in synergistic effects on growth inhibition and apoptosis. Finally, combined treatment of FLT3D835H PDX-ALL with the ATP-competitive group I PAK inhibitor FRAX486 and midostaurin in vivo significantly prolonged leukemia progression-free survival compared with midostaurin monotherapy or control. Our study establishes PAKs as potential downstream targets in RTK-dependent ALL of childhood, the inhibition of which might help prevent the selection or acquisition of resistance mutations toward tyrosine kinase inhibitors.

Cytotoxic Desulfated Saponin from Holothuria atra Predicted to Have High Binding Affinity to the Oncogenic Kinase PAK1: A Combined In Vitro and In Silico Study.

Sea cucumbers have long been utilized in foods and Asiatic folk medicines for their nutritive and health benefits. Herein, three sea cucumber species were investigated and Holothuria atra showed the highest cytotoxicity among these. Next, a desulfated saponin, desulfated echinoside B (DEB), was purified from H. atra through bioassay-guided fractionation. LC-ESI-MS (Liquid chromatography-electrospray ionization mass spectrometry) analysis also showed H. atra to be a rich source of saponins. DEB showed cytotoxicity on cancer cells with IC50 values of 0.5⁻2.5 µM, and on brine shrimps with an IC50 value of 9.2 µM. In molecular docking studies, DEB was found to bind strongly with the catalytic domain of PAK1 (p21-activated kinase 1) and it showed binding energy of -8.2 kcal/mol compared to binding energy of -7.7 kcal/mol for frondoside A (FRA). Both of them bind to the novel allosteric site close to the ATP-binding cleft. Molecular dynamics (MD) simulation demonstrated that DEB can form a more stable complex with PAK1, remaining inside the allosteric binding pocket and forming the maximum number of hydrogen bonds with the surrounding residues. Moreover, important ligand binding residues were found to be less fluctuating in the DEB-PAK1 complex than in the FRA-PAK1 complex throughout MD simulation. Our experimental and computational studies showed that both DEB and FRA can act as natural allosteric PAK1 inhibitors and DEB appeared to be more promising than FRA.

Inhibition of p21-activated kinase 1 attenuates the cardinal features of asthma through suppressing the lymph node homing of dendritic cells

Dendritic cell (DC) trafficking from lung to the draining mediastinal lymph nodes (MLNs) is a key step for initiation of T cell responses in allergic asthma. In the present study, we investigate the role of DC-mediated airway inflammation after inhibition of p21-activated kinase-1 (PAK1), an effector of Rac and Cdc42 small GTPases, in the allergen-induced mouse models of asthma. Systemic administration of PAK1 specific inhibitor IPA-3 significantly attenuates not only the airway inflammation but also the airway hyperresponsiveness in a mouse model of ovalbumin-induced asthma. Specifically, intratracheal administration of low dosage of IPA-3 consistently decreases not only the airway inflammation but also the DC trafficking from lung to the MLNs. Importantly, intratracheal instillation of IPA-3-treated and ovalbumin-pulsed DCs behaves largely the same as that of either Rac inhibitor-treated and ovalbumin-pulsed DCs or Cdc42 inhibitor-treated and ovalbumin-pulsed DCs in attenuation of the airway inflammation in ovalbumin-challenged mice. Mechanistically, PAK1 is not involved in the maturation, apoptosis, antigen uptake, and T cell activation of cultured DCs, but PAK1 dose lie on the downstream of Rac and Cdc42 to regulate the DC migration toward the chemokine C–C motif chemokine ligand 19. Taken together, this study demonstrates that inhibition of PAK1 attenuates the cardinal features of asthma through suppressing the DC trafficking from lung to the MLN, and that interfere with DC trafficking by a PAK1 inhibitor thus holds great promise for the therapeutic intervention of allergic diseases.

IPA-3 (PAK1 inhibitor) or OTSSP167 (MELK inhibitor) plus auranofin (PKCι inhibitor), a therapeutic option for EGFR mutant, KRAS mutant and squamous cell non-small cell lung cancer (NSCLC).

e24001Background: p21-activated kinase 1 (PAK1) stimulates growth and metastasis in several types of tumors, including NSCLC. Protein kinase C iota (PKCi) is highly expressed in NSCLC and regulates...

Inhibitors of the p21 Activated Kinases

This review article provides an overview of some of the inhibitors that have been generated against the p21-activated kinases (PAKs). Immunohistopathological evaluation, gene profiling studies, and functional assays have indicated that PAKs play important roles in human diseases, particularly cancer. Many tumor samples that have been evaluated have shown overexpression or amplification of PAK genes. Furthermore, several studies have also shown that PAKs are involved in brain diseases as well as infectious diseases. Since the PAK kinases have been linked to cancer and other diseases, they are often considered to be valuable therapeutic targets. A number of PAK inhibitors are currently under study for their use in research or as therapeutic agents. In some cases, promising results have been obtained from both in vivo and in vitro studies. Studies are ongoing to assess the specificity of PAK inhibitors toward specific PAK isoforms, to determine their pharmacokinetic profiles, and to determine optimal doses. Future studies will determine which PAK inhibitors will be the most promising candidates for clinical development.

Regulation of Insulin Sensitivity by Skeletal Muscle Specific Modulation Of p21‐Activated Kinase 1 (PAK1)

Impaired skeletal muscle (skm) insulin signaling results in insulin resistance and the development of type 2 diabetes (T2D). Insulin binding to its receptors on skm cells leads to an intracellular signaling cascade resulting in glucose transporter type 4 (GLUT4) translocation to the plasma membrane (PM) and glucose uptake. The p21–activated kinase 1 (PAK1) is a required element for skm insulin sensitivity. It has been shown that whole body PAK1−/− knock‐out (KO) mice exhibit glucose intolerance and insulin resistance. In clonal skm cells (L6‐GLUT4myc) PAK1 inhibition was shown to impair insulin‐induced filamentous actin (F‐actin) remodeling and GLUT4 translocation; coupled with a loss of normal insulin‐stimulated cofilin dephosphorylation (i.e. activation). Recently, we have shown that PAK1 is essential in the process of actin polymerization via p41‐ARC (Actin‐related protein 2/3 complex subunit 1B). However, the effect of skm specific PAK1 modulations on in vivo glucose homeostasis, and downstream effectors of PAK1 are yet to be fully determined.We hypothesize that PAK1 is essential for insulin signaling in skm, and that upon diabetogenic stress, i) reduced PAK1 levels in skm will adversely affect whole body insulin sensitivity, whereas ii) PAK1 enrichment can preserve or restore insulin sensitivity. To study the effects of PAK1 modulation on insulin sensitivity in vivo, state‐of‐the‐art inducible skm‐specific PAK1 KO and overexpressing mouse models are used. L6‐GLUT4myc clonal cells are used to identify key downstream effectors of PAK1 using cutting‐edge imaging, mass spectrometry, and metabolomic analyzes.Importantly, both PAK1 and p41‐ARC protein levels were found to be significantly reduced in the skm of human T2D donors compared to healthy human donors (n=3–4). We have generated a doxycycline‐inducible skm‐specific, i) PAK1 depletion mouse model (skm‐PAK1‐KO) and ii) PAK1 overexpressing transgenic mouse model (skm‐PAK1‐Tg). PAK1 deletion in skm resulted into impaired in vivo insulin sensitivity in skm‐PAK1‐KO mice compared to control mice (n=10, per group), studied by intraperitoneal insulin tolerance test (IPITT). Moreover, this was coupled with reduced insulin‐induced GLUT4 translocation in skm of skm‐PAK1‐KO mice compared to the control mice (n=2). Conversely, overexpression of PAK1 in L6‐GLUT4myc cells significantly improved GLUT4 translocation under insulin resistance condition (5 nM of insulin for 12 h) compared to control cells (n=4). Altogether, these findings emphasize the i) importance of PAK1 in skm and thereby whole body glucose homeostasis and ii) potential protective effect of PAK1 enrichment on skm insulin sensitivity under diabetogenic conditions. Moreover, this study in future is aimed at determining key downstream effectors of PAK1, which might serve as insulin sensitizers.Support or Funding InformationThis study was supported by grants from the National Institutes of Health (DK067912 and DK102233 to D.C.T.), the American Heart Association (15PRE21970002 to RT, 17POST33661194 to JZ), as well a gift from the Ruth and Robert Lanman Endowment (to D.C.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P1-01-08: DPYSL3 modulates proliferation and migration in claudin-low breast cancer

Abstract Cancer is often driven by deregulated protein kinases pathways including breast cancer. To profile the kinome at the protein level we are developing a kinase pulldown (KiP) proteomic platform that uses multiple kinase inhibitors conjugated to sepharose beads for protein kinase profiling. Mass spectrometry-base tryptic peptide sequencing is used to identify and quantify the kinome in an unbiased manner. To date, we have profiled and analyzed the kinomes of more than 50 breast cancer PDX model systems that include all five breast cancer subtypes. In these analyses we have begun to explore the kinome interactome because we have observed that KiP also pulls down breast cancer subtype specific kinase interacting proteins that may represent new biological insights. For example we have consistently identified dihydropyrimidinase-like 3 (DPYSL3) in p21-activated kinase (PAK) inhibitor KiP interactome in the claudin-low WHIM12 PDX model. DPYSL3 is an intracellular phosphoprotein known to play a role in cell migration and metastasis. However it remains to be seen if DPYSL3 functions as a suppressor or a promoter of metastasis. We therefore generated DPYSL3 knock-down cell lines to evaluate function in claudin-low breast cancer. Proliferation levels of DPYSL3 depleted Claudin-low WHIM12 cells (DPYSL3-) were lower than those of control WHIM 12 (DPYSL3+) cells. In contrast, migration levels of DPYSL3- WHIM12 were greater than those of control cells, suggesting a pivotal role for DPYSL3 in several cellular physiologies. Additionally the activity of a PAK inhibitor (FRAX597) on migration appeared to require DPYSL3, as the inhibitory effect of FRAX579 on migration in WHIM12 was lost when DPYSL3 expression was depleted. Additionally we find that only the long isoform of DPYSL3, not a short isoform, interacts with PAK to regulate migration, suggesting that alternative splicing of DPYSL3 may an important to the control of migration. Furthermore Snail is also negatively regulated DPYSL3 with elevated expression in DYPSL3- cells. In conclusion, the study of the kinome interactome in breast cancer PDX identified DPYSL3 as regulator of the PAK family of kinases in a claudin low breast cancer. DPYSL3 is necessary for PAK to promote migration and may play a pivotal role in complex cellular transitions from a proliferative state versus an EMT/cell migratory state. Citation Format: Matsunuma R, Chan DW, Kim B-J, Singh P, Han A, Malovannaya A, Perou CM, Ellis MJ. DPYSL3 modulates proliferation and migration in claudin-low breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-08.

Discovery of 2-(4-Substituted-piperidin/piperazine-1-yl)-N-(5-cyclopropyl-1H-pyrazol-3-yl)-quinazoline-2,4-diamines as PAK4 Inhibitors with Potent A549 Cell Proliferation, Migration, and Invasion Inhibition Activity.

A series of novel 2,4-diaminoquinazoline derivatives were designed, synthesized, and evaluated as p21-activated kinase 4 (PAK4) inhibitors. All compounds showed significant inhibitory activity against PAK4 (half-maximal inhibitory concentration IC50 < 1 μM). Among them, compounds 8d and 9c demonstrated the most potent inhibitory activity against PAK4 (IC50 = 0.060 μM and 0.068 μM, respectively). Furthermore, we observed that compounds 8d and 9c displayed potent antiproliferative activity against the A549 cell line and inhibited cell cycle distribution, migration, and invasion of this cell line. In addition, molecular docking analysis was performed to predict the possible binding mode of compound 8d. This series of compounds has the potential for further development as PAK4 inhibitors for anticancer activity.

PAK1 overexpression promotes cell proliferation in cutaneous T cell lymphoma via suppression of PUMA and p21

BackgroundCutaneous T cell lymphoma (CTCL) comprises a heterogeneous group of skin-homing T cell tumors. The small guanosine triphosphate effector p21-activated kinase 1 (PAK1) plays an important role in many fundamental cellular functions, including cell motility, proliferation, and apoptosis. The expression of PAK1 is up-regulated in several types of human cancers. However, little is known about the role of PAK1 in the pathogenesis of CTCL. ObjectiveThe aim of this study was to evaluate the expression pattern and underlying mechanism of PAK1 in CTCL. MethodsQuantitative real-time polymerase chain reaction(qRT-PCR) was used to detect PAK1 mRNA expression in the peripheral blood mononuclear cells (PBMCs) of patients with CTCL. The expression of PAK1 protein in CTCL tumor tissues was determined by immunohistochemistry. CTCL cell lines were treated with a small molecule inhibitor of PAK1, p21-activated kinase inhibitor III (IPA3), at concentrations of 2, 3.5 and 5 μM for 24 h. Hut 78 and HH CTCL cells were transfected with lentiviral-based PAK1 gene knockdown vectors. We determined the effects of PAK1 knockdown on cell proliferation and apoptosis in CTCL cells by MTS assay and flow cytometry. Animal experiments were performed to investigate the effects of PAK1 knockdown on the growth of tumors in vivo. Transcriptomic sequencing was performed to detect the direct downstream targets of PAK1 silencing. Reverse transcription polymerase chain reaction and western blot analysis were applied to verify the results of the transcriptomic analysis. ResultsWe detected PAK1 overexpression in PBMCs and skin lesions from patients with CTCL compared with benign inflammatory dermatoses (BID). Knockdown of PAK1 inhibited cell proliferation and promoted spontaneous apoptosis. In addition, the inhibitory effect of IPA3 was validated in the CTCL cell lines. Additionally, mice injected with PAK1-silenced cells presented with a decreased rate of tumor growth compared with the control groups. Moreover, the mRNA and protein expression of PUMA (BBC3) and p21 (CDKN1A) were increased in PAK1-silenced Hut 78 and HH cells. ConclusionsOur data indicated that PAK1 is upregulated in CTCL. PAK1 silencing induced apoptosis and inhibited cell growth by stimulating the expression of PUMA and p21. Thus, PAK1 may be a potential tumor marker and therapeutic target of CTCL.

Abstract B066: Unique roles of group I PAK isoforms in regulating MPNST cell viability

Abstract Group I p21-activated kinases (PAKs) include three members (PAK1, PAK2, and PAK3) that represent key components of several cell signaling networks. By transmitting growth factor-induced signals from Rho family GTPases, PAK1/2/3 regulate cytoskeletal dynamics, cell shape, and motility. Group I PAKs modulate several oncogenic and survival pathways and are thought to play a critical role in initiation and progression of certain tumor types. Although group I PAK members share significant sequence similarity, mechanism of regulation, and substrates, PAK isoforms show different gene expression patterns in tissues and play unique functions in development and immune responses. While the described PAK1/2/3 functions in tumorigenesis are generally assigned to PAK1, there are increasing hints of isoform-specific roles for group I PAKs in cancer. We have recently demonstrated that PAK1/2/3 signaling is upregulated in malignant peripheral nerve sheath tumors (MPNSTs) and that small-molecule PAK inhibitors suppress MPNST growth and dissemination. The selective group I PAK inhibitors (Frax1036 and GST-PID) tested in this study target all three isoforms and exhibit moderate effects on MPNST cell viability as single agents. We have now used an RNAi approach to knock down individual group I PAK isoforms and their combinations to show that PAK1 and PAK3 knockdown inhibits MPNST cell proliferation, while PAK2 knockdown does not impede the growth of most MPNST cell lines, and in some cases, augments it. Interestingly, combined depletion of PAK1/2/3 has less profound cytotoxic effects compared to single knockdowns of PAK1 or PAK3, suggesting the potential need for isoform-selective PAK inhibitors as therapeutic agents for MPNSTs. Citation Format: Galina Semenova, Dina Stepanova, Sofiia Karchugina, Cara Dubyk, Sergey Deyev, Alexander Lazar, Jonathan Chernoff. Unique roles of group I PAK isoforms in regulating MPNST cell viability [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B066.

Computer-aided drug design, synthesis and identification of disulfide compounds as novel and potential allosteric PAK1 inhibitors.

p21-activated kinase 1 (PAK1) is an evolutionarily conserved serine/threonine protein kinase, which has been considered as one of the key regulatory factors in signaling network of tumor cells. Therefore, inhibition of PAK1 may be a potential approach to treat many types of solid tumors. Several allosteric inhibitors of PAK1 have been identified, and the most well known one is IPA-3. But its biological activity is not satisfied, and the structure activity relationship (SAR) of PAK1 allosteric inhibitors is unclear. In this study, we designed and synthesized 13 potential allosteric inhibitors by using computer-aided drug design based on the structure of the existing PAK1 allosteric inhibitors. All the compounds were characterized by 1H-NMR and 13C-NMR, among which six were not reported previously. SAR was investigated by pharmacological studies and In03 and In06 showed increased PAK1 inhibition than previously reported IPA-3. These findings could guide further structure optimization of PAK1 inhibitors.

Structure-Based Design of 6-Chloro-4-aminoquinazoline-2-carboxamide Derivatives as Potent and Selective p21-Activated Kinase 4 (PAK4) Inhibitors.

Herein, we report the discovery and characterization of a novel class of PAK4 inhibitors with a quinazoline scaffold. Based on the shape and chemical composition of the ATP-binding pocket of PAKs, we chose a 2,4-diaminoquinazoline series of inhibitors as a starting point. Guided by X-ray crystallography and a structure-based drug design (SBDD) approach, a series of novel 4-aminoquinazoline-2-carboxamide PAK4 inhibitors were designed and synthesized. The inhibitors' selectivity, therapeutic potency, and pharmaceutical properties were optimized. One of the best compounds, 31 (CZh226), showed remarkable PAK4 selectivity (346-fold vs PAK1) and favorable kinase selectivity profile. Moreover, this compound potently inhibited the migration and invasion of A549 tumor cells by regulating the PAK4-directed downstream signaling pathways in vitro. Taken together, these data support the further development of 31 as a lead compound for PAK4-targeted anticancer drug discovery and as a valuable research probe for the further biological investigation of group II PAKs.

Inhibition of p21 activated kinase enhances tumour immune response and sensitizes pancreatic cancer to gemcitabine.

Pancreatic ductal adenocarcinoma (PDA) is one of the major types of cancer that exhibit high mortality worldwide because of the late diagnosis and the lack of effective treatment. Immunotherapy appears to be ineffective in PDA treatment due to the existence of a unique immune-suppressive microenvironment in PDA. Gemcitabine-based therapy is still the most commonly used chemotherapy to treat PDA patients with only marginal increased survival rates. This prompted us to continue the search for more effective therapy for PDA treatment. The effects of p21 activated kinases (PAKs) on tumour immune response and gemcitabine response were examined in PDA. An orthotopic murine PDA model, in which pancreatic cancer cells were injected to the tail of pancreas, was used. The mice were treated with PAK inhibitor, PF‑3758309, plus or minus gemcitabine. Tumour growth was measured by volume and weight. Tumour immune response was determined by flow cytometry analysis of splenic cells and immunohistochemical staining of intratumoural lymphocytes. Inhibition of PAKs by PF‑3758309, not only suppressed tumour growth, but also stimulated tumour immune response by increasing the numbers of splenic and intratumoural Tlymphocytes. Furthermore, inhibition of PAKs decreased PDA cell growth synergistically with gemcitabine invitro and invivo. The dual effects of inhibition of PAKs make PAK-targeted therapy more potent for the treatment of PDA. The combination of PAK inhibitors with gemcitabine may be a more effective therapeutic approach in PDA treatment.

Affecting long-term fear memory formation through optical control of Rac1 GTPase and PAK activity in lateral amygdala

Fear conditioning, a behavioral model for studying fear-related disorders, is believed to be formed by alterations of synaptic efficacy mediated by changes in synaptic transmission and neuronal morphology in lateral amygdala (LA). Rac GTPase and its downstream effector p21-activated kinase (PAK) are involved in such key neuronal functions. Here we show that optical activation of Rac1 GTPase using photoactivatable form of Rac1 (PA-Rac1) in amygdala led to phosphorylation of PAK and inhibition of long-term but not short-term auditory fear conditioning memory formation. Activation of PA-Rac1 in LA one day after fear conditioning had no effect on long-term fear memory tested 24 hrs after PA-Rac1 activation. Inhibition of PAK in LA by microinjection of the PAK inhibitor IPA-3 30 minutes before fear conditioning enhanced long-term but not short-term fear memory formation. Our results demonstrate that photoactivation of Rac1 GTPase in lateral amygdala impairs fear memory formation. Moreover, Rac1 effector PAK activity during fear conditioning constrains the formation of fear memory in LA. Thus, Rac GTPase and PAK proteins may serve as targets for treatment of fear and anxiety disorders.

Suppression of RAC1-driven malignant melanoma by group A PAK inhibitors.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1.In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival, and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1 mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

Targeting the vulnerability to NAD+ depletion in B-cell acute lymphoblastic leukemia.

Although substantial progress has been made in the treatment of B-cell acute lymphoblastic leukemia (B-ALL), the prognosis of patients with either refractory or relapsed B-ALL remains dismal. Novel therapeutic strategies are needed to improve the outcome of these patients. KPT-9274 is a novel dual inhibitor of p21-activated kinase 4 (PAK4) and nicotinamide phosphoribosyltransferase (NAMPT). PAK4 is a serine/threonine kinase that regulates a variety of fundamental cellular processes. NAMPT is a rate-limiting enzyme in the salvage biosynthesis pathway of nicotinamide adenine dinucleotide (NAD) that plays a vital role in energy metabolism. Here, we show that KPT-9274 strongly inhibits B-ALL cell growth regardless of cytogenetic abnormalities. We also demonstrate the potent in vivo efficacy and tolerability of KPT-9274 in a patient-derived xenograft murine model of B-ALL. Interestingly, although KPT-9274 is a dual PAK4/NAMPT inhibitor, B-ALL cell growth inhibition by KPT-9274 was largely abolished with nicotinic acid supplementation, indicating that the inhibitory effects on B-ALL cells are mainly exerted by NAD+ depletion through NAMPT inhibition. Moreover, we have found that the extreme susceptibility of B-ALL cells to NAMPT inhibition is related to the reduced cellular NAD+ reserve. NAD+ depletion may be a promising alternative approach to treating patients with B-ALL.

Protein kinase D1 (PKD1) phosphorylation on Ser203 by type I p21-activated kinase (PAK) regulates PKD1 localization

Although PKC-mediated phosphorylation of protein kinase D1 (PKD1) has been extensively characterized, little is known about PKD1 regulation by other upstream kinases. Here we report that stimulation of epithelial or fibroblastic cells with G protein-coupled receptor agonists, including angiotensin II or bombesin, induced rapid and persistent PKD1 phosphorylation at Ser203, a highly conserved residue located within the PKD1 N-terminal domain. Exposure to PKD or PKC family inhibitors did not prevent PKD1 phosphorylation at Ser203, indicating that it is not mediated by autophosphorylation. In contrast, several lines of evidence indicated that the phosphorylation of PKD1 at Ser203 is mediated by kinases of the class I PAK subfamily, specifically 1) exposing cells to four structurally unrelated PAK inhibitors (PF-3758309, FRAX486, FRAX597, and IPA-3) that act via different mechanisms abrogated PKD1 phosphorylation at Ser203, 2) siRNA-mediated knockdown of PAK1 and PAK2 in IEC-18 and Swiss 3T3 cells blunted PKD1 phosphorylation at Ser203, 3) phosphorylation of Ser203 markedly increased in vitro when recombinant PKD1 was incubated with either PAK1 or PAK2 in the presence of ATP. PAK inhibitors did not interfere with G protein-coupled receptor activation-induced rapid translocation of PKD1 to the plasma membrane but strikingly prevented the dissociation of PKD1 from the plasma membrane and blunted the phosphorylation of nuclear targets, including class IIa histone deacetylases. We conclude that PAK-mediated phosphorylation of PKD1 at Ser203 triggers its membrane dissociation and subsequent entry into the nucleus, thereby regulating the phosphorylation of PKD1 nuclear targets, including class IIa histone deacetylases.