rho - family gtpases signal through a variety of well - studied  effector protein kinases ( reviewed in ) . the p21-activated kinases ( paks ) contribute to cell cycle progression and the plasticity of the actin cytoskeleton and thus can contribute to a number of steps of cancer initiation and progression . the mammalian pak kinases fall into two distinct subgroups based on distinct catalytic and regulatory domain homology ( figure 1 ) . mammalian pak1 , pak2 , and pak3 comprise the better - understood group i paks that are activated by rac1 and cdc42 and can be purified with a rac1 and the guanine nucleotide exchange factor ( gef ) termed pix ( pak - interacting exchange factor ) . in contrast , pak4 , pak5 , and pak6 ( group ii ) are not activated by cdc42 binding to the crib ( cdc42/rac interaction and binding ) region . both group i and group ii paks contain related catalytic and cdc42/rac interaction and binding ( crib ) domains . pak1 , pak2 , and pak3 are inhibited in trans via an auto - inhibitory domain ( aid ) , which when expressed as a glutathione s - transferase ( gst ) fusion protein can effectively inhibit the kinase . the pak1- , pak2- , and pak3-binding partner pix ( pak - interacting exchange factor ) interacts via a highly unusual sh3-interacting region . the small - molecule inhibitor ipa-3 ( p21-activated kinase inhibitor 3 ) is a symmetric dimer joined by a disulphide bond   which targets the crib region . the structure of pf-3758309 is shown for reference ; pf-3758309 binds the atp - binding pocket of paks as revealed by the x - ray structure of the pak4-pf-3758309 complex . it is notable that both pak1 and the pix - associated git1 protein   co - localize with huntingtin inclusions and that constitutively active pak1 enhances the huntingtin aggregation . the group 1 paks may be oncogenic only in specific backgrounds such as with loss of nf2 ( neurofibromatosis type 2 ) . inhibition of pak could be beneficial in immune systems in which pak1 macrophages have an attenuated erk ( extracellular signal - regulated kinase ) response . there is general evidence for misregulated pak4 in human tumor cell lines   and pak5 somatic mutations as a cancer  driver . experiments involving upregulation or downregulation of pak kinase activity are the basis for most of our current understanding of the pak kinases . it is therefore unfortunate that a widely used activation loop , phospho - mimetic mutant pak1 ( t423e ) , is actually not active in vivo . aside from sirna ( short interfering rna)-mediated knockdown of one or multiple pak isoforms , most credible information regarding pak function has been gleaned through the use of the pak1 auto - inhibitory domain ( aid ) , which exhibits a dissociation constant ( ki ) of approximately 90 nm toward pak1 activation in vitro . the glutathione s - transferase ( gst ) aid does exert effects independent of pak1 inhibition : some rationale for this off - target effect of pak1 aid comes from the observation that it avidly binds fragile - x proteins . fortunately , the pak2 aid does not have this unwanted side effect as a genetically encoded gst fusion protein ( zhao z - s , manser e , unpublished observations ) . there has been significant interest in development of drugs against paks as therapeutic agents , and these would greatly aid in elucidating the biology of paks . aside from sirna ( short interfering rna)-mediated knockdown of one or multiple pak isoforms , most credible information regarding pak function has been gleaned through the use of the pak1 auto - inhibitory domain ( aid ) , which exhibits a dissociation constant ( ki ) of approximately 90 nm toward pak1 activation in vitro . the glutathione s - transferase ( gst ) aid does exert effects independent of pak1 inhibition : some rationale for this off - target effect of pak1 aid comes from the observation that it avidly binds fragile - x proteins . fortunately , the pak2 aid does not have this unwanted side effect as a genetically encoded gst fusion protein ( zhao z - s , manser e , unpublished observations ) . there has been significant interest in development of drugs against paks as therapeutic agents , and these would greatly aid in elucidating the biology of paks . the only reported pak inhibitor for this group in which selectivity has been assessed against a wide spectrum of kinases is ipa-3 ( p21-activated kinase inhibitor 3 ) . this small - molecule pak inhibitor was developed from a screen designed to detect an inhibition of cdc42-mediated pak1 activation rather than kinase activity . this allosteric inhibitor of pak kinase activation does not target the atp - binding pocket but rather the regulatory interface where cdc42 or rac1 binds pak ( figure 1 ) . the mechanism of action of ipa-3 involves binding and covalent attachment to the pak1 regulatory domain , but this reaction is reversible . since the compound contains a disulphide bond , it is inactivated inside cells , and this makes it unsuitable for therapeutic use . ( new york , ny , usa ) , termed pf-3758309 , which exhibits potent binding and inhibition ( ki of ~5 - 15 nm ) of paks in an atp - competitive manner ( figure 1 ) . pf-3758309 was profiled for its growth - inhibitory activity in a panel of 92 tumor cell lines , half of which exhibited ic50 ( inhibitory concentration for 50% of maximal effect ) values of less than 10 nm . pathway profiling of this inhibitor has suggested links to apoptosis and the cytoskeleton as expected for pak4 . in all , this paper gives support to the notion of paks as therapeutic targets in cancer . however , pf-3758309 acts on both group i and ii paks as well as ampk ( amp - dependent kinase ) and rsk ( ribosomal s6 kinase ) at nanomolar concentrations . the in vivo inhibition of gef - h1 phosphorylation at ser-810 indicates pak4 or pak1 inhibition ( or both ) . measured in vitro , the ki toward pak4 is approximately 19 nm whereas the value quoted for pak2 is 10 times higher ( ~190 nm ) and that for pak1 is approximately 14 nm . given that the active sites of the group i pak kinases are essentially identical , this is unexpected . the only reported pak inhibitor for this group in which selectivity has been assessed against a wide spectrum of kinases is ipa-3 ( p21-activated kinase inhibitor 3 ) . this small - molecule pak inhibitor was developed from a screen designed to detect an inhibition of cdc42-mediated pak1 activation rather than kinase activity . this allosteric inhibitor of pak kinase activation does not target the atp - binding pocket but rather the regulatory interface where cdc42 or rac1 binds pak ( figure 1 ) . the mechanism of action of ipa-3 involves binding and covalent attachment to the pak1 regulatory domain , but this reaction is reversible . since the compound contains a disulphide bond , it is inactivated inside cells , and this makes it unsuitable for therapeutic use . ( new york , ny , usa ) , termed pf-3758309 , which exhibits potent binding and inhibition ( ki of ~5 - 15 nm ) of paks in an atp - competitive manner ( figure 1 ) . pf-3758309 was profiled for its growth - inhibitory activity in a panel of 92 tumor cell lines , half of which exhibited ic50 ( inhibitory concentration for 50% of maximal effect ) values of less than 10 nm . pathway profiling of this inhibitor has suggested links to apoptosis and the cytoskeleton as expected for pak4 . in all , this paper gives support to the notion of paks as therapeutic targets in cancer . however , pf-3758309 acts on both group i and ii paks as well as ampk ( amp - dependent kinase ) and rsk ( ribosomal s6 kinase ) at nanomolar concentrations . the in vivo inhibition of gef - h1 phosphorylation at ser-810 indicates pak4 or pak1 inhibition ( or both ) . measured in vitro , the ki toward pak4 is approximately 19 nm whereas the value quoted for pak2 is 10 times higher ( ~190 nm ) and that for pak1 is approximately 14 nm . given that the active sites of the group i pak kinases are essentially identical , this is unexpected . the data on pf-3758309 as described   indicate that this drug is an excellent inhibitor of both group i and ii paks . the two pak classes can have somewhat different substrate specificities in vitro , although thus far we are not aware of any that are validated in vivo . comparing the effects of ipa-3 versus pf-3758309 would allow one to differentiate between these targets . with respect to genuine isoform - specific pak inhibitors , targeting the kinase domain would seem to be problematic . since pak1 knockout mice have only minor abnormalities in the central nervous system and immune systems , compensation in any case between pak1 and pak2 is likely in most tissues . the complication with comparing knockout models with inhibitors is that paks have kinase - independent activities . at present , pak - oriented therapy seems obvious for oncology in which paks contribute to progression and persistence of the disease ; the xenograft models in mice suggest that pf-3758309 does not impact animal survival over the period of the experiments . the potential of pak inhibitors such as pf-3758309 for treatment of nf2 patients is quite exciting . nf2 is usually diagnosed in young adults by tinnitus or imbalance and is characterized by bilateral vestibular schwannomas growing on the eighth cranial nerve . the nf2 gene product termed merlin is thought to be activated by pak or pka ( protein kinase a ) phosphorylation at ser-518 . merlin is proposed to negatively regulate pak1 , and thus primary schwannoma samples derived from nf2 patients have elevated pak1 activity . the small g - protein signalling and kinases ( sgsk ) group has no direct affiliation with glaxosmithkline ( uxbridge , middlesex , uk ) .
p21-activated kinases ( paks ) act downstream of rho - family gtpase and are linked to steps in both cancer initiation and progression . 
 there are six mammalian pak isoforms that are divided into two groups , and for different reasons both groups are attractive targets for cancer therapy . 
 we describe the background and recent development of a pak inhibitor , pf-3758309 , which exhibits relatively good selectivity and high potency for paks . 
 experiments using pf-3758309 confirm that inhibiting pak is a beneficial strategy to combat some tumors , and this activity is likely related to modulation of both cell proliferation and survival . 
 the genetic loss of nf2 ( neurofibromatosis type 2 ) leading to increased cell proliferation through a ras - rac - pak pathway may represent a good test system to analyze this new pak inhibitor .