Protein kinase D1 (PKD1/PKCμ; GenBank: ABE96833.1) is a member of a novel family of serine/threonine kinases characterized by their diacylglycerol-dependent regulation. PKD1 is one of three PKD isoforms (PKD1–3), which have well recognized roles in cell proliferation, survival, invasion and protein transport 
. Although functional redundancy has been documented among PKD1–3, evidence suggests specialized roles for each isoform, most likely due to differences in protein structure as well as expression patterns, protein localization and substrate proximity 
. Extensive studies have documented a role of PKD proteins in cancer and cardiac cell model systems; however, considerable evidence supports roles for PKDs in neuronal signaling pathways, DNA damage, growth factor signaling, embryogenesis, multi-drug resistance, metabolic disorders, inflammation and immune responses, further emphasizing the critical role of PKDs across multiple biological systems 
. Although loss of PKD function experiments (i.e.
, dominant negative mutants, antisense oligonucleotides and RNA interference) have been used to characterize the functional role of PKDs in various model systems, studies had been hampered by the lack of readily available potent and PKD specific/selective small molecule inhibitors 
. The lack of structural data for PKDs hinder our ability to exploit traditional structure-based drug design approaches to expedite the identification of more potent and highly specific PKD inhibitors. Nevertheless, previous comparative studies of the kinase family using sequence similarity revealed that kinases share a conserved catalytic core where ATP and substrate binding and phosphate transfer occurs 
. Moreover, Thompson et al. 
reported the three-dimensional structure of the catalytic domain of the protein serine/threonine kinase, cAMP-dependent protein kinase (PKA) and compared it to a set of 10 active kinase crystal structures spanning the kinome. Using a unique pocket clustering methodology, they found there is little deviation with respect to the spatial conservation within kinase active site clefts 
. Thus, this model may be exploitable to help characterize ATP competitive kinase inhibitors in the absence of crystal structures.
We previously identified and characterized the first selective in vitro
PKD1 inhibitor with cellular activity: CID 755673 
. This compound is a non-ATP competitive, pan-PKD inhibitor discovered through implementation of in vitro
immobilized metal affinity for phosphochemicals (IMAP) PKD1 fluorescence polarization high throughput screening (HTS) assay 
. Using CID 755673 as a parental structure, subsequent analogue development resulted in PKD1 inhibitors with increased potency in both in vitro
and cell-based models 
. Additional compounds, also initially identified by others from in vitro
HTS assays, have been reported as PKD inhibitors, including CRT5 as well as novel 3,5-diarylazole and 2,6-naphthyridine compounds 
. Similar to CID 755673, these compounds are pan-PKD small molecule inhibitors with cellular inhibitory activity. Moreover, subsequent chemical modifications of these parental chemotypes have improved their in vitro
and cellular potency and specificity, yielding, for example, the PKD inhibitors BPKDi and CRT0066101 
. However, direct comparison of these latter PKD inhibitors is limited, as precise structural information is not available for CRT0066101. Currently, it appears that all reported PKD small molecule inhibitors have some form of liability, including physicochemical (i.e.
, poor potency or specificity), off-target effects or limited availability to the scientific community.
Herein, we describe 11 novel PKD1 inhibitory chemotypes identified through our previous HTS campaigns. All compounds displayed in vitro PKD1 activity in two independent in vitro assay formats and were characterized with a series of secondary assays. Three novel chemotypes inhibited phorbol ester-induced endogenous PKD1 activation (i.e., PKD Ser916 phosphorylation) in LNCaP prostate cancer cells and were structurally dissimilar to CID 755673, BKPDi and CRT5, revealing an expanded diversity of PKD1 small molecule inhibitors.