Abstract We describe a novel series of small molecule ATP-competitive inhibitors of the atypical class of Protein Kinase C (PKC). The PKC family of serine/threonine kinases are divided into four structurally and functionally distinct sub-types: classic (PKCα, PKCβ, PKCγ); novel (PKCδ, PKCε, PKCη, PKCθ); atypical (PKCι,PKCζ); and PKNs (PKN1, PKN2, PKN3). The PKCs have been defined as key regulators in a multitude of signal transduction pathways that impinge on diverse cellular processes such as proliferation, differentiation, and survival. The regulation and functions of the atypical PKCs are distinct from the classic and novel PKCs.Both atypical isoforms have been implicated in various models of cancer, and PKCι in particular has been described as a potential oncogene. Its abundance is frequently increased, primarily through PKCι gene amplification, in many epithelial tumours including: subsets of squamous non-small cell lung carcinoma, serous ovarian carcinoma, and squamous esophageal carcinoma. It has been widely demonstrated that deregulation of PKCι signalling leads to unconstrained cell growth, increased migratory and invasive behaviour, and aberrant cellular polarity, which is a hallmark of aggressive cancers. Together, these data make a strong case for the inhibition of PKCι as a novel therapeutic strategy. We have discovered a series of ATP-competitive thieno[3,2-d]pyrimidine- based PKCι inhibitors from a high throughput screen. Using structure-based design we have optimized the series and demonstrated potent and selective inhibition of PKCι in biochemical and cellular models. The biochemical, structural, and cellular characterization of these compounds will be described herein. Citation Format: Christelle Soudy, Emma Stanway, Bhavisha Patel, Caroline Barton, Michelle Barnard, Leon Pang, Paul Owen, Andrew Turnbull, Bruce A. Ruggeri, Bruce D. Dorsey, Mark A. Ator, Greg R. Ott, Mark Linch, Philippe Riou, Peter J. Parker, Sven Kjaer, Christian Dillon, Neil Q. McDonald, Jon Roffey. Identification and characterization of small molecule thieno[3,2-d]pyrimidine inhibitors of Protein Kinase C iota (PKCι). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-99. doi:10.1158/1538-7445.AM2014-LB-99