Structural model of PORCN illuminates disease-associated variants and drug
binding sites
Jia Yu
1,*
, Pei Ju Liao
1,*
, Weijun Xu
2
, Julie R. Jones
3
, David B. Everman
3
, Heather Flanagan-
Steet
3
,
Thomas H. Keller
2
and David M. Virshup
1,4,‡
*
These authors contributed equally to this work
‡
Author for correspondence
Affiliations:
1- Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore
169857
2- Experimental Drug Development Centre, 10 Biopolis Road, Chromos, Singapore 138670
3- JC Self Research Institute, Greenwood Genetic Center, Greenwood, SC 29646
USA
4-Department of Pediatrics, Duke University School of Medicine, Durham, NC, USA 27710
ORCiD IDs:
JY: 0000-0002-7004-9104
WX: 0000-0002-3566-1082
DBE: 0000-0001-8331-992X
HFS:0000-0001-8295-0453
TK: 0000-0002-7553-6235
DMV 0000-0001-6976-850X
Structural model of PORCN
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Correspondence: David M. Virshup
Programme in Cancer and Stem Cell Biology, Duke-NUS Medical School, Singapore
Email: david.virshup@duke-nus.edu.sg
Funding Information: The National Research Foundation, administered by the Singapore Ministry
of Health’s National Medical Research Council under Singapore Translational Research (STaR)
Award MOH-000155 to D.M.V.
Competing Interests: WX and TK are employees of the Experimental Drug Development Centre.
This institute has a commercial interest in the development of ETC-159. DMV also has financial
interest in ETC-159. Other authors declare no competing interests.
Running Title: PORCN model predicts disease and drug mechanism (47 characters)
Structural model of PORCN
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ABSTRACT
(179 words)
Wnt signaling is essential for normal development and is a therapeutic target in cancer. The
enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required
for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic
acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal
hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Here,
based on homology to mammalian MBOAT proteins we develop and validate a molecular structural
model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in
the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how
previously uncharacterized human variants of uncertain significance can alter PORCN function.
Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing
insights into PORCN pharmacologic inhibition. This structural model provides mechanistic insights
into PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and
can facilitate the development of improved inhibitors and the understanding of disease relevant
PORCN mutants.
Keywords: PORCN, Wnt signaling, Focal Dermal Hypoplasia, SHFM, MBOAT, Wnt
inhibitors
Structural model of PORCN
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INTRODUCTION
Wnts are secreted fatty acid-modified glycoproteins that play essential roles in both
development and adult homeostasis (Loh et al., 2016; Zhong et al., 2020). Wnt ligand binding to
receptors and coreceptors on the surface of target cells initiates an array of downstream signaling
events, most notably β-catenin mediated transcription (Jung and Park, 2020; Nusse and Clevers,
2017; Yu and Virshup, 2014). Due to its important functions in diverse processes, the Wnt pathway
is tightly regulated at multiple levels. One essential control point is the production and transport of
Wnt ligands from Wnt-producing cells to target cells in the local environment.
There are 19 Wnt genes in the mammalian genome, and each requires a post-translational
acylation on an essential serine positioned at the tip of disulfide-bond stabilized Wnt hairpin 2
(Bazan et al., 2012). This unique modification is catalyzed by the enzyme Porcupine (PORCN)
which is present in essentially all metazoans. PORCN is a member of the family of membrane-bound
O-acyltransferases (MBOATs) (Hofmann, 2000), and catalyzes the transfer of an monounsaturated
16:1 palmitoleate from Coenzyme A, S-(9Z)-9-hexadecenoate (PAM-CoA) to the hydroxyl of the
serine residue (see model, Figure 1A) (Heuvel et al., 1993; Riggleman et al., 1990; Takada et al.,
2006). Acylated Wnts are then able to transfer to the Wnt transport protein WLS and be transported
to the plasma membrane (Coombs et al., 2010; Nygaard et al., 2021). The palmitoleate modification
is also required for Wnt binding to its receptor Frizzled (FZD) (Hirai et al., 2019; Janda et al., 2012;
Nile et al., 2017).
Because Wnt signaling drives an important subset of cancers and PORCN function is
essential for Wnt activity, a growing number of small molecules that potently inhibit the catalytic
activity of PORCN have been developed (Shah et al., 2021; Zhong and Virshup, 2020). These drugs
block the palmitoleation of Wnts and inhibit the growth of Wnt-addicted cancers. Several of these
PORCN inhibitors are in human clinical trials. How and where these drugs interact with the PORCN
Structural model of PORCN
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structure to block its activity is not known, although pharmacophore models have been developed to
identify critical features of the drugs (Poulsen et al., 2015).
Wnt signaling is also critically important during embryonic development (Loh et al., 2016).
Loss of function mutations of PORCN and WLS that block the acylation and delivery of Wnts cause
early embryonic lethality in mouse knockout models (Barrott et al., 2011; Biechele et al., 2011; Fu et
al., 2009). Multiple congenital malformation syndromes are due to partial loss of function mutations
in Wnt production. Among those, diverse germ line mutations in PORCN are associated with Goltz
syndrome, an X-linked genetic disorder also known as focal dermal hypoplasia (FDH; OMIM
305600) (Bostwick et al., 2016; Goltz, 1992; Grzeschik et al., 2007; Wang et al., 2007). This rare
disorder affects the development of ectodermal and mesodermal tissues, and affected individuals
have variable defects in limbs, skin, and bone as well as other abnormalities in internal organs. FDH
is usually found in heterozygous females where the severity of the disease depends on the pattern of
X-inactivation or the presence of somatic mosaicism. Males may also have FDH, usually in the
setting of a mosaic mutation, although germline mutations in PORCN have been identified in
hemizygous males with colobomata, microphthalmia, anophthalmia or other findings of FDH (Brady
et al., 2015; Madan et al., 2017; Wawrocka et al., 2021).
Overall, more than 170 unique genomic variants in the PORCN locus linked to FDH have
been listed in the Global Variome shared LOVD database
(https://databases.lovd.nl/shared/genes/PORCN). Many of these are classified as Variants of
Uncertain Significance (VUS) because they are missense mutations located outside of the known
functional features. Determining if these variants are deleterious can be facilitated by analysis of
comparative evolutionary and where available, structural features and computationally can be
estimated by online tools such as PolyPhen-2 (Adzhubei et al., 2010), Missense3D (Khanna et al.,
2021), and PROVEAN (Choi and Chan, 2015). Laboratory-based assessment of PORCN function,
an approach we and others have used previously, is significantly more laborious (Proffitt and
Virshup, 2012; Rios-Esteves et al., 2014). Prioritizing which mutants to test in cell-based assays
Structural model of PORCN
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was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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