Integration of Wnt-inhibitory activity and structural novelty scoring results to uncover novel bioactive natural products: new Bicyclo[3.3.1]non-3-ene-2,9-diones from the leaves of Hymenocardia punctata.

In natural products (NPs) research, methods for the efficient prioritization of natural extracts (NEs) are key for discovering novel bioactive NPs. In this study a biodiverse collection of 1,600 NEs, previously analyzed by UHPLC-HRMS2 metabolite profiling was screened for Wnt pathway regulation. The results of the biological screening drove the selection of a subset of 30 non-toxic NEs with an inhibitory IC50 ≤ 5 µg/mL. To increase the chance of finding structurally novel bioactive NPs, Inventa, a computational tool for automated scoring of NEs based on structural novelty was used to mine the HRMS2 analysis and dereplication results. After this, four out of the 30 bioactive NEs were shortlisted by this approach. The most promising sample was the ethyl acetate extract of the leaves of Hymenocardia punctata (Phyllanthaceae). Further phytochemical investigations of this species resulted in the isolation of three known prenylated flavones (3, 5, 7) and ten novel bicyclo[3.3.1]non-3-ene-2,9-diones (1, 2, 4, 6, 8-13), named Hymenotamayonins. Assessment of the Wnt inhibitory activity of these compounds revealed that two prenylated flavones and three novel bicyclic compounds showed interesting activity without apparent cytotoxicity. This study highlights the potential of combining Inventa's structural novelty scores with biological screening results to effectively discover novel bioactive NPs in large NE collections.

Cis-regulatory polymorphism at fiz ecdysone oxidase contributes to polygenic evolutionary response to malnutrition in Drosophila.

We investigate the contribution of a candidate gene, fiz (fezzik), to complex polygenic adaptation to juvenile malnutrition in Drosophila melanogaster. Experimental populations maintained for >250 generations of experimental evolution to a nutritionally poor larval diet (Selected populations) evolved several-fold lower fiz expression compared to unselected Control populations. Here we show that this divergence in fiz expression is mediated by a cis-regulatory polymorphism. This polymorphism, originally sampled from a natural population in Switzerland, is distinct from a second cis-regulatory SNP previously identified in non-African D. melanogaster populations, implying that two independent cis-regulatory variants promoting high fiz expression segregate in non-African populations. Enzymatic analyses of Fiz protein expressed in E. coli demonstrate that it has ecdysone oxidase activity acting on both ecdysone and 20-hydroxyecdysone. Four of five fiz paralogs annotated to ecdysteroid metabolism also show reduced expression in Selected larvae, implying that malnutrition-driven selection favored general downregulation of ecdysone oxidases. Finally, as an independent test of the role of fiz in poor diet adaptation, we show that fiz knockdown by RNAi results in faster larval growth on the poor diet, but at the cost of greatly reduced survival. These results imply that downregulation of fiz in Selected populations was favored by selection on the nutritionally poor diet because of its role in suppressing growth in response to nutrient shortage. However, they suggest that fiz downregulation is only adaptive in combination with other changes evolved by Selected populations, which ensure that the organism can sustain the faster growth promoted by fiz downregulation.

GNAO1 Mutations Affecting the N-Terminal α-Helix of Gαo Lead to Parkinsonism.

BACKGROUND: Patients carrying pathogenic variants in GNAO1 present a phenotypic spectrum ranging from severe early-onset epileptic encephalopathy and developmental delay to mild adolescent/adult-onset dystonia. Genotype-phenotype correlation and molecular mechanisms underlying the disease remain understudied. METHODS: We analyzed the clinical course of a child carrying the novel GNAO1 mutation c.38T>C;p.Leu13Pro, and structural, biochemical, and cellular properties of the corresponding mutant Gαo-GNAO1-encoded protein-alongside the related mutation c.68T>C;p.Leu23Pro. RESULTS: The main clinical feature was parkinsonism with bradykinesia and rigidity, unlike the hyperkinetic movement disorder commonly associated with GNAO1 mutations. The Leu ➔ Pro substitutions have no impact on enzymatic activity or overall folding of Gαo but uniquely destabilize the N-terminal α-helix, blocking formation of the heterotrimeric G-protein and disabling activation by G-protein-coupled receptors. CONCLUSIONS: Our study defines a parkinsonism phenotype within the spectrum of GNAO1 disorders and suggests a genotype-phenotype correlation by GNAO1 mutations targeting the N-terminal α-helix of Gαo. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Clinical Cases and the Molecular Profiling of a Novel Childhood Encephalopathy-Causing GNAO1 Mutation P170R.

De novo mutations in GNAO1, the gene encoding the major neuronal G protein Gαo, cause a spectrum of pediatric encephalopathies with seizures, motor dysfunction, and developmental delay. Of the >80 distinct missense pathogenic variants, many appear to uniformly destabilize the guanine nucleotide handling of the mutant protein, speeding up GTP uptake and deactivating GTP hydrolysis. Zinc supplementation emerges as a promising treatment option for this disease, as Zn2+ ions reactivate the GTP hydrolysis on the mutant Gαo and restore cellular interactions for some of the mutants studied earlier. The molecular etiology of GNAO1 encephalopathies needs further elucidation as a prerequisite for the development of efficient therapeutic approaches. In this work, we combine clinical and medical genetics analysis of a novel GNAO1 mutation with an in-depth molecular dissection of the resultant protein variant. We identify two unrelated patients from Norway and France with a previously unknown mutation in GNAO1, c.509C>G that results in the production of the Pro170Arg mutant Gαo, leading to severe developmental and epileptic encephalopathy. Molecular investigations of Pro170Arg identify this mutant as a unique representative of the pathogenic variants. Its 100-fold-accelerated GTP uptake is not accompanied by a loss in GTP hydrolysis; Zn2+ ions induce a previously unseen effect on the mutant, forcing it to lose the bound GTP. Our work combining clinical and molecular analyses discovers a novel, biochemically distinct pathogenic missense variant of GNAO1 laying the ground for personalized treatment development.

Clofazimine: A journey of a drug.

Among different strategies to develop novel therapies, drug repositioning (aka repurposing) aims at identifying new uses of an already approved or investigational drug. This approach has the advantages of availability of the extensive pre-existing knowledge of the drug's safety, pharmacology and toxicology, manufacturing and formulation. It provides advantages to the risk-versus-rewards trade-off as compared to the costly and time-consuming de novo drug discovery process. Clofazimine, a red-colored synthetic derivative of riminophenazines initially isolated from lichens, was first synthesized in the 1950 s, and passed through several phases of repositioning in its history as a drug. Being initially developed as an anti-tuberculosis treatment, it was repurposed for the treatment of leprosy, prior to re-repositioning for the treatment of multidrug-resistant tuberculosis and other infections. Since 1990 s, reports on the anticancer properties of clofazimine, both in vitro and in vivo, started to appear. Among the diverse mechanisms of action proposed, the activity of clofazimine as a specific inhibitor of the oncogenic Wnt signaling pathway has recently emerged as the promising targeting mechanism of the drug against breast, colon, liver, and other forms of cancer. Seventy years after the initial discovery, clofazimine's journey as a drug finding new applications continues, serving as a colorful illustration of drug repurposing in modern pharmacology.

Improved approaches to channel capacity estimation discover compromised GPCR signaling in diverse cancer cells.

Intracellular signaling orchestrates an organism's development and functioning and underlies various pathologies, such as cancer, when aberrant. A universal cell signaling characteristic is channel capacity - the measure of how much information a given transmitting system can reliably transduce. Here, we describe improved approaches to quantify GPCR signaling channel capacity in single cells, averaged across cell population. We assess the channel capacity based on distribution of residuals by the cellular response amplitude. We further develop means to handle irregularly responding cancer cells using the integral values of their response to different agonist concentrations. These approaches enabled us to analyze, for the first time, channel capacity in single cancer cells. A universal feature emerging for different cancer cell types is a decreased channel capacity of their GPCR signaling. These findings provide experimental validation to the hypothesis that cancer is an information disease, bearing importance for basic cancer biology and anticancer drug discovery.

Wnt-Independent SARS-CoV-2 Infection in Pulmonary Epithelial Cells.

The Wnt signaling pathway within host cells regulates infections by several pathogenic bacteria and viruses. Recent studies suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on ß-catenin and can be inhibited by the antileprotic drug clofazimine. Since clofazimine has been identified by us as a specific inhibitor of Wnt/ß-catenin signaling, these works could indicate a potential role of the Wnt pathway in SARS-CoV-2 infection. Here, we show that the Wnt pathway is active in pulmonary epithelial cells. However, we find that in multiple assays, SARS-CoV-2 infection is insensitive to Wnt inhibitors, including clofazimine, acting at different levels within the pathway. Our findings assert that endogenous Wnt signaling in the lung is unlikely required or involved in the SARS-CoV-2 infection and that pharmacological inhibition of this pathway with clofazimine or other compounds is not a universal way to develop treatments against the SARS-CoV-2 infection. IMPORTANCE The development of inhibitors of the SARS-CoV-2 infection remains a need of utmost importance. The Wnt signaling pathway in host cells is often implicated in infections by bacteria and viruses. In this work, we show that, despite previous indications, pharmacological modulation of the Wnt pathway does not represent a promising strategy to control SARS-CoV-2 infection in lung epithelia.

In-depth molecular profiling of an intronic GNAO1 mutant as the basis for personalized high-throughput drug screening.

BACKGROUND: The GNAO1 gene, encoding the major neuronal G protein Gαo, is mutated in a subset of pediatric encephalopathies. Most such mutations consist of missense variants. METHODS: In this study, we present a precision medicine workflow combining next-generation sequencing (NGS) diagnostics, molecular etiology analysis, and personalized drug discovery. FINDINGS: We describe a patient carrying a de novo intronic mutation (NM_020988.3:c.724-8G>A), leading to epilepsy-negative encephalopathy with motor dysfunction from the second decade. Our data show that this mutation creates a novel splice acceptor site that in turn causes an in-frame insertion of two amino acid residues, Pro-Gln, within the regulatory switch III region of Gαo. This insertion misconfigures the switch III loop and creates novel interactions with the catalytic switch II region, resulting in increased GTP uptake, defective GTP hydrolysis, and aberrant interactions with effector proteins. In contrast, intracellular localization, Gßγ interactions, and G protein-coupled receptor (GPCR) coupling of the Gαo[insPQ] mutant protein remain unchanged. CONCLUSIONS: This in-depth analysis characterizes the heterozygous c.724-8G>A mutation as partially dominant negative, providing clues to the molecular etiology of this specific pathology. Further, this analysis allows us to establish and validate a high-throughput screening platform aiming at identifying molecules that could correct the aberrant biochemical functions of the mutant Gαo. FUNDING: This work was supported by the Joint Seed Money Funding scheme between the University of Geneva and the Hebrew University of Jerusalem.

Shallow- and Deep-Water Ophiura Species Produce a Panel of Chlorin Compounds with Potent Photodynamic Anticancer Activities.

A Pacific brittle star Ophiura sarsii has previously been shown to produce a chlorin (3S,4S)-14-Ethyl-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (ETPA) (1) with potent phototoxic activities, making it applicable to photodynamic therapy. Using extensive LC-MS metabolite profiling, molecular network analysis, and targeted isolation with de novo NMR structure elucidation, we herein identify five additional chlorin compounds from O. sarsii and its deep-sea relative O. ooplax: 10S-Hydroxypheophorbide a (2), Pheophorbide a (3), Pyropheophorbide a (4), (3S,4S,21R)-14-Ethyl-9-(hydroxymethyl)-21-(methoxycarbonyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (5), and (3S,4S,21R)-14-Ethyl-21-hydroxy-9-(hydroxymethyl)-4,8,13,18-tetramethyl-20-oxo-3-phorbinepropanoic acid (6). Chlorins 5 and 6 have not been previously reported in natural sources. Interestingly, low amounts of chlorins 1-4 and 6 could also be identified in a distant species, the basket star Gorgonocephalus cf. eucnemis, demonstrating that chlorins are produced by a wide spectrum of marine invertebrates of the class Ophiuroidea. Following the purification of these major Ophiura chlorin metabolites, we discovered the significant singlet oxygen quantum yield upon their photoinduction and the resulting phototoxicity against triple-negative breast cancer BT-20 cells. These studies identify an arsenal of brittle star chlorins as natural photosensitizers with potential photodynamic therapy applications.

Wnt-pathway inhibitors with selective activity against triple-negative breast cancer: From thienopyrimidine to quinazoline inhibitors.

The Wnt-pathway has a critical role in development and tissue homeostasis and has attracted increased attention to develop anticancer drugs due to its aberrant activation in many cancers. In this study, we identified a novel small molecule series with a thienopyrimidine scaffold acting as a downstream inhibitor of the ß-catenin-dependent Wnt-pathway. This novel chemotype was investigated using Wnt-dependent triple-negative breast cancer (TNBC) cell lines. Structure activity relationship (SAR) exploration led to identification of low micromolar compounds such as 5a, 5d, 5e and a novel series with quinazoline scaffold such as 9d. Further investigation showed translation of activity to inhibit cancer survival of HCC1395 and MDA-MB-468 TNBC cell lines without affecting a non-cancerous breast epithelial cell line MCF10a. This anti-proliferative effect was synergistic to docetaxel treatment. Collectively, we identified novel chemotypes acting as a downstream inhibitor of ß-catenin-dependent Wnt-pathway that could expand therapeutic options to manage TNBC.

Restoration of the GTPase activity and cellular interactions of Gαo mutants by Zn2+ in GNAO1 encephalopathy models.

De novo point mutations in GNAO1, gene encoding the major neuronal G protein Gαo, have recently emerged in patients with pediatric encephalopathy having motor, developmental, and epileptic dysfunctions. Half of clinical cases affect codons Gly203, Arg209, or Glu246; we show that these mutations accelerate GTP uptake and inactivate GTP hydrolysis through displacement Gln205 critical for GTP hydrolysis, resulting in constitutive GTP binding by Gαo. However, the mutants fail to adopt the activated conformation and display aberrant interactions with signaling partners. Through high-throughput screening of approved drugs, we identify zinc pyrithione and Zn2+ as agents restoring active conformation, GTPase activity, and cellular interactions of the encephalopathy mutants, with negligible effects on wild-type Gαo. We describe a Drosophila model of GNAO1 encephalopathy where dietary zinc restores the motor function and longevity of the mutant flies. Zinc supplements are approved for diverse human neurological conditions. Our work provides insights into the molecular etiology of GNAO1 encephalopathy and defines a potential therapy for the patients.

Chemoenzymatic Synthesis of Original Stilbene Dimers Possessing Wnt Inhibition Activity in Triple-Negative Breast Cancer Cells Using the Enzymatic Secretome of Botrytis cinerea Pers.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancer and triple-negative breast cancer (TNBC) in particular depend upon Wnt pathway overactivation. Despite this importance, no Wnt pathway-targeting drugs are currently available, which necessitates novel approaches to search for therapeutically relevant compounds targeting this oncogenic pathway. Stilbene analogs represent an under-explored field of therapeutic natural products research. In the present work, a library of complex stilbene derivatives was obtained through biotransformation of a mixture of resveratrol and pterostilbene using the enzymatic secretome of Botrytis cinerea. To improve the chemodiversity, the reactions were performed using i-PrOH, n-BuOH, i-BuOH, EtOH, or MeOH as cosolvents. Using this strategy, a series of 73 unusual derivatives was generated distributed among 6 scaffolds; 55 derivatives represent novel compounds. The structure of each compound isolated was determined by nuclear magnetic resonance and high-resolution mass spectrometry. The inhibitory activity of the isolated compounds against the oncogenic Wnt pathway was comprehensively quantified and correlated with their capacity to inhibit the growth of the cancer cells, leading to insights into structure-activity relationships of the derivatives. Finally, we have dissected mechanistic details of the stilbene derivatives activity within the pathway.

Gαi2-induced conductin/axin2 condensates inhibit Wnt/ß-catenin signaling and suppress cancer growth.

Conductin/axin2 is a scaffold protein negatively regulating the pro-proliferative Wnt/ß-catenin signaling pathway. Accumulation of scaffold proteins in condensates frequently increases their activity, but whether condensation contributes to Wnt pathway inhibition by conductin remains unclear. Here, we show that the Gαi2 subunit of trimeric G-proteins induces conductin condensation by targeting a polymerization-inhibiting aggregon in its RGS domain, thereby promoting conductin-mediated ß-catenin degradation. Consistently, transient Gαi2 expression inhibited, whereas knockdown activated Wnt signaling via conductin. Colorectal cancers appear to evade Gαi2-induced Wnt pathway suppression by decreased Gαi2 expression and inactivating mutations, associated with shorter patient survival. Notably, the Gαi2-activating drug guanabenz inhibited Wnt signaling via conductin, consequently reducing colorectal cancer growth in vitro and in mouse models. In summary, we demonstrate Wnt pathway inhibition via Gαi2-triggered conductin condensation, suggesting a tumor suppressor function for Gαi2 in colorectal cancer, and pointing to the FDA-approved drug guanabenz for targeted cancer therapy.

Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation.

GNAO1 encephalopathy characterized by a wide spectrum of neurological deficiencies in pediatric patients originates from de novo heterozygous mutations in the gene encoding Gαo, the major neuronal G protein. Efficient treatments and even the proper understanding of the underlying etiology are currently lacking for this dominant disease. Adequate animal models of GNAO1 encephalopathy are urgently needed. Here we describe establishment and characterization of mouse models of the disease based on two point mutations in GNAO1 with different clinical manifestations. One of them is G203R leading to the early-onset epileptic seizures, motor dysfunction, developmental delay and intellectual disability. The other is C215Y producing much milder clinical outcomes, mostly-late-onset hyperkinetic movement disorder. The resultant mouse models show distinct phenotypes: severe neonatal lethality in GNAO1[G203R]/ + mice vs. normal vitality in GNAO1[C215Y]/ + . The latter model further revealed strong hyperactivity and hyperlocomotion in a panel of behavioral assays, without signs of epilepsy, recapitulating the patients' manifestations. Importantly, despite these differences the two models similarly revealed prenatal brain developmental anomalies, such as enlarged lateral ventricles and decreased numbers of neuronal precursor cells in the cortex. Thus, our work unveils GNAO1 encephalopathy as to a large extent neurodevelopmental malady. We expect that this understanding and the tools we established will be instrumental for future therapeutic developments.

Unlocking the Wnt pathway: Therapeutic potential of selective targeting FZD7 in cancer.

The Wnt signaling is of paramount pathophysiological importance. Despite showing promising anticancer activities in pre-clinical studies, current Wnt pathway inhibitors face complications in clinical trials resulting from on-target toxicity. Hence, the targeting of pathway component(s) that are essential for cancer but dispensable for normal physiology is key to the development of a safe Wnt signaling inhibitor. Frizzled7 (FZD7) is a Wnt pathway receptor that is redundant in healthy tissues but crucial in various cancers. FZD7 modulates diverse aspects of carcinogenesis, including cancer growth, metastasis, maintenance of cancer stem cells, and chemoresistance. In this review, we describe state-of-the-art knowledge of the functions of FZD7 in carcinogenesis and adult tissue homeostasis. Next, we overview the development of small molecules and biomolecules that target FZD7. Finally, we discuss challenges and possibilities in developing FZD7-selective antagonists.

Pediatric Encephalopathy: Clinical, Biochemical and Cellular Insights into the Role of Gln52 of GNAO1 and GNAI1 for the Dominant Disease.

Heterotrimeric G proteins are immediate transducers of G protein-coupled receptors-the biggest receptor family in metazoans-and play innumerate functions in health and disease. A set of de novo point mutations in GNAO1 and GNAI1, the genes encoding the α-subunits (Gαo and Gαi1, respectively) of the heterotrimeric G proteins, have been described to cause pediatric encephalopathies represented by epileptic seizures, movement disorders, developmental delay, intellectual disability, and signs of neurodegeneration. Among such mutations, the Gln52Pro substitutions have been previously identified in GNAO1 and GNAI1. Here, we describe the case of an infant with another mutation in the same site, Gln52Arg. The patient manifested epileptic and movement disorders and a developmental delay, at the onset of 1.5 weeks after birth. We have analyzed biochemical and cellular properties of the three types of dominant pathogenic mutants in the Gln52 position described so far: Gαo[Gln52Pro], Gαi1[Gln52Pro], and the novel Gαo[Gln52Arg]. At the biochemical level, the three mutant proteins are deficient in binding and hydrolyzing GTP, which is the fundamental function of the healthy G proteins. At the cellular level, the mutants are defective in the interaction with partner proteins recognizing either the GDP-loaded or the GTP-loaded forms of Gαo. Further, of the two intracellular sites of Gαo localization, plasma membrane and Golgi, the former is strongly reduced for the mutant proteins. We conclude that the point mutations at Gln52 inactivate the Gαo and Gαi1 proteins leading to aberrant intracellular localization and partner protein interactions. These features likely lie at the core of the molecular etiology of pediatric encephalopathies associated with the codon 52 mutations in GNAO1/GNAI1.

Isolation and Identification of Isocoumarin Derivatives With Specific Inhibitory Activity Against Wnt Pathway and Metabolome Characterization of Lasiodiplodia venezuelensis.

The Wnt signaling pathway controls multiple events during embryonic development of multicellular animals and is carcinogenic when aberrantly activated in adults. Breast cancers are dependent on Wnt pathway overactivation mostly through dysregulation of pathway component protein expression, which necessitates the search for therapeutically relevant compounds targeting them. Highly diverse microorganisms as endophytes represent an underexplored field in the therapeutic natural products research. In the present work, the objective was to explore the chemical diversity and presence of selective Wnt inhibitors within a unique collection of fungi isolated as foliar endophytes from the long-lived tropical palm Astrocaryum sciophilum. The fungi were cultured, extracted with ethyl acetate, and screened for their effects on the Wnt pathway and cell proliferation. The endophytic strain Lasiodiplodia venezuelensis was prioritized for scaled-up fractionation based on its selective activity. Application of geometric transfer from analytical HPLC conditions to semi-preparative scale and use of dry load sample introduction enabled the isolation of 15 pure compounds in a single step. Among the molecules identified, five are original natural products described for the first time, and six are new to this species. An active fraction obtained by semi-preparative HPLC was re-purified by UHPLC-PDA using a 1.7 µm phenyl column. 75 injections of 8 µg were necessary to obtain sufficient amounts of each compound for structure elucidation and bioassays. Using this original approach, in addition to the two major compounds, a third minor compound identified as (R)-(-)-5-hydroxymellein (18) was obtained, which was found to be responsible for the significant Wnt inhibition activity recorded. Further studies of this compound and its structural analogs showed that only 18 acts in a highly specific manner, with no acute cytotoxicity. This compound is notably selective for upstream components of the Wnt pathway and is able to inhibit the proliferation of three triple negative breast cancer cell lines. In addition to the discovery of Wnt inhibitors of interest, this study contributes to better characterize the biosynthetic potential of L. venezuelensis.

Mining Natural Compounds to Target WNT Signaling: Land and Sea Tales.

WNT signaling plays paramount roles in organism development, physiology, and disease, representing a highly attractive target for drug development. However, no WNT-modulating drugs have been approved, with several candidates trudging through the early clinical trials. This delay instigates alternative approaches to discover WNT-modulating drugs. Natural products were the source of therapeutics for centuries, but the chemical diversity they offer, especially when looking at different taxonomic groups and habitats, is still to a large extent unexplored. These considerations urge researchers to screen natural compounds for the WNT-modulatory activities. Since several reviews on such endeavors exist, we here have attempted to present these efforts as "Land and sea tales" (citing the book title by Rudyard Kipling) superimposing them onto the traditional pipeline of drug discovery and early development. In doing so, we illustrate each step of the pipeline with case studies stemming from our own research. It will become obvious that several steps of the pipeline need to be modified when applied to natural products rather than to synthetic libraries. Yet the main message of this chapter is that natural compounds represent a powerful source for the WNT signaling modulators and can be developed towards drug candidates against WNT-dependent maladies.

Short stature and combined immunodeficiency associated with mutations in RGS10.

We report the clinical and molecular phenotype of three siblings from one family, who presented with short stature and immunodeficiency and carried uncharacterized variants in RGS10 (c.489_491del:p.E163del and c.G511T:p.A171S). This gene encodes regulator of G protein signaling 10 (RGS10), a member of a large family of GTPase-activating proteins (GAPs) that targets heterotrimeric G proteins to constrain the activity of G protein-coupled receptors, including receptors for chemoattractants. The affected individuals exhibited systemic abnormalities directly related to the RGS10 mutations, including recurrent infections, hypergammaglobulinemia, profoundly reduced lymphocyte chemotaxis, abnormal lymph node architecture, and short stature due to growth hormone deficiency. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant patterns of PKA-mediated phosphorylation and increased cytosolic and cell membrane localization and activity compared to the wild-type protein. We propose that the RGS10 p.E163del and p.A171S mutations lead to mislocalization of the RGS10 protein in the cytosol, thereby resulting in attenuated chemokine signaling. This study suggests that RGS10 is critical for both immune competence and normal hormonal metabolism in humans and that rare RGS10 variants may contribute to distinct systemic genetic disorders.