Macrocephaly and developmental delay caused by missense variants in RAB5C.

Rab GTPases are important regulators of intracellular vesicular trafficking. RAB5C is a member of the Rab GTPase family that plays an important role in the endocytic pathway, membrane protein recycling and signaling. Here we report on 12 individuals with nine different heterozygous de novo variants in RAB5C. All but one patient with missense variants (n = 9) exhibited macrocephaly, combined with mild-to-moderate developmental delay. Patients with loss of function variants (n = 2) had an apparently more severe clinical phenotype with refractory epilepsy and intellectual disability but a normal head circumference. Four missense variants were investigated experimentally. In vitro biochemical studies revealed that all four variants were damaging, resulting in increased nucleotide exchange rate, attenuated responsivity to guanine exchange factors and heterogeneous effects on interactions with effector proteins. Studies in C. elegans confirmed that all four variants were damaging in vivo and showed defects in endocytic pathway function. The variant heterozygotes displayed phenotypes that were not observed in null heterozygotes, with two shown to be through a dominant negative mechanism. Expression of the human RAB5C variants in zebrafish embryos resulted in defective development, further underscoring the damaging effects of the RAB5C variants. Our combined bioinformatic, in vitro and in vivo experimental studies and clinical data support the association of RAB5C missense variants with a neurodevelopmental disorder characterized by macrocephaly and mild-to-moderate developmental delay through disruption of the endocytic pathway.

A dominant negative variant of RAB5B disrupts maturation of surfactant protein B and surfactant protein C.

Pathogenic variants in surfactant proteins SP-B and SP-C cause surfactant deficiency and interstitial lung disease. Surfactant proteins are synthesized as precursors (proSP-B, proSP-C), trafficked, and processed via a vesicular-regulated secretion pathway; however, control of vesicular trafficking events is not fully understood. Through the Undiagnosed Diseases Network, we evaluated a child with interstitial lung disease suggestive of surfactant deficiency. Variants in known surfactant dysfunction disorder genes were not found in trio exome sequencing. Instead, a de novo heterozygous variant in RAB5B was identified in the Ras/Rab GTPases family nucleotide binding domain, p.Asp136His. Functional studies were performed in Caenorhabditis elegans by knocking the proband variant into the conserved position (Asp135) of the ortholog, rab-5 Genetic analysis demonstrated that rab-5[Asp135His] is damaging, producing a strong dominant negative gene product. rab-5[Asp135His] heterozygotes were also defective in endocytosis and early endosome (EE) fusion. Immunostaining studies of the proband's lung biopsy revealed that RAB5B and EE marker EEA1 were significantly reduced in alveolar type II cells and that mature SP-B and SP-C were significantly reduced, while proSP-B and proSP-C were normal. Furthermore, staining normal lung showed colocalization of RAB5B and EEA1 with proSP-B and proSP-C. These findings indicate that dominant negative-acting RAB5B Asp136His and EE dysfunction cause a defect in processing/trafficking to produce mature SP-B and SP-C, resulting in interstitial lung disease, and that RAB5B and EEs normally function in the surfactant secretion pathway. Together, the data suggest a noncanonical function for RAB5B and identify RAB5B p.Asp136His as a genetic mechanism for a surfactant dysfunction disorder.

CemOrange2 fusions facilitate multifluorophore subcellular imaging in C. elegans.

Due to its ease of genetic manipulation and transparency, Caenorhabditis elegans (C. elegans) has become a preferred model system to study gene function by microscopy. The use of Aequorea victoria green fluorescent protein (GFP) fused to proteins or targeting sequences of interest, further expanded upon the utility of C. elegans by labeling subcellular structures, which enables following their disposition during development or in the presence of genetic mutations. Fluorescent proteins with excitation and emission spectra different from that of GFP accelerated the use of multifluorophore imaging in real time. We have expanded the repertoire of fluorescent proteins for use in C. elegans by developing a codon-optimized version of Orange2 (CemOrange2). Proteins or targeting motifs fused to CemOrange2 were distinguishable from the more common fluorophores used in the nematode; such as GFP, YFP, and mKate2. We generated a panel of CemOrange2 fusion constructs, and confirmed they were targeted to their correct subcellular addresses by colocalization with independent markers. To demonstrate the potential usefulness of this new panel of fluorescent protein markers, we showed that CemOrange2 fusion proteins could be used to: 1) monitor biological pathways, 2) multiplex with other fluorescent proteins to determine colocalization and 3) gain phenotypic knowledge of a human ABCA3 orthologue, ABT-4, trafficking variant in the C. elegans model organism.

Enantioselective total synthesis of macrolide (+)-neopeltolide.

The asymmetric total synthesis of the anti-proliferative macrolide (+)-neopeltolide has been completed. The stereochemically defined trisubstituted tetrahydropyran ring was constructed via a catalytic hetero-Diels-Alder reaction creating two new chiral centers in a highly diastereoselective manner. The other key features of this synthesis included Brown's asymmetric allylation to install the requisite C-11 and C-13 stereocenters. The synthesis of the oxazole side chain consisted of a hydrozirconation of an alkynyl stannane to establish the Z stereochemistry, followed by a palladium catalyzed cross coupling to introduce the desired Z olefin in the oxazole side chain.

Synthesis and biological evaluation of new jasplakinolide (jaspamide) analogs.

Synthesis and biological evaluation of jasplakinolide analogs are described. The synthesis of analogs utilized a diastereoselective syn-aldol reaction and an orthoester Claisen rearrangement as key steps. All synthetic analogs were evaluated for their ability to disrupt the actin cytoskeleton. Compounds 2, 3, and 4 essentially displayed similar activity to jasplakinolide.

Synthesis of Bioactive Natural Products by Asymmetric syn- and anti-Aldol Reactions.

The use of several variants of the asymmetric aldol reaction as key steps in the syntheses of bioactive target molecules is described.

Darunavir, a conceptually new HIV-1 protease inhibitor for the treatment of drug-resistant HIV.

Our structure-based design strategies which specifically target the HIV-1 protease backbone, resulted in a number of exceedingly potent nonpeptidyl inhibitors. One of these inhibitors, darunavir (TMC114), contains a privileged, structure-based designed high-affinity P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF). Darunavir has recently been approved for the treatment of HIV/AIDS patients harboring multidrug-resistant HIV-1 variants that do not respond to previously existing HAART regimens.