Photoswitchable Isoprenoid Lipids Enable Optical Control of Peptide Lipidation.

Photoswitchable lipids have emerged as attractive tools for the optical control of lipid bioactivity, metabolism, and biophysical properties. Their design is typically based on the incorporation of an azobenzene photoswitch into the hydrophobic lipid tail, which can be switched between its trans- and cis-form using two different wavelengths of light. While glycero- and sphingolipids have been successfully designed to be photoswitchable, isoprenoid lipids have not yet been investigated. Herein, we describe the development of photoswitchable analogs of an isoprenoid lipid and systematically assess their potential for the optical control of various steps in the isoprenylation processing pathway of CaaX proteins in Saccharomyces cerevisiae. One photoswitchable analog of farnesyl diphosphate (AzoFPP-1) allowed effective optical control of substrate prenylation by farnesyltransferase. The subsequent steps of isoprenylation processing (proteolysis by either Ste24 or Rce1 and carboxyl methylation by Ste14) were less affected by photoisomerization of the group introduced into the lipid moiety of the substrate a-factor, a mating pheromone from yeast. We assessed both proteolysis and methylation of the a-factor analogs in vitro and the bioactivity of a fully processed a-factor analog containing the photoswitch, exogenously added to cognate yeast cells. Combined, these data describe the first successful conversion of an isoprenoid lipid into a photolipid and suggest the utility of this approach for the optical control of protein prenylation.

The roles of the human ATP-binding cassette transporters P-glycoprotein and ABCG2 in multidrug resistance in cancer and at endogenous sites: future opportunities for structure-based drug design of inhibitors.

The ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and ABCG2 are multidrug transporters that confer drug resistance to numerous anti-cancer therapeutics in cell culture. These findings initially created great excitement in the medical oncology community, as inhibitors of these transporters held the promise of overcoming clinical multidrug resistance in cancer patients. However, clinical trials of P-gp and ABCG2 inhibitors in combination with cancer chemotherapeutics have not been successful due, in part, to flawed clinical trial designs resulting from an incomplete molecular understanding of the multifactorial basis of multidrug resistance (MDR) in the cancers examined. The field was also stymied by the lack of high-resolution structural information for P-gp and ABCG2 for use in the rational structure-based drug design of inhibitors. Recent advances in structural biology have led to numerous structures of both ABCG2 and P-gp that elucidated more clearly the mechanism of transport and the polyspecific nature of their substrate and inhibitor binding sites. These data should prove useful helpful for developing even more potent and specific inhibitors of both transporters. As such, although possible pharmacokinetic interactions would need to be evaluated, these inhibitors may show greater effectiveness in overcoming ABC-dependent multidrug resistance in combination with chemotherapeutics in carefully selected subsets of cancers. Another perhaps even more compelling use of these inhibitors may be in reversibly inhibiting endogenously expressed P-gp and ABCG2, which serve a protective role at various blood-tissue barriers. Inhibition of these transporters at sanctuary sites such as the brain and gut could lead to increased penetration by chemotherapeutics used to treat brain cancers or other brain disorders and increased oral bioavailability of these agents, respectively.

Potential Tools for Eradicating HIV Reservoirs in the Brain: Development of Trojan Horse Prodrugs for the Inhibition of P-Glycoprotein with Anti-HIV-1 Activity.

Combination antiretroviral therapy is the mainstay of HIV treatment, lowering plasma viral levels below detection. However, eradication of HIV is a major challenge due to cellular and anatomical viral reservoirs that are often protected from treatment by efflux transporters, such as P-glycoprotein (P-gp) at the blood-brain barrier (BBB). Herein we described a Trojan horse approach to therapeutic evasion of P-gp based on a reversibly linked combination of HIV reverse transcriptase and protease inhibitors. Potent inhibition of P-gp efflux in cells, including human brain endothelial cells, was observed with the linked heterodimeric compounds. In vitro regeneration of active monomeric drugs was observed in a reducing environment with these dimeric prodrugs, with the superior leaving group promoting more facile release from the tether. These release trends were mirrored in the efficacy of the in cyto anti-HIV-1 activity of the Trojan horse heterodimers.

Methyltransferase-like 21c methylates and stabilizes the heat shock protein Hspa8 in type I myofibers in mice.

Protein methyltransferases mediate posttranslational modifications of both histone and nonhistone proteins. Whereas histone methylation is well-known to regulate gene expression, the biological significance of nonhistone methylation is poorly understood. Methyltransferase-like 21c (Mettl21c) is a newly classified nonhistone lysine methyltransferase whose in vivo function has remained elusive. Using a Mettl21cLacZ knockin mouse model, we show here that Mettl21c expression is absent during myogenesis and restricted to mature type I (slow) myofibers in the muscle. Using co-immunoprecipitation, MS, and methylation assays, we demonstrate that Mettl21c trimethylates heat shock protein 8 (Hspa8) at Lys-561 to enhance its stability. As such, Mettl21c knockout reduced Hspa8 trimethylation and protein levels in slow muscles, and Mettl21c overexpression in myoblasts increased Hspa8 trimethylation and protein levels. We further show that Mettl21c-mediated stabilization of Hspa8 enhances its function in chaperone-mediated autophagy, leading to degradation of client proteins such as the transcription factors myocyte enhancer factor 2A (Mef2A) and Mef2D. In contrast, Mettl21c knockout increased Mef2 protein levels in slow muscles. These results identify Hspa8 as a Mettl21c substrate and reveal that nonhistone methylation has a physiological function in protein stabilization.

A Quantitative FRET Assay for the Upstream Cleavage Activity of the Integral Membrane Proteases Human ZMPSTE24 and Yeast Ste24.

The integral membrane protease ZMPSTE24 plays an important role in the lamin A maturation pathway. ZMPSTE24 is the only known enzyme to cleave the last 15 residues from the C-terminus of prelamin A, including a farnesylated and carboxyl methylated cysteine. Mutations in ZMPSTE24 lead to progeroid diseases with abnormal prelamin A accumulation in the nucleus. Ste24 is the yeast functional homolog of ZMPSTE24 and similarly cleaves the a-factor pheromone precursor during its posttranslational maturation. To complement established qualitative techniques used to detect the upstream enzymatic cleavage by ZMPSTE24 and Ste24, including gel-shift assays and mass spectrometry analyses, we developed an enzymatic in vitro FRET-based assay to quantitatively measure the upstream cleavage activities of these two enzymes. This assay uses either purified enzyme or enzyme in crude membrane preparations and a 33-amino acid a-factor analog peptide that is a substrate for both Ste24 and ZMPSTE24. This peptide contains a fluorophore (2-aminobenzoic acid-Abz) at its N-terminus and a quencher moiety (dinitrophenol-DNP) positioned four residues downstream from the cleavage site. Upon cleavage, a fluorescent signal is generated in real time at 420 nm that is proportional to cleavage of the peptide and these kinetic data are used to quantify activity. This assay should provide a useful tool for kinetic analysis and for studying the catalytic mechanism of both ZMPSTE24 and Ste24.

Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

Skeletal muscles contain heterogeneous myofibers that are different in size and contractile speed, with type IIb myofiber being the largest and fastest. Here, we identify methyltransferase-like 21e (Mettl21e), a member of newly classified nonhistone methyltransferases, as a gene enriched in type IIb myofibers. The expression of Mettl21e was strikingly up-regulated in hypertrophic muscles and during myogenic differentiation in vitro and in vivo. Knockdown (KD) of Mettl21e led to atrophy of cultured myotubes, and targeted mutation of Mettl21e in mice reduced the size of IIb myofibers without affecting the composition of myofiber types. Mass spectrometry and methyltransferase assay revealed that Mettl21e methylated valosin-containing protein (Vcp/p97), a key component of the ubiquitin-proteasome system. KD or knockout of Mettl21e resulted in elevated 26S proteasome activity, and inhibition of proteasome activity prevented atrophy of Mettl21e KD myotubes. These results demonstrate that Mettl21e functions to maintain myofiber size through inhibiting proteasome-mediated protein degradation.-Wang, C., Zhang, B., Ratliff, A. C., Arrington, J., Chen, J., Xiong, Y., Yue, F., Nie, Y., Hu, K., Jin, W., Tao, W. A., Hrycyna, C. A., Sun, X., Kuang, S. Methyltransferase-like 21e inhibits 26S proteasome activity to facilitate hypertrophy of type IIb myofibers.

ZMPSTE24 missense mutations that cause progeroid diseases decrease prelamin A cleavage activity and/or protein stability.

The human zinc metalloprotease ZMPSTE24 is an integral membrane protein crucial for the final step in the biogenesis of the nuclear scaffold protein lamin A, encoded by LMNA After farnesylation and carboxyl methylation of its C-terminal CAAX motif, the lamin A precursor (prelamin A) undergoes proteolytic removal of its modified C-terminal 15 amino acids by ZMPSTE24. Mutations in LMNA or ZMPSTE24 that impede this prelamin A cleavage step cause the premature aging disease Hutchinson-Gilford progeria syndrome (HGPS), and the related progeroid disorders mandibuloacral dysplasia type B (MAD-B) and restrictive dermopathy (RD). Here, we report the development of a 'humanized yeast system' to assay ZMPSTE24-dependent cleavage of prelamin A and examine the eight known disease-associated ZMPSTE24 missense mutations. All mutations show diminished prelamin A processing and fall into three classes, with defects in activity, protein stability or both. Notably, some ZMPSTE24 mutants can be rescued by deleting the E3 ubiquitin ligase Doa10, involved in endoplasmic reticulum (ER)-associated degradation of misfolded membrane proteins, or by treatment with the proteasome inhibitor bortezomib. This finding may have important therapeutic implications for some patients. We also show that ZMPSTE24-mediated prelamin A cleavage can be uncoupled from the recently discovered role of ZMPSTE24 in clearance of ER membrane translocon-clogged substrates. Together with the crystal structure of ZMPSTE24, this humanized yeast system can guide structure-function studies to uncover mechanisms of prelamin A cleavage, translocon unclogging, and membrane protein folding and stability.

a-Factor Analogues Containing Alkyne- and Azide-Functionalized Isoprenoids Are Efficiently Enzymatically Processed and Retain Wild-Type Bioactivity.

Protein prenylation is a post-translational modification that involves the addition of one or two isoprenoid groups to the C-terminus of selected proteins using either farnesyl diphosphate or geranylgeranyl diphosphate. Three crucial enzymatic steps are involved in the processing of prenylated proteins to yield the final mature product. The farnesylated dodecapeptide, a-factor, is particularly useful for studies of protein prenylation because it requires the identical three-step process to generate the same C-terminal farnesylated cysteine methyl ester substructure present in larger farnesylated proteins. Recently, several groups have developed isoprenoid analogs bearing azide and alkyne groups that can be used in metabolic labeling experiments. Those compounds have proven useful for profiling prenylated proteins and also show great promise as tools to study how the levels of prenylated proteins vary in different disease models. Herein, we describe the preparation and use of prenylated a-factor analogs, and precursor peptides, to investigate two key questions. First, a-factor analogues containing modified isoprenoids were prepared to evaluate whether the non-natural lipid group interferes with the biological activity of the a-factor. Second, a-factor-derived precursor peptides were synthesized to evaluate whether they can be efficiently processed by the yeast proteases Rce1 and Ste24 as well as the yeast methyltransferase Ste14 to yield mature a-factor analogues. Taken together, the results reported here indicate that metabolic labeling experiments with azide- and alkyne-functionalized isoprenoids can yield prenylated products that are fully processed and biologically functional. Overall, these observations suggest that the isoprenoids studied here that incorporate bio-orthogonal functionality can be used in metabolic labeling experiments without concern that they will induce undesired physiological changes that may complicate data interpretation.

Dual inhibitors of the human blood-brain barrier drug efflux transporters P-glycoprotein and ABCG2 based on the antiviral azidothymidine.

The brain provides a sanctuary site for HIV due, in part, to poor penetration of antiretroviral agents at the blood-brain barrier. This lack of penetration is partially attributed to drug efflux transporters such as P-glycoprotein (P-gp) and ABCG2. Inhibition of both ABCG2 and P-gp is critical for enhancing drug accumulation into the brain. In this work, we have developed a class of homodimers based on the HIV reverse transcriptase inhibitor azidothymidine (AZT) that effectively inhibits P-gp and ABCG2. These agents block transporter mediated efflux of the P-gp substrate calcein-AM and the ABCG2 substrate mitoxantrone. The homodimers function by interacting with the transporter drug binding sites as demonstrated by competition studies with the photo-affinity agent and P-gp/ABCG2 substrate [125I]iodoarylazidoprazosin. As such, these dual inhibitors of both efflux transporters provide a model for the future development of delivery vehicles for antiretroviral agents to the brain.

Dual Modulation of Human P-Glycoprotein and ABCG2 with Prodrug Dimers of the Atypical Antipsychotic Agent Paliperidone in a Model of the Blood-Brain Barrier.

Many atypical antipsychotic drugs currently prescribed for the treatment of schizophrenia have limited brain penetration due to the efflux activity of ATP-binding cassette (ABC) transporters at the blood-brain barrier (BBB), including P-glycoprotein (P-gp) and ABCG2. Herein, we describe the design and synthesis of the first class of homodimeric prodrug dual inhibitors of P-gp and ABCG2. These inhibitors are based on the structure of the atypical antipsychotic drug paliperidone (Pal), a transport substrate for both transporters. We synthesized and characterized a small library of homodimeric bivalent Pal inhibitors that contain a variety of tethers joining the two monomers via ester linkages. The majority of our compounds were low micromolar to sub-micromolar inhibitors of both P-gp and ABCG2 in cells overexpressing these transporters and in immortalized human hCMEC/D3 cells that are derived from the BBB. Our most potent dual inhibitor also contained an internal disulfide bond in the tether (Pal-8SS) that allowed for rapid reversion to monomer in the presence of reducing agents or plasma esterases. To increase stability against these esterases, we further engineered Pal-8SS to contain two hindering methyl groups alpha to the carbonyl of the ester moiety within the tether. The resulting dimer, Pal-8SSMe, was also a potent dual inhibitor that remained susceptible to reducing conditions but was more resistant to breakdown in human plasma. Importantly, Pal-8SSMe both accumulated and subsequently reverted to the therapeutic Pal monomer in the reducing environment of BBB cells. Thus, these molecules serve two purposes, acting as both inhibitors of P-gp and ABCG2 at the BBB and as prodrugs, effectively delivering therapies to the brain that would otherwise be precluded.

Non-Substrate Based, Small Molecule Inhibitors of the Human Isoprenylcysteine Carboxyl Methyltransferase.

Activating mutations of human K-Ras proteins are among the most common oncogenic mutations, present in approximately 30% of all human cancers. Posttranslational modifications to K-Ras guide it to the plasma membrane and disruption of this localization inhibits the growth of Ras-driven cancers. The human isoprenylcysteine carboxyl methyltransferase (hIcmt) enzyme catalyzes the final α-carboxyl methylesterification of the C-terminal farnesyl cysteine of K-Ras, which is necessary for its proper localization. Thus, hIcmt inhibition is a regarded as a promising cancer therapy. A high quality inhibitor of hIcmt with in vivo activity would advance hIcmt research and drug development. Herein, Wwe report the results of a screen for small molecule hIcmt inhibitors in a library of molecules that were not hIcmt substrate analogs. The lead compound identified by this screen (1) was modified to remove chemical liabilities and to increase potency. The most potent resulting compound (5) inhibited hIcmt in vitro with low micromolar potency (IC50 = 1.5 ± 0.2 µM) and was kinetically characterized as a competitive inhibitor for prenylated substrates and a non-competitive inhibitor for the cofactor and methyl donor S-adenosylmethionine (SAM). These inhibitors offer important structure activity relationships for the future development of hIcmt inhibitors with in vivo activity.

AIM2 inflammasome is activated by pharmacological disruption of nuclear envelope integrity.

Inflammasomes are critical sensors that convey cellular stress and pathogen presence to the immune system by activating inflammatory caspases and cytokines such as IL-1ß. The nature of endogenous stress signals that activate inflammasomes remains unclear. Here we show that an inhibitor of the HIV aspartyl protease, Nelfinavir, triggers inflammasome formation and elicits an IL-1R-dependent inflammation in mice. We found that Nelfinavir impaired the maturation of lamin A, a structural component of the nuclear envelope, thereby promoting the release of DNA in the cytosol. Moreover, deficiency of the cytosolic DNA-sensor AIM2 impaired Nelfinavir-mediated inflammasome activation. These findings identify a pharmacologic activator of inflammasome and demonstrate the role of AIM2 in detecting endogenous DNA release upon perturbation of nuclear envelope integrity.

Diazirine-containing photoactivatable isoprenoid: synthesis and application in studies with isoprenylcysteine carboxyl methyltransferase.

Photoaffinity labeling is a useful technique employed to identify protein-ligand and protein-protein noncovalent interactions. Photolabeling experiments have been particularly informative for probing membrane-bound proteins where structural information is difficult to obtain. The most widely used classes of photoactive functionalities include aryl azides, diazocarbonyls, diazirines, and benzophenones. Diazirines are intrinsically smaller than benzophenones and generate carbenes upon photolysis that react with a broader range of amino acid side chains compared with the benzophenone-derived diradical; this makes diazirines potentially more general photoaffinity-labeling agents. In this article, we describe the development and application of a new isoprenoid analogue containing a diazirine moiety that was prepared in six steps and incorporated into an a-factor-derived peptide produced via solid-phase synthesis. In addition to the diazirine moiety, fluorescein and biotin groups were also incorporated into the peptide to aid in the detection and enrichment of photo-cross-linked products. This multifuctional diazirine-containing peptide was a substrate for Ste14p, the yeast homologue of the potential anticancer target Icmt, with K(m) (6.6 µM) and V(max) (947 pmol min(-1) mg(-1)) values comparable or better than a-factor peptides functionalized with benzophenone-based isoprenoids. Photo-cross-linking experiments demonstrated that the diazirine probe photo-cross-linked to Ste14p with observably higher efficiency than benzophenone-containing a-factor peptides.

Reversible dimers of the atypical antipsychotic quetiapine inhibit p-glycoprotein-mediated efflux in vitro with increased binding affinity and in situ at the blood-brain barrier.

The multidrug resistance transporter P-glycoprotein (P-gp) is highly expressed in the capillary endothelial cells of the blood-brain barrier (BBB) where it functions to limit the brain penetration of many drugs, including antipsychotic agents used to treat schizophrenia. Therefore, in an effort to inhibit the transporter, we designed dimers of the antipsychotic drug and P-gp substrate quetiapine (QT), linked by variable length tethers. In P-gp overexpressing cells and in human brain capillary endothelial hCMEC/D3 cells, the dimer with the shortest tether length (QT2C2) (1) was the most potent inhibitor showing >80-fold better inhibition of P-gp-mediated transport than monomeric QT. The dimers, which are linked via ester moieties, are designed to revert to the therapeutic monomer once inside the target cells. We demonstrated that the addition of two sterically blocking methyl groups to the linker (QT2C2Me2, 8) increased the half-life of the molecule in plasma 10-fold as compared to the dimer lacking methyl groups (QT2C2, 1), while retaining inhibitory potency for P-gp transport and sensitivity to cellular esterases. Experiments with purified P-gp demonstrated that QT2C2 (1) and QT2C2Me2 (8) interacted with both the H- and R-binding sites of the transporter with binding affinities 20- to 30-fold higher than that of monomeric QT. Using isolated rat brain capillaries, QT2C2Me2 (8) was a more potent inhibitor of P-gp transport than QT. Lastly, we showed that QT2C2Me2 (8) increased the accumulation of the P-gp substrate verapamil in rat brain in situ three times more than QT. Together, these results indicate that the QT dimer QT2C2Me2 (8) strongly inhibited P-gp transport activity in human brain capillary endothelial cells, in rat brain capillaries, and at the BBB in an animal model.

Quinine dimers are potent inhibitors of the Plasmodium falciparum chloroquine resistance transporter and are active against quinoline-resistant P. falciparum.

Chloroquine (CQ) resistance in the human malaria parasite Plasmodium falciparum is primarily conferred by mutations in the "chloroquine resistance transporter" (PfCRT). The resistance-conferring form of PfCRT (PfCRT(CQR)) mediates CQ resistance by effluxing the drug from the parasite's digestive vacuole, the acidic compartment in which CQ exerts its antiplasmodial effect. PfCRT(CQR) can also decrease the parasite's susceptibility to other quinoline drugs, including the current antimalarials quinine and amodiaquine. Here we describe interactions between PfCRT(CQR) and a series of dimeric quinine molecules using a Xenopus laevis oocyte system for the heterologous expression of PfCRT and using an assay that detects the drug-associated efflux of H(+) ions from the digestive vacuole in parasites that harbor different forms of PfCRT. The antiplasmodial activities of dimers 1 and 6 were also examined in vitro (against drug-sensitive and drug-resistant strains of P. falciparum) and in vivo (against drug-sensitive P. berghei). Our data reveal that the quinine dimers are the most potent inhibitors of PfCRT(CQR) reported to date. Furthermore, the lead compounds (1 and 6) were not effluxed by PfCRT(CQR) from the digestive vacuole but instead accumulated to very high levels within this organelle. Both 1 and 6 exhibited in vitro antiplasmodial activities that were inversely correlated with CQ. Moreover, the additional parasiticidal effect exerted by 1 and 6 in the drug-resistant parasites was attributable, at least in part, to their ability to inhibit PfCRT(CQR). This highlights the potential for devising new antimalarial therapies that exploit inherent weaknesses in a key resistance mechanism of P. falciparum.

Homodimers of the Antiviral Abacavir as Modulators of P-glycoprotein Transport in Cell Culture: Probing Tether Length.

A major hurdle in permanently eliminating HIV from the body is the persistence of viral reservoirs, including those of the brain. One potential strategy towards eradicating HIV reservoirs of the brain is to block efflux transporters, such as P-glycoprotein (P-gp), that contribute to the limited penetration of antiviral agents across the blood-brain barrier (BBB). Herein, we described a series of dimeric inhibitors of P-gp based on the nucleoside reverse transcriptase inhibitor and P-gp substrate, abacavir. Varying tether lengths were used to generate abacavir dimers to probe tether requirements for inhibitory potency. These dimeric agents were evaluated in two cell lines that express P-gp at varying levels: a P-gp over-expressing CD4+ T-lymphocyte cell line (12D7-MDR) and a human brain capillary endothelial cell line as an in vitro model of the BBB (hCMEC/D3) that expresses endogenous levels of P-gp. All dimeric abacavir analogs were inhibitors of P-gp efflux in the two cell lines with potencies that varied with tether length; the most potent agents displayed low micromolar inhibition. P-gp inhibition in a highly P-gp over-expressing cell line (MCF-7/DX1) was also observed with a range of therapeutic substrates. Competition studies with the photoaffinity substrate [125I]iodoarylazidoprazosin demonstrated that abacavir dimers act by competing for the substrate binding sites of P-gp. These data demonstrate that the tether length of dimeric abacavir derivatives has a significant effect on inhibition of P-gp drug efflux, with up to a 35-fold increase in potency observed with longer tether linkages.

Competency-based reforms of the undergraduate biology curriculum: integrating the physical and biological sciences.

The National Experiment in Undergraduate Science Education project funded by the Howard Hughes Medical Institute is a direct response to the Scientific Foundations for Future Physicians report, which urged a shift in premedical student preparation from a narrow list of specific course work to a more flexible curriculum that helps students develop broad scientific competencies. A consortium of four universities is working to create, pilot, and assess modular, competency-based curricular units that require students to use higher-order cognitive skills and reason across traditional disciplinary boundaries. Purdue University; the University of Maryland, Baltimore County; and the University of Miami are each developing modules and case studies that integrate the biological, chemical, physical, and mathematical sciences. The University of Maryland, College Park, is leading the effort to create an introductory physics for life sciences course that is reformed in both content and pedagogy. This course has prerequisites of biology, chemistry, and calculus, allowing students to apply strategies from the physical sciences to solving authentic biological problems. A comprehensive assessment plan is examining students' conceptual knowledge of physics, their attitudes toward interdisciplinary approaches, and the development of specific scientific competencies. Teaching modules developed during this initial phase will be tested on multiple partner campuses in preparation for eventual broad dissemination.

S-Farnesyl-Thiopropionic Acid (FTPA) Triazoles as Potent Inhibitors of Isoprenylcysteine Carboxyl Methyltransferase.

We report the design and synthesis of novel FTPA-triazole compounds as potent inhibitors of isoprenylcysteine carboxyl methyltransferase (Icmt), through a focus on thioether and isoprenoid mimetics. These mimetics were coupled utilizing a copper-assisted cycloaddition to assemble the potential inhibitors. Using the resulting triazole from the coupling as an isoprenyl mimetic resulted in the biphenyl substituted FTPA triazole 10n. This lipid-modified analog is a potent inhibitor of Icmt (IC(50) = 0.8 ± 0.1 µM; calculated K(i) = 0.4 µM).

Human ZMPSTE24 disease mutations: residual proteolytic activity correlates with disease severity.

The zinc metalloprotease ZMPSTE24 plays a critical role in nuclear lamin biology by cleaving the prenylated and carboxylmethylated 15-amino acid tail from the C-terminus of prelamin A to yield mature lamin A. A defect in this proteolytic event, caused by a mutation in the lamin A gene (LMNA) that eliminates the ZMPSTE24 cleavage site, underlies the premature aging disease Hutchinson-Gilford Progeria Syndrome (HGPS). Likewise, mutations in the ZMPSTE24 gene that result in decreased enzyme function cause a spectrum of diseases that share certain features of premature aging. Twenty human ZMPSTE24 alleles have been identified that are associated with three disease categories of increasing severity: mandibuloacral dysplasia type B (MAD-B), severe progeria (atypical 'HGPS') and restrictive dermopathy (RD). To determine whether a correlation exists between decreasing ZMPSTE24 protease activity and increasing disease severity, we expressed mutant alleles of ZMPSTE24 in yeast and optimized in vivo yeast mating assays to directly compare the activity of alleles associated with each disease category. We also measured the activity of yeast crude membranes containing the ZMPSTE24 mutant proteins in vitro. We determined that, in general, the residual activity of ZMPSTE24 patient alleles correlates with disease severity. Complete loss-of-function alleles are associated with RD, whereas retention of partial, measureable activity results in MAD-B or severe progeria. Importantly, our assays can discriminate small differences in activity among the mutants, confirming that the methods presented here will be useful for characterizing any new ZMPSTE24 mutations that are discovered.