Design and synthesis of C-aryl angular luotonins via a one-pot aza-Nazarov-Friedlander sequence and their Topo-I inhibition studies along with C-aryl vasicinones and luotonins.

A facile one-pot synthesis of C-ring substituted angular luotonins has been realized via a methanesulfonic acid mediated aza-Nazarov-Friedlander condensation sequence on quinazolinonyl enones. Topoisomerase I (topo-I) inhibition studies revealed that the angular luotonin library (7a-7l) and their regioisomeric analogs (linear luotonins, 8a-8l) are weak negative modulators, compared to camptothecin. These results would fare well for the design of topo-I-inert luotonins for non-oncological applications such as anti-fungal and insecticide lead developments. Surprisingly, the tricyclic vasicinones (9h, 9i, and 9j) showed better topo-I inhibition compared to pentacyclic C-aryl luotonins providing a novel pharmacophore for further explorations.

Ruthenium coordination preferences in imidazole-containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)-aminomethane/imidazole complexes.

Ruthenium is a platinoid that exhibits a range of unique chemical properties in solution, which are exploited in a variety of applications, including luminescent probes, anticancer therapies, and artificial photosynthesis. This paper focuses on a recently demonstrated ability of this metal in its +3 oxidation state to form highly stable complexes with tris (hydroxymethyl)aminomethane (H2 NC(CH2 OH)3 , Tris-base or T) and imidazole (Im) ligands, where a single RuIII cation is coordinated by two molecules of each T and Im. High-resolution electrospray ionization mass spectrometry (ESI MS) is used to characterize RuIII complexes formed by placing a RuII complex [(NH3 )5 RuII Cl]Cl in a Tris buffer under aerobic conditions. The most abundant ionic species in ESI MS represent mononuclear complexes containing an oxidized form of the metal, ie, [Xn RuIII T2 - 2H]+ , where X could be an additional T (n = 1) or NH3 (n = 0-2). Di- and tri-metal complexes also give rise to a series of abundant ions, with the highest mass ion representing a metal complex with an empirical formula Ru3 C24 O21 N6 H66 (interpreted as cyclo(T2 RuO)3 , a cyclic oxo-bridged structure, where the coordination sphere of each metal is completed by two T ligands). The empirical formulae of the binuclear species are consistent with the structures representing acyclic fragments of cyclo(T2 RuO)3 with addition of various combinations of ammonia and dioxygen as ligands. Addition of histidine in large molar excess to this solution results in complete disassembly of poly-nuclear complexes and gives rise to a variety of ionic species in the ESI mass spectrum with a general formula [RuIII Hisk Tm (NH3 )n - 2H]+ , where k = 0 to 2, m = 0 to 3, and n = 0 to 4. Ammonia adducts are present for all observed combinations of k and m, except k = m = 2, suggesting that [His2 RuIII T2 - 2H]+ represents a complex with a fully completed coordination sphere. The observed cornucopia of RuIII complexes formed in the presence of histidine is in stark contrast to the previously reported selective reactivity of imidazole, which interacts with the metal by preserving the RuT2 core and giving rise to a single abundant ruthenium complex (represented by [Im2 RuIII T2 - 2H]+ in ESI mass spectra). Surprisingly, the behavior of a hexa-histidine peptide (HHHHHH) is similar to that of a single imidazole, rather than a single histidine amino acid: The RuT2 core is preserved, with the following ionic species observed in ESI mass spectra: [HHHHHH·(RuIII T2 )m - (3m-1)H]+ (m = 1-3). The remarkable selectivity of the imidazole interaction with the RuIII T2 core is rationalized using energetic considerations at the quantum mechanical level of theory.

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional in vitro studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.

Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity.

The cysteine protease Caspase-6 (Casp6) is a potential therapeutic target of Alzheimer Disease (AD) and age-dependent cognitive impairment. To assess if Casp6 is essential to human health, we investigated the effect of CASP6 variants sequenced from healthy humans on Casp6 activity. Here, we report the effects of two rare Casp6 amino acid polymorphisms, R65W and G66R, on the catalytic function and structure of Casp6. The G66R substitution eliminated and R65W substitution significantly reduced Casp6 catalytic activity through impaired substrate binding. In contrast to wild-type Casp6, both Casp6 variants were unstable and inactive in transfected mammalian cells. In addition, Casp6-G66R acted as a dominant negative inhibitor of wild-type Casp6. The R65W and G66R substitutions caused perturbations in substrate recognition and active site organization as revealed by molecular dynamics simulations. Our results suggest that full Casp6 activity may not be essential for healthy humans and support the use of Casp6 inhibitors against Casp6-dependent neurodegeneration in age-dependent cognitive impairment and AD. Furthermore, this work illustrates that studying natural single amino acid polymorphisms of enzyme drug targets is a promising approach to uncover previously uncharacterized regulatory sites important for enzyme activity.

Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding.

Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a ß-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK a values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.

Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP.

In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).

Chemical and biological mechanisms of phytochemical activation of Nrf2 and importance in disease prevention.

Plants are an incredibly rich source of compounds that activate the Nrf2 transcription factor, leading to upregulation of a battery of cytoprotective genes. This perspective surveys established and proposed molecular mechanisms of Nrf2 activation by phytochemicals with a special emphasis on a common chemical property of Nrf2 activators: the ability as "soft" electrophiles to modify cellular thiols, either directly or as oxidized biotransformants. In addition, the role of reactive oxygen/nitrogen species as secondary messengers in Nrf2 activation is discussed. While the uniquely reactive C151 of Keap1, an Nrf2 repressor protein, is highlighted as a key target of cytoprotective phytochemicals, also reviewed are other stress-responsive proteins, including kinases, which play non-redundant roles in the activation of Nrf2 by plant-derived agents. Finally, the perspective presents two key factors accounting for the enhanced therapeutic windows of effective phytochemical activators of the Keap1-Nrf2 axis: enhanced selectivity toward sensor cysteines and reversibility of addition to thiolate molecules.

Intracellular detection and evolution of site-specific proteases using a genetic selection system.

Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.

Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system.

Upregulation of structurally homologous oncoproteins Hdm2 and Hdmx has been linked to the depletion or inactivation of their common regulation target the tumor suppressor p53 protein leading to the progression of cancer. The restoration of the p53 function, rendered suppressed or dormant by these negative regulators, establishes, therefore, a unique opportunity for a targeted induction of apoptosis in cancers that retain wild-type p53. While several small molecules have been reported to rescue the tumor suppressor by antagonizing the Hdm2-p53 interaction, these agents displayed limited application scope by being ineffective in tumors enriched with active Hdmx. Here, we describe the use of a genetic selection system and encoded library of conformationally pre-organized peptides to perform functional profiling of each regulator revealing specific recognition features that guide the antagonism of Hdm2-p53 and Hdmx-p53 interactions. Structure-activity relationship analysis of the most effective leads identified functional and structural elements mediating selective recognition of the two structurally related regulators, while providing convenient starting points for further activity optimization.

Chemoselective nitration of phenols with tert-butyl nitrite in solution and on solid support.

tert-Butyl nitrite was identified as a safe and chemoselective nitrating agent that provides preferentially mononitro derivatives of phenolic substrates in the presence of potentially competitive functional groups. On the basis of our control experiments, we propose that the reaction proceeds through the formation of O-nitrosyl intermediates prior to C-nitration via homolysis and oxidation. The reported nitration method is compatible with tyrosine-containing peptides on solid support in the synthesis of fluorogenic substrates for characterization of proteases.

Structure-activity relationships in the binding of chemically derivatized CD4 to gp120 from human immunodeficiency virus.

The first step in HIV infection is the binding of the envelope glycoprotein gp120 to the host cell receptor CD4. An interfacial "Phe43 cavity" in gp120, adjacent to residue Phe43 of gp120-bound CD4, has been suggested as a potential target for therapeutic intervention. We designed a CD4 mutant (D1D2F43C) for site-specific coupling of compounds for screening against the cavity. Altogether, 81 cysteine-reactive compounds were designed, synthesized, and tested. Eight derivatives exceeded the affinity of native D1D2 for gp120. Structure-activity relationships (SAR) for derivatized CD4 binding to gp120 revealed significant plasticity of the Phe43 cavity and a narrow entrance. The primary contacts for compound recognition inside the cavity were found to be van der Waals interactions, whereas hydrophilic interactions were detected in the entrance. This first SAR on ligand binding to an interior cavity of gp120 may provide a starting point for structure-based assembly of small molecules targeting gp120-CD4 interaction.

Investigation of an antibody-ligase. Evidence for strain-induced catalysis.

Catalytic antibody 16G3, a peptide ligase with extended substrate scope has been characterized mechanistically exploiting a set of systematically designed perspective substrates 6-9, two of which, thioesters 8 and 9 act instead as inhibitors. Taken together the structure/activity relationships suggest a catalytic mechanism dependent on induction of strain, programmed via specific structural deviations between the hapten and the substrates. General mechanistic implications for de novo induced catalysis are presented.

Peptide-small molecule hybrids via orthogonal deprotection-chemoselective conjugation to cysteine-anchored scaffolds. A model study.

The feasibility of an orthogonal deprotection-conjugation protocol, holding the promise of libraries of functionally diverse chemical probes attached to cysteine-anchored peptide scaffolds, has been explored with a model system. The necessary tools for assembly of the hybrid libraries have been prepared and the tandem procedure optimized. S-alkylation and S-sulfenylation are featured as the chemoselective ligation reactions. [reaction: see text]