A lack of target specificity has greatly hindered the success of inhibitor development against matrix metalloproteinases (MMPs) for the treatment of various cancers. The MMP catalytic domains are highly conserved, whereas the hemopexin-like domains of MMPs are unique to each family member. The hemopexin-like domain of MMP-9 enhances cancer cell migration through self-interaction and heterointeractions with cell surface proteins including CD44 and α4β1 integrin. These interactions activate EGFR-MAP kinase dependent signaling that leads to cell migration. In this work, we generated a library of compounds, based on hit molecule N-[4-(difluoromethoxy)phenyl]-2-[(4-oxo-6-propyl-1H-pyrimidin-2-yl)sulfanyl]-acetamide, that target the hemopexin-like domain of MMP-9. We identify N-(4-fluorophenyl)-4-(4-oxo-3,4,5,6,7,8-hexahydroquinazolin-2-ylthio)butanamide, 3c, as a potent lead (Kd = 320 nM) that is specific for binding to the proMMP-9 hemopexin-like domain. We demonstrate that 3c disruption of MMP-9 homodimerization prevents association of proMMP-9 with both α4β1 integrin and CD44 and results in the dissociation of EGFR. This disruption results in decreased phosphorylation of Src and its downstream target proteins focal adhesion kinase (FAK) and paxillin (PAX), which are implicated in promoting tumor cell growth, migration, and invasion. Using a chicken chorioallantoic membrane in vivo assay, we demonstrate that 500 nM 3c blocks cancer cell invasion of the basement membrane and reduces angiogenesis. In conclusion, we present a mechanism of action for 3c whereby targeting the hemopexin domain results in decreased cancer cell migration through simultaneous disruption of α4β1 integrin and EGFR signaling pathways, thereby preventing signaling bypass. Targeting through the hemopexin-like domain is a powerful approach to antimetastatic drug development.

As a prerequisite to mammalian fertilization, the sperm acrosomal vesicle fuses with the plasma membrane and the acrosome contents are exocytosed. Induction occurs through engagement of the sperm receptors by multiple sugar residues. Multivalent polymers displaying mannose, fucose, or GlcNAc are effective synthetic inducers of mouse sperm acrosomal exocytosis (AE). Each carbohydrate is proposed to have a distinct binding site on the sperm cell surface. To determine the role of the scaffold structure in the efficiency of AE induction, different polymer backbones were employed to display the different activating sugar residues. These glycopolymers were prepared by ruthenium-catalyzed ring-opening metathesis of 5-substituted norbornene or cyclooctene. The conformations of the glycopolymers were characterized by small-angle X-ray scattering. Polynorbornene displaying mannose, fucose, or GlcNAc forms flexible cylinders in aqueous solution. However, polycyclooctenes displaying any of these same sugars are much more flexible and form random coils. The flexible polycyclooctenes displaying fucose or GlcNAc were less effective inducers of AE than their norbornene counterparts. In contrast, polycyclooctene displaying mannose was the most effective AE inducer and had a more collapsed spherelike structure. Our results suggest that the AE efficacy of fucose, GlcNAc, and mannose polymers relies on a relatively rigid polymer that can stabilize receptor signaling complexes.

Investigation of complex molecular systems depends on our ability to correlate physical measurements with molecular structure. Interpretation of studies that rely on synthetic polymers is generally limited by their heterogeneity; i.e., there is variation in the number and arrangement of the monomeric building blocks that have been incorporated. Superior physics and biology can be performed with materials and tools that exert precise control over the sequence and spacing of functional groups.An interest in functional ligands combined with a desire to control the orientation and stereochemistry of monomer incorporation led to the design of new substrates for ruthenium-catalyzed ring-opening metathesis polymerization (ROMP). We discovered that ROMP of cyclobutene-1-carboxamides provides uniform and translationally invariant polymers. In contrast, cyclobutene-1-carboxylate esters ring open upon treatment with ruthenium catalyst, but they are stable to homopolymerization. However, in the presence of cyclohexene monomers, they undergo alternating ROMP (AROMP or alt-ROMP) to give copolymers with a precisely controlled sequence.The alternating cyclobutene ester/cyclohexene pair provides access to functional group spacing larger than is possible with homopolymers. This can be desirable; for example, polymers with a regular 8–10 Å backbone spacing of cationic charge and with between four and eight cationic groups were the most effective antibacterial agents and had low cytotoxicity.Moreover, the AROMP chemistry allows alternation of two functional moieties: one associated with the cyclohexene and one attached to the cyclobutene. In the case of antibacterial copolymers, the alternating chemistry allowed variation of hydrophobicity via the cyclohexene while maintaining a constant cation spacing through the cyclobutene. In the case of copolymers that bear donor and acceptor groups, strict alternation of the groups increased intrachain charge transfer.Like cyclobutene-1-carboxylate esters, bicyclo[4.2.0]oct-7-ene-7-carboxylate esters ring open upon treatment with ruthenium catalyst and undergo ring opening cross-metathesis with cyclohexene to form alternating copolymers. The corresponding bicyclo[4.2.0]oct-7-ene-7-carboxyamides isomerize to the bicyclo[4.2.0]oct-1(8)-ene-8-carboxamides before they can ring open. However, the isomerized amides undergo ruthenium-catalyzed ring opening metathesis and rapidly AROMP with cyclohexene.Our alternating copolymer systems allow functionality to be placed along a polymer chain with larger than typical spacing. We have used both homopolymers and alternating copolymers for defining the functional group density required for targeting a cell surface and for the exploration of functional group positioning within a polymer chain. These polymer systems provide access to new materials with previously inaccessible types of nanoscale structures.

Labeling proteins with biotin is a widely used method to identify target proteins due to biotin's strong binding affinity for streptavidin. Combined with alkyne–azide cycloaddition, which enables the coupling of probes to targeted proteins, biotin tags linked to an alkyne or azide have become a powerful tool for purification and analysis of proteins in proteomics. However, biotin requires harsh elution conditions to release the captured protein from the bead matrix. Use of these conditions reduces signal to noise and complicates the analysis. To improve affinity capture, cleavable linkers have been introduced. Here, we demonstrate the use of a cyclic acetal biotin probe that is prepared easily from commercially available starting materials, is stable to cell lysates, yet is cleaved under mildly acidic conditions, and which provides an aldehyde for further elaboration of the protein, if desired.

Metabolism of cholesterol by Mycobacterium tuberculosis (Mtb) contributes to its pathogenesis. We show that ChsE4-ChsE5 (Rv3504/Rv3505) specifically catalyzes dehydrogenation of the (25S)-3-oxo-cholest-4-en-26-oyl-CoA diastereomer in cholesterol side chain β-oxidation. Thus, a dichotomy between the supply of both 25R and 25S metabolic precursors by upstream cytochrome P450s and the substrate stereospecificity of ChsE4-ChsE5 exists. We reconcile the dilemma of 25R metabolite production by demonstrating that mycobacterial MCR (Rv1143) can efficiently epimerize C25 diastereomers of 3-oxo-cholest-4-en-26-oyl-CoA. Our data suggest that cholesterol and cholesterol ester precursors can converge into a single catabolic pathway, thus widening the metabolic niche in which Mtb survives.

We describe an isomerization–alternating ROMP protocol that gives linear copolymers with rigorous sequence alternation. Bicyclo[4.2.0]oct-7-ene-7-carboxamides of primary amines are isomerized in the presence of (3-BrPyr)2Cl2(H2IMes)Ru═CHPh to the corresponding bicyclo[4.2.0]oct-1(8)-ene-8-carboxamides in which the olefinic bond is tetrasubstituted. The isomerized amides undergo alternating ring-opening metathesis polymerization with cyclohexene to provide soluble and linear copolymers with molecular weights up to ∼130 kDa. This process provides efficient entry to strictly alternating copolymers that can display diverse functional groups.

The metabolism of host cholesterol by Mycobacterium tuberculosis (Mtb) is an important factor for both its virulence and pathogenesis, although how and why cholesterol metabolism is required is not fully understood. Mtb uses a unique set of catabolic enzymes that are homologous to those required for classical β-oxidation of fatty acids but are specific for steroid-derived substrates. Here, we identify and assign the substrate specificities of two of these enzymes, ChsE4-ChsE5 (Rv3504-Rv3505) and ChsE3 (Rv3573c), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The functional redundancy of ChsE4-ChsE5 explains the in vivo phenotype of the igr knockout strain of Mycobacterium tuberculosis; the loss of ChsE1-ChsE2 can be compensated for by ChsE4-ChsE5 during the chronic phase of infection. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. A comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side chain degradation by Mtb.Keywords: 17-hydroxy-3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA; 3-oxo-23,24-bisnorchol-4,17-dien-22-oyl-CoA; 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA; cholesterol metabolism; drug target; tuberculosis

The sperm acrosome reaction (AR), an essential exocytosis step in mammalian fertilization, is mediated by a species-specific interaction of sperm surface molecules with glycans on the egg. Previous studies indicate that a subset of terminal carbohydrates on the mouse egg zona pellucida (ZP) trigger the AR by cross-linking or aggregating receptors on the sperm membrane. However, the exact role of those carbohydrates in AR has not been identified and the mechanism underlying the AR still needs further investigation. To study this process, a series of glycopolymers was synthesized. The glycopolymers are composed of a multivalent scaffold (norbornene), a functional ligand (previously identified ZP terminal monosaccharides), and a linker connecting the ligand and the scaffold. The polymers were tested for their ability to initiate AR and through which signaling pathways AR induction occurred. Our data demonstrate that mannose, fucose, and β-N-acetylglucosamine 10-mers and 100-mers initiate AR in a dose-dependent manner, and the 100-mers are more potent on a per monomer basis than the 10-mers. Although nearly equipotent in inducing the AR at the optimal concentrations, their AR activation kinetics are not identical. Similar to mouse ZP3, all 100-mer-activated AR are sensitive to guanine-binding regulatory proteins (G-proteins), tyrosine kinase, protein kinase A, protein kinase C, and Ca2+-related antagonists. Thus, the chemotypes of synthetic glycopolymers imitate the physiologic AR-activation agents and provide evidence that occupation of one of at least three different receptor binding sites is sufficient to initiate the AR.

The Mycobacterium tuberculosis (Mtb) igr operon plays an essential role in Mtb cholesterol metabolism, which is critical for pathogenesis during the latent stage of Mtb infection. Here we report the first structure of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, 3-oxo-4,17-pregnadiene-20-carboxyl-CoA, in the modified β-oxidation pathway for cholesterol side chain degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2N reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. The unique αβ heterodimer architecture utilized by ChsH1-ChsH2 to bind its distinctive substrate highlights an opportunity for the development of new antimycobacterial drugs that target a pathway specific to Mtb.

The Mycobacterium tuberculosis Rv3409c gene is required for modulation of the Toll-like receptor 2 (TLR-2) signaling response in infected macrophages. Although each is annotated as encoding a cholesterol oxidase, neither Rv3409c nor its ortholog MSMEG1604 is required for the metabolism of cholesterol in mycobacteria. Here we report that a unique lipid, L1334, accumulates in a MSMEG1604 transposon mutant in the Mycobacterium smegmatis cell envelope. L1334 is a polar glycopeptidolipid that is hyperrhamnosylated and in which the 6-deoxytalose moiety is not acetylated. The alteration of L1334 acetylation is consistent with a reduced level of interference with TLR-2 signaling in mutant infected macrophages.

Strained bicyclic carbomethoxy olefins were utilized as substrates in alternating ring-opening metathesis polymerization and found to provide low-dispersity polymers with novel backbones. The polymerization of methyl bicyclo[4.2.0]oct-7-ene-7-carboxylate with cyclohexene in the presence of the fast-initiating Grubbs catalyst (H2IMes)(3-Br-Pyr)2Cl2Ru═CHPh leads to a completely linear as well as alternating copolymer, as demonstrated by NMR spectroscopy, isotopic labeling, and gel permeation chromatography. In contrast, intramolecular chain-transfer reactions were observed with [5.2.0] and [3.2.0] bicyclic carbomethoxy olefins, although to a lesser extent than with the previously reported monocyclic cyclobutenecarboxylic ester monomers [Song, A.; Parker, K. A.; Sampson, N. S. J. Am. Chem. Soc. 2009, 131, 3444]. Inclusion of cyclohexyl rings fused to the copolymer backbone minimizes intramolecular chain-transfer reactions and provides a framework for creating alternating functionality in a one-step polymerization.

Alternating copolymers derived from the ring-opening metathesis polymerization (ROMP) of functionalized cyclobutene esters (CBEs) and cyclohexenes (CHs) are described. The copolymer poly(CBE-alt-CH)n contains alternating side chains based on dialkoxynaphthalene (DAN) and pyromellitic dianhydride (PDI). Characterization by UV–vis spectroscopy showed that the copolymers exhibit an increase in charge-transfer intensity in comparison to previously reported alternating copolymers. The bulky side chains inhibit backbiting during the polymerization and allow for enhanced control over the polymerization in comparison to copolymers functionalized with linear alkyl groups.

The plasma membrane contains a diverse array of proteins, including receptors, channels, and signaling complexes, that serve as decision-making centers. Investigation of membrane protein topology is important for understanding the function of these types of protein. Here, we report a method to determine protein topology in the membrane that utilizes labeling of cysteine with isotope-coded mass tags. The mass tags contain a thiol reactive moiety, linker, and a quaternary ammonium group to aid ionization in the mass spectrometer and were synthesized in both light and heavy (deuterated) forms. The probes were found to be membrane impermeable when applied to lipid vesicles. To assess the utility of the probes for mapping peptide thiol topology, we employed a two-step labeling procedure. Vesicles containing α-helical transmembrane peptides were labeled with heavy (or light) probe, solubilized by detergent, and then labeled by an excess of the complementary probe. Peptide for which the cysteine was oriented in the center of the lipid bilayer was not labeled until the lipid vesicles were lysed with detergent, consistent with the membrane impermeability of the probes and reduced ionization of the thiol in the hydrophobic membrane. Peptide for which the cysteine was positioned in the headgroup zone of the lipid bilayer was labeled rapidly. Peptide for which the cysteine was positioned below the headgroup abutting the hydrocarbon region was labeled at a reduced rate compared to the fully accessible cysteine. Moreover, the effect of lipid bilayer structure on the kinetics of peptide and lipid flipping in the bilayer was readily measured with our two-step labeling method. The small sample size required, the ease and rapidity of sample preparation, and the amenability of MALDI-TOF mass spectral analysis to the presence of lipids will enable future facile investigation of membrane proteins in a cellular context.

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. On the basis of early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2, respectively, in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and liquid chromatography–ultraviolet experiments establish that ChsE1–ChsE2 forms an α2β2 heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1–ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1–ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system versus hexahydroindanone substrates with specificity constants (kcat/KM) of (2.5 ± 0.5) × 105 s–1 M–1 versus 9.8 × 102 s–1 M–1, respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol-metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.

Antibacterial polymers have potential as pharmaceuticals and as coatings for implantation devices. The design of these materials will be optimized when we have a complete understanding of the structural features that impart activity toward target organisms and those that are benign with respect to the mammalian host. In this work, four series of polymers in which cationic and hydrophobic groups were distributed along the backbone were tested against six different bacterial species (both Gram-positive and Gram-negative) and for host cytotoxicities (red blood cell lysis). The most effective of the polymers studied are regularly spaced, featuring a 6−8 carbon stretch along the backbone between side chains that present positively charged groups. They cause potassium efflux, disorder the bacterial cytoplasmic membrane, and disrupt the membrane potential. These polymers, available from alternating ring-opening metathesis polymerization (AROMP), offer proof of principle for the importance of regular spacing in antibacterial polymers and for the synthesis of additional functional materials based on regularly spaced scaffolds.

The cholesterol metabolism pathway in Mycobacterium tuberculosis (M. tb) is a potential source of energy as well as secondary metabolite production that is important for survival of M. tb in the host macrophage. Oxidation and isomerization of 3β-hydroxysterols to 4-en-3-ones is requisite for sterol metabolism and the reaction is catalyzed by 3β-hydroxysteroid dehydrogenase (Rv1106c). Three series of 6-azasteroids and 4-azasteroids were employed to define the substrate preferences of M. tb 3β-hydroxysteroid dehydrogenase. 6-Azasteroids with large, hydrophobic side chains at the C17 position are the most effective inhibitors. Substitutions at C1, C2, C4 and N6 were poorly tolerated. Our structure–activity studies indicate that the 6-aza version of cholesterol is the best and tightest binding competitive inhibitor (Ki = 100 nM) of the steroid substrate and are consistent with cholesterol being the preferred substrate of M. tb 3β-hydroxysteroid dehydrogenase.

The reactivities of a series of 1-substituted cyclobutene derivatives (carboxylate esters, carboxamides, and carbinol esters) were investigated as substrates for ring-opening metathesis polymerization (ROMP) with [(H2IMes)(3-Br-pyridine)2(Cl)2Ru═CHPh]. Both the secondary amides of 1-cyclobutenecarboxylic acid and the esters of 1-cyclobutene-1-methanol undergo polymerization. The secondary amides provide translationally invariant polymers (E-olefins). Although the carbinol esters yield stereo- and regiochemically heterogeneous polymers, the 1-cyclobutenecarboxylic acid esters and tertiary amides undergo ring-opening metathesis (ROM) but not ROMP. The regio- and stereochemical outcomes of these ROMP and ROM reactions were analyzed at the B3LYP/6-31G* and LANL2DZ levels of theory. Calculations suggest that the regiochemistry and stereochemistry of the addition to the propagating carbene to form the metallocyclobutane intermediate depend on both charge distribution and steric interactions.

Catalysis of alternating ROMP with (H2IMes)Cl2Ru═CHPh(OiPr), the second generation Hoveyda−Grubbs catalyst, provided an entirely cyclic alternating polymer. Conditions for the cyclic AROMP were used to prepare a polymer in which one of the repeat units bore a primary alkyl chloride that was used for further elaboration.

We investigated the role of β1 integrin in mammalian fertilization and the mode of inhibition of fertilinβ-derived polymers. We determined that polymers displaying the Glu-Cys-Asp peptide from the fertilinβ disintegrin domain mediate inhibition of mammalian fertilization through a β1 integrin receptor on the egg surface. Inhibition of fertilization is a consequence of competition with sperm binding to the cell surface, not activation of an egg-signaling pathway. The presence of the β1 integrin on the egg surface increases the rate of sperm attachment but does not alter the total number of sperm that can attach or fuse to the egg. We conclude that the presence of β1 integrin enhances the initial adhesion of sperm to the egg plasma membrane and that subsequent attachment and fusion are mediated by additional egg and sperm proteins present in the β1 integrin complex. Therefore, the mechanisms by which sperm fertilize wild-type and β1 knockout eggs are different.

Mycobacterium tuberculosis can metabolize cholesterol to both acetate and propionate. The mass of isolated phthiocerol dimycoserate, a methyl-branched fatty acylated polyketide, was used as a reporter for intracellular propionate metabolic flux. When M. tuberculosis is grown using cholesterol as the only source of carbon, a 42 amu increase in average phthiocerol dimycoserate molecular weight is observed, consistent with the cellular pool of propionate and, thus, methylmalonyl CoA increasing upon cholesterol metabolism. In contrast, no shift in phthiocerol dimycoserate molecular weight is observed upon supplementation of medium containing glycerol and glucose with cholesterol. We conclude that cholesterol is a significant source of propionate only in the absence of sugar carbon sources.

The usage by enzymes of specific binding pathways for gaseous substrates or products is debated. The crystal structure of the redox enzyme cholesterol oxidase, determined at sub-angstrom resolution, revealed a hydrophobic tunnel that may serve as a binding pathway for oxygen and hydrogen peroxide. This tunnel is formed by a cascade of conformational rearrangements and connects the active site with the exterior surface of the protein. To elucidate the relationship between this tunnel and gas binding and release, three mutant enzymes were constructed to block the tunnel or its putative gate. Mutation of the proposed gating residue Asn485 to Asp or tunnel residue Phe359 or Gly347 to Trp or Asn reduces the catalytic efficiency of oxidation. The KmO2 increases from 300 ± 35 µM for the wild-type enzyme to 617 ± 15 µM for the F359W mutant. The kcat for the F359W mutant-catalyzed reaction decreases 13-fold relative to that of the wild-type-catalyzed reaction. The N485D and G347N mutants could not be saturated with oxygen. Transfer of hydride from the sterol to the flavin prosthetic group is no longer rate-limiting for these tunnel mutants. The steady-state kinetics of both wild-type and tunnel mutant enzymes are consistent with formation of a ternary complex of steroid and oxygen during catalysis. Furthermore, kinetic cooperativity with respect to molecular oxygen is observed with the tunnel mutants, but not with the wild-type enzyme. A rate-limiting conformational change for binding and release of oxygen and hydrogen peroxide, respectively, is consistent with the cooperative kinetics. In the atomic-resolution structure of F359W, the indole ring of the tryptophan completely fills the tunnel and is observed in only a single conformation. The size of the indole is proposed to limit conformational rearrangement of residue 359 that leads to tunnel opening in the wild-type enzyme. Overall, these results substantiate the functional importance of the tunnel for substrate binding and product release.

In this issue of Chemistry & Biology, Aparicio and coworkers report that secreted bacterial cholesterol oxidase is required for the biosynthesis of the antifungal polyene pimaricin by Streptomyces natalensis [1]. Their discovery expands the inventory of tasks this biotechnologically important enzyme performs.

The sperm protein fertilinβ, a member of the ADAM family of proteins, is implicated in sperm-egg binding in all mammals studied to date. Multivalent inhibitors containing the three amino acid binding sequence of fertilinβ, ECD, have been shown previously to be more effective inhibitors of fertilization than their monovalent counterparts. Here, we probed sperm-egg interactions with ruthenium-catalyzed ring-opening metathesis polymers that contained from 3 to 70 ECD pharmacophores in densities ranging from 10% to 100%. Evaluation of the polymer potencies, and synthesis of a triblock copolymer from two building blocks, revealed that two multivalent contacts are sufficient for maximal inhibition, and that the distance between ECD pharmacophores required is 7–9 monomers spanning 4–5 nm. We conclude that inhibition requires recruitment of two receptors on the egg surface into an inhibitory complex.

•Structural characterization of the mycobacterial FadA5 thiolase•Structure of a thiolase in complex with a bound steroid•Elucidation of preference for specific steroids•Similarity analysis with human thiolases and feasibility of targeting FadA5With the exception of HIV, tuberculosis (TB) is the leading cause of mortality among infectious diseases. The urgent need to develop new antitubercular drugs is apparent due to the increasing number of drug-resistant Mycobacterium tuberculosis (Mtb) strains. Proteins involved in cholesterol import and metabolism have recently been discovered as potent targets against TB. FadA5, a thiolase from Mtb, is catalyzing the last step of the β-oxidation reaction of the cholesterol side-chain degradation under release of critical metabolites and was shown to be of importance during the chronic stage of TB infections. To gain structural and mechanistic insight on FadA5, we characterized the enzyme in different stages of the cleavage reaction and with a steroid bound to the binding pocket. Structural comparisons to human thiolases revealed that it should be possible to target FadA5 specifically, and the steroid-bound structure provides a solid basis for the development of inhibitors against FadA5.Download high-res image (558KB)Download full-size image

Labeling proteins with biotin is a widely used method to identify target proteins due to biotin's strong binding affinity for streptavidin. Combined with alkyne–azide cycloaddition, which enables the coupling of probes to targeted proteins, biotin tags linked to an alkyne or azide have become a powerful tool for purification and analysis of proteins in proteomics. However, biotin requires harsh elution conditions to release the captured protein from the bead matrix. Use of these conditions reduces signal to noise and complicates the analysis. To improve affinity capture, cleavable linkers have been introduced. Here, we demonstrate the use of a cyclic acetal biotin probe that is prepared easily from commercially available starting materials, is stable to cell lysates, yet is cleaved under mildly acidic conditions, and which provides an aldehyde for further elaboration of the protein, if desired.