Designing Protease-Triggered Protein Cages.

Proteins that self-assemble into enclosed polyhedral cages, both naturally and by design, are garnering attention for their prospective utility in the fields of medicine and biotechnology. Notably, their potential for encapsulation and surface display are attractive for experiments that require protection and targeted delivery of cargo. The ability to control their opening or disassembly would greatly advance the development of protein nanocages into widespread molecular tools. Toward the development of protein cages that disassemble in a systematic manner and in response to biologically relevant stimuli, here we demonstrate a modular protein cage system that is opened by highly sequence-specific proteases, based on sequence insertions at strategically chosen loop positions in the protein cage subunits. We probed the generality of the approach in the context of protein cages built using the two prevailing methods of construction: genetic fusion between oligomeric components and (non-covalent) computational interface design between oligomeric components. Our results suggest that the former type of cage may be more amenable than the latter for endowing proteolytically controlled disassembly. We show that a successfully designed cage system, based on oligomeric fusion, is modular with regard to its triggering protease. One version of the cage is targeted by an asparagine protease implicated in cancer and Alzheimer's disease, whereas the second version is responsive to the blood-clotting protease, thrombin. The approach demonstrated here should guide future efforts to develop therapeutic vectors to treat disease states where protease induction or mis-regulation occurs.

Tensor-structured decomposition improves systems serology analysis.

Systems serology provides a broad view of humoral immunity by profiling both the antigen-binding and Fc properties of antibodies. These studies contain structured biophysical profiling across disease-relevant antigen targets, alongside additional measurements made for single antigens or in an antigen-generic manner. Identifying patterns in these measurements helps guide vaccine and therapeutic antibody development, improve our understanding of diseases, and discover conserved regulatory mechanisms. Here, we report that coupled matrix-tensor factorization (CMTF) can reduce these data into consistent patterns by recognizing the intrinsic structure of these data. We use measurements from two previous studies of HIV- and SARS-CoV-2-infected subjects as examples. CMTF outperforms standard methods like principal components analysis in the extent of data reduction while maintaining equivalent prediction of immune functional responses and disease status. Under CMTF, model interpretation improves through effective data reduction, separation of the Fc and antigen-binding effects, and recognition of consistent patterns across individual measurements. Data reduction also helps make prediction models more replicable. Therefore, we propose that CMTF is an effective general strategy for data exploration in systems serology.

The impact of lysyl-phosphatidylglycerol on the interaction of daptomycin with model membranes.

Daptomycin is an important clinical antibiotic for which resistance is rising. Daptomycin resistant strains of S. aureus often have increased 1,2-diacyl-sn-glycero-3-[phospho-1-(3-lysyl(1-glycerol))] (lysyl-PG) and mutations to the proteins directly involved in the synthesis and translocation of lysyl-PG are implicated in resistance mechanisms. To study the interaction of daptomycin with lysyl-DMPG-containing model membranes a new stereospecific and regioselective synthesis of lysyl-DMPG was developed. Studies on model membranes containing lysyl-DMPG demonstrate that: (1) daptomycin is not significantly repelled by the cationic charge of lysyl-DMPG; (2) daptomycin binds less avidly to lysyl-DMPG compared to DMPG; (3) the presence of lysyl-DMPG does not impact the membrane bound backbone conformation of daptomycin in a significant way; (4) lysyl-DMPG increases oligomer formation; (5) lysyl-DMPG does not impact model membrane fluidity at lysyl-PG : PG ratios that are relevant to daptomycin resistance. The results of these studies suggest that increased lysyl-PG content does not confer resistance to daptomycin by altering membrane fluidity or reducing membrane affinity but may confer resistance by altering the structure of daptomycin oligomers.

The Chiral Target of Daptomycin Is the 2R,2'S Stereoisomer of Phosphatidylglycerol.

Daptomycin (dap) is an important antibiotic that interacts with the bacterial membrane lipid phosphatidylglycerol (PG) in a calcium-dependent manner. The enantiomer of dap (ent-dap) was synthesized and was found to be 85-fold less active than dap against B. subtilis, indicating that dap interacts with a chiral target as part of its mechanism of action. Using liposomes containing enantiopure PG, we demonstrate that the binding of dap to PG, the structural transition that occurs upon dap binding to PG, and the subsequent oligomerization of dap, depends upon the configuration of PG, and that dap prefers the 1,2-diacyl-sn-glycero-3-phospho-1'-sn-glycerol stereoisomer (2R,2'S configuration). Ent-dap has a lower affinity for 2R,2'S liposomes than dap and cannot oligomerize to the same extent as dap, which accounts for why ent-dap is less active than dap. To our knowledge, this is the first example whereby the activity of an antibiotic depends upon the configuration of a lipid head group.

Enantioselective Synthesis and Application of Small and Environmentally Sensitive Fluorescent Amino Acids for Probing Biological Interactions.

Environmentally sensitive fluorescent amino acids (FlAAs) have been used extensively to probe biological interactions. However, most of these amino acids are large and do not resemble amino acid side chains. Here, we report the enantioselective synthesis of two small and environmentally sensitive fluorescent amino acids bearing 7-dialkylaminocoumarin side chains by alkylation of a Ni(II) glycine Schiff base complex. These amino acids exhibit a large increase in fluorescence as environment polarity decreases. One of these FLAAs was incorporated into a highly active analog of the cyclic lipopeptide antibiotic paenibacterin by Fmoc solid-phase peptide synthesis via a new and very efficient route. This peptide was used to probe the interaction of the antibiotic with model liposomes, lipopolysaccharides, and live bacteria.

Synthesis of ß-Hydroxy-α,α-difluorosulfonamides from Carbanions of Difluoromethanesulfonamides.

The synthesis of ß-hydroxy-α,α-difluorosulfonamides was achieved by reacting difluoromethanesulfonamides with KHMDS in the presence of an aldehyde or ketone. The reaction exhibited a dramatic counterion effect with KHMDS or NaHMDS usually giving excellent yields in minutes, while lithium bases gave little or no product. Excellent yields and high diastereomeric ratios were achieved with Nα-benzyl-Nα-phenylfluorenyl (PhF)-protected chiral amino aldehydes derived from amino acids. Following deprotection, a ß-hydroxy-α,α-sulfonamide reacted under peptide coupling and Mitsunobu conditions to furnish a peptidomimetic in an excellent overall yield.

A high-yielding solid-phase total synthesis of daptomycin using a Fmoc SPPS stable kynurenine synthon.

A high-yielding total synthesis of daptomycin, an important clinical antibiotic, is described. Key to the development of this synthesis was the elucidation of a Camps cyclization reaction that occurs in the solid-phase when conventionally used kynurenine (Kyn) synthons, such as Fmoc-l-Kyn(Boc,CHO)-OH and Fmoc-l-Kyn(CHO,CHO)-OH, are exposed to 20% 2-methylpiperidine (2MP)/DMF. During the synthesis of daptomycin, this side reaction was accompanied by intractable peptide decomposition, which resulted in a low yield of Dap and a 4-quinolone containing peptide. The Camps cyclization was found to occur in solution when Boc-l-Kyn(Boc,CHO)-Ot-Bu and Boc-l-Kyn(CHO,CHO)-OMe were exposed to 20% 2MP/DMF giving the corresponding 4-quinolone amino acid. In contrast, Boc-l-Kyn(CHO)-OMe was stable under these conditions, demonstrating that removing one of the electron withdrawing groups from the aforementioned building blocks prevents enolization in 2MP/DMF. Hence, a new synthesis of daptomycin was developed using Fmoc-l-Kyn(Boc)-OH, which is prepared in two steps from Fmoc-l-Trp(Boc)-OH, that proceeded with an unprecedented 22% overall yield. The simplicity and efficiency of this synthesis will facilitate the preparation of analogs of daptomycin. In addition, the elucidation of this side reaction will simplify preparation of other Kyn-containing natural products via Fmoc SPPS.

Highly efficient and enantioselective syntheses of (2S,3R)-3-alkyl- and alkenylglutamates from Fmoc-protected Garner's aldehyde.

A 6-step enantioselective synthesis of (2S,3R)-3-alkyl/alkenylglutamates, including the biologically significant amino acid, (2S,3R)-3-methylglutamate, protected for Fmoc SPPS, is reported. Overall yields range from 52-65%. Key to the success of these syntheses was the development of a high-yielding 2-step synthesis of Fmoc Garner's aldehyde followed by a Horner-Wadsworth-Emmons reaction to give the corresponding Fmoc Garner's enoate in a 94% yield. The diastereoselective 1,4-addition of lithium dialkylcuprates to the Fmoc Garner's enoate was explored. Significant decomposition occurred when using lithium diethylcuprate and conditions previously reported for the 1,4-addition of lithium dialkylcuprates to Boc or Cbz-protected Garner's enoate. An optimization study of this reaction resulted in a robust set of conditions that addressed the shortcomings of previously reported conditions. Under these conditions, highly diastereoselective (> 20:1 in most cases) 1,4-addition reactions of lithium dialkyl/dialkenylcuprates to the Fmoc Garner's enoate were achieved in 76-99% yield. The resulting 1,4-addition products were easily converted into the Fmoc-(2S,3R)-3-alkyl/alkenylglutamates in two steps.

Mild, Rapid, and Chemoselective Procedure for the Introduction of the 9-Phenyl-9-fluorenyl Protecting Group into Amines, Acids, Alcohols, Sulfonamides, Amides, and Thiols.

The 9-phenyl-9-fluorenyl (PhF) group has been used as an Nα protecting group of amino acids and their derivatives mainly as a result of its ability to prevent racemization. However, installing this group using the standard protocol, which employs 9-bromo-9-phenylfluorene/K3PO4/Pb(NO3)2, often takes days and yields can be variable. Here, we demonstrate that the PhF group can be introduced into the amino group of Weinreb's amides and methyl esters of amino acids, as well as into alcohols and carboxylic acids, rapidly and in excellent yields, using 9-chloro-9-phenylfluorene (PhFCl)/N-methylmorpholine (NMM)/AgNO3. Nα-PhF-protected amino acids can be prepared from unprotected α-amino acids, rapidly and often in near quantitative yields, by treatment with N,O-bis(trimethylsilyl)acetamide (BSA) and then PhFCl/NMM/AgNO3. Primary alcohols can be protected with the PhF group in the presence of secondary alcohols in moderate yield. Using PhFCl/AgNO3, a primary alcohol can be protected in good yield in the presence of a primary ammonium salt or a carboxylic acid. Primary sulfonamides and amides can be protected in moderate to good yields using phenylfluorenyl alcohol (PhFOH)/BF3·OEt2/K3PO4, while thiols can be protected in good to excellent yield using PhFOH/BF3·OEt2 even in the presence of a carboxylic acid or primary ammonium group.

Total Synthesis of Analogs of A54145D and A54145A1 for Structure-Activity Relationship Studies.

The total solid-phase synthesis and in vitro biological activity of a series of analogs of A54145 factor D (A5D) and A54145 factor A1 (A5A1), two cyclic lipodepsipeptide antibiotics, are reported. An on-resin cyclization strategy was employed to prepare A5A1 analogs in which Thr4, the residue involved in the depsi (ester) bond, was replaced with either diaminopropionic acid (DAPA), (2S,3R)-diaminobutyric acid (DABA), or serine, effectively replacing the ring-closing ester bond with an amide linkage or with a primary ester. Antibacterial studies with these four analogs revealed that, contrary to a previous report, replacing the ester bond with an amide bond significantly reduces biological activity, and that both the ester bond and the methyl group at the γ-position of Thr4 are crucial for activity. Consistent with literature reports, we found that the single substitution of either 3-hydroxyasparagine (HOAsn) or 3-methoxyaspartate (MeOAsp) with Asn or Asp, respectively, in A5D is more detrimental to activity than the double substitution where both HOAsn and MeOAsp are replaced with Asn or Asp, respectively.

Thermoresponsive Starch for the Flocculation of Oil Sands Mature Fine Tailings.

The reclamation of land and recovery of water from tailing ponds created during bitumen extraction from oil sands is a major technical and environmental challenge. In the current study, thermoresponsive hydroxybutylated (HB) corn starch (HB-CS) and potato starch (HB-PS), with lower critical solution temperatures (LCSTs) ranging from 36 to 45 °C, were examined as flocculants for oil sands mature fine tailings (MFT). The ability of different doses of the HB-CS and HB-PS to flocculate 2 and 10 wt % MFT, prepared by diluting 35 wt % MFT in tap water, in terms of the initial settling rate (ISR), supernatant turbidity (ST), sediment solids content (SSC), and water recovery (WR), was examined at temperatures below and above their LCSTs. The thermoresponsive HB-CS and HB-PS were good flocculants of MFT, and their thermoresponsive behavior was essential for optimal results in that they were considerably more effective in several aspects at temperatures above their LCSTs than below. In terms of ISRs, the HB-PS was a considerably better flocculant than the HB-CS, and this was especially so with the 10 wt % MFT. With the HB-PS, the ISR was lower when using diluted MFT prepared with tap water as opposed to simulated oil sands process water.

Discovery of 5-aryl-3-thiophen-2-yl-1H-pyrazoles as a new class of Hsp90 inhibitors in hepatocellular carcinoma.

Although hepatocellular carcinoma (HCC)-related mortality has increased over the past decades, treatment options are still very limited, underlining the need for developing new therapeutic strategies. The molecular chaperone heat shock protein 90 (Hsp90) plays a key role in post-translational maturation of many oncogenic client proteins that are important for survival and proliferation of cancer cells. Thus, inhibitors of Hsp90 are promising targets for many cancer types. In this study, 15 diarylpyrazole compounds were screened against MCF7 and HepG2 cell lines. Compound 8, which contained a thiophene group, demonstrated the highest antiproliferative activity against HepG2 cells having an IC50 of 0.083 µM. Four additional diarylpyrazoles, each containing a thiophene group, were prepared and screened for antiproliferative activity. None of these four compounds exhibited superior activity to compound 8 on HepG2 cells. Therefore, compound 8 was selected for further in vitro assays. Cell cycle arrest was observed at the G2 phase in compound 8-treated cells. Compound 8 also caused a 7.7-fold increase in caspase-3. These results confirm the apoptotic effect of compound 8 on HepG2 cells. Moreover, compound 8 inhibited Hsp90 (IC50 = 2.67 ±â€¯0.18 µM) in an in vitro assay and caused a 70.8% reduction in Hsp90 levels in a HepG2 cell-based assay. Additionally, compound 8 caused significant reduction in the levels of Hsp90 client proteins (Akt, c-Met, c-Raf, and EGFR) and a 1.57-fold increase in Hsp70. Molecular docking studies were also performed to predict the binding mode of compound 8 and followed by molecular dynamics simulations to give further insights into the binding mode of 8.

Synthesis of Fmoc-Protected Amino Alcohols via the Sharpless Asymmetric Aminohydroxylation Reaction Using FmocNHCl as the Nitrogen Source.

The aminohydroxylation of various alkenes using FmocNHCl as a nitrogen source is reported. In general, in the absence of a ligand, the reaction provided racemic Fmoc-protected amino alcohols with excellent regioselectivity but in low to moderate yields. However, in some instances, the yield of an amino alcohol product and the regioselectivity could be altered by the addition of a catalytic amount of triethylamine (TEA). The Sharpless asymmetric variant of this reaction (Sharpless asymmetric aminohydroxylation (SAAH)), using (DHQD)2PHAL (DHQD) or (DHQ)2PHAL (DHQ) as chiral ligands, proceeded more readily and in higher yield compared to the same reaction in the absence of a chiral ligand. The enantiomeric ratios (er) of all but two examples exceeded 90:10 with many examples giving er values of 95:5 or higher, making FmocNHCl a highly practical reagent for preparing chiral amino alcohols. The SAAH reaction using FmocNHCl was used for the preparation of d-threo-ß-hydroxyasparagine and d-threo-ß-methoxyaspartate, suitably protected for Fmoc solid phase peptide synthesis.

Total Synthesis of A54145 Factor D.

An efficient total synthesis of A54145 factor D (A5D), a member of the A54145 family of cyclic lipodepsipeptide antibiotics, is reported. The peptide was constructed by attaching the peptide to the 2'-chlorotrityl polystyrene resin via Sar5 and developing conditions that avoided diketopiperazine formation upon subsequent elaboration using 9-fluorenylmethoxycarbonyl solid-phase peptide synthesis. This route allowed for facile formation of the crucial depsi bond. A branched acyclic precursor was cyclized off-resin and then globally deprotected to obtain A5D. Consistent with recent studies by others, we found that the MeOAsp residue has the 2S,3R configuration. We also established that the configuration of the stereocenter in the anteiso-undecanoyl lipid tail does not affect biological activity.

Total Synthesis of Paenibacterin and Its Analogues.

Paenibacterin is a recently discovered cyclic lipodepsipeptide antibiotic produced by the soil bacterium Paenibacillus thiaminolyticus. It is produced as a mixture of three compounds with isomeric 15-carbon acyl lipids, designated P-A1 (linear lipid), P-A2 (anteiso lipid), and P-A3 (iso lipid). Here, we report the total synthesis of P-A1 and P-A2, as well as two analogues of P-A1 in which the threonine residue in P-A1 was replaced with l-2,3-diaminopropionic acid (P-A1-Dapa) and (2 S,3 R)-2,3-diaminobutyric acid (P-A1-Daba), converting the ring-closing ester bond to an amide bond. Solid phase peptide chemistry was used to prepare branched precursors which were cyclized off-resin to obtain the target peptides in good to excellent overall yields. The use of a pseudoproline dipeptide building block was found to be important for obtaining good yields. The antibacterial activity of the peptides was determined against Escherichia coli K-12 (G-) and Bacillus subtilis 1046 (G+). The minimum inhibitory concentrations of P-A1 and P-A2 were the same despite the variation in the structure of the acyl tail. Replacing the ring-closing ester bond with an amide bond had little or no effect on activity. The synthetic routes developed here should prove to be useful for creating new antibiotics based on the structure of paenibacterin.

The effect of replacing the ester bond with an amide bond and of overall stereochemistry on the activity of daptomycin.

Daptomycin, a cyclic lipodepsipeptide antibiotic, has been used clinically since 2003 to treat serious infections caused by Gram-positive bacteria. Although 37 years have passed since daptomycin's discovery, its mechanism of action is still debated. In this report, the effect of replacing the ester bond with an amide bond, and overall stereochemistry, on daptomycin's biological activity was examined. Two peptides were prepared in which the threonine4 residue in the active daptomycin analog, Dap-K6-E12-W13, was replaced with (2S,3R)-diaminobutyric acid ((2S,3R)-DABA) or its epimer (2S,3S-DABA) converting the ring-closing ester bond to an amide bond. Both of these peptides were found to be considerably less active than Dap-K6-E12-W13. These results, along with our previous studies on other daptomycin analogs, enabled us to conclude that the ester bond is crucial to daptomycin's activity. ent-Dap-K6-E12-W13 was found to be at least 133-fold less active than Dap-K6-E12-W13, indicating that a chiral interaction with a chiral target is essential to daptomycin's activity. Studies examining the binding of Dap-K6-E12-W13 and ent-Dap-K6-E12-W13 to model liposomes consisting of phosphatidylglycerol (PG) and phosphatidylcholine suggest that the stereochemistry of PG plays a crucial role in daptomycin-membrane interactions.

An entirely fmoc solid phase approach to the synthesis of daptomycin analogs.

Daptomycin is an important Ca2+ -dependent cyclic lipodepsipeptide antibiotic used to treat serious gram-positive infections. The search for daptomycin analogs with improved activity and their application as tools for studying its mechanism of action has prompted us to develop an entirely Fmoc solid phase approach to the synthesis of daptomycin analogs. Key to the success of this approach was the development of conditions that allowed for the formation of the ester bond on resin-bound peptides consisting of residues 1-10 and the decanoyl lipid tail. The esterification reaction proceeded more efficiently on Tentagel resin as opposed to standard polystyrene resin. This approach was used to synthesize a series of analogs in which each position of Dap-E12-W13, a relatively active daptomycin analog, was individually substituted by alanine. Only positions 2, 6, and 11 were found to be amenable to substitution by alanine in that the corresponding alanine analogs were only 1.5- to 4-fold less active than Dap-E12-W13. We also found that the daptomycin analog, Dap-K6-E12-W13, exhibits in vitro activity approaching that of daptomycin at physiological Ca2+ concentration. Studies with Dap-K6-E12-W13 and model liposomes indicate that this analog interacts with membranes by the same mechanism as daptomycin. This analog is currently being used as a lead for the development daptomycin analogs with improved activity.

Synthesis of ß-Ketosulfonamides Derived from Amino Acids and Their Conversion to ß-Keto-α,α-difluorosulfonamides via Electrophilic Fluorination.

ß-Ketosulfonamides derived from Boc or Cbz-protected amino acids bearing hydrophobic side chains were prepared in good to excellent yield by treating N-allyl, N-alkyl methanesulfonamides with n-BuLi, followed by reaction of the resulting carbanion with methyl esters of N-protected l-amino acids. The analogous reaction using the dianion derived from an N-alkyl methanesulfonamide proceeded in much lower yield. Electrophilic fluorination of the ß-ketosulfonamides using Selectfluor in the presence of CsF in DMF at room temperature for 15-60 min provided ß-keto-α,α-difluorosulfonamides in good to excellent yields. The allyl protecting group could be removed in good yield using cat. Pd(PPh)3)4 and dimethyl barbituric acid. When the fluorination reaction was performed with Cs2CO3 as base, ß-ketosulfonamides derived from Val, Leu or Ile gave the expected ß-keto-α,α-difluorosulfonamides, while ß-ketosulfonamides derived from Ala, Phe, or hPhe gave the hydrates of the imino ß-keto-α,α-difluorosulfonamides.

Membrane Binding and Oligomerization of the Lipopeptide A54145 Studied by Pyrene Fluorescence.

A54145 is a lipopeptide antibiotic related to daptomycin that permeabilizes bacterial cell membranes. Its action requires both calcium and phosphatidylglycerol in the target membrane, and it is accompanied by the formation of membrane-associated oligomers. We here probed the interaction of A54145 with model membranes composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, using the steady-state and time-resolved fluorescence of a pyrene-labeled derivative (Py-A54145). In solution, the labeled peptide was found to exist as a monomer. Its membrane interaction occurred in two stages that could be clearly distinguished by varying the calcium concentration. In the first stage, which was observed between 0.15 and 1 mM calcium, Py-A54145 bound to the membrane, as indicated by a strong increase in pyrene monomer emission. At the same calcium concentration, excimer emission increased also, suggesting that Py-A54145 had oligomerized. A global analysis of the time-resolved pyrene monomer and excimer fluorescence confirmed that Py-A54145 forms oligomers quantitatively and concomitantly with membrane binding. When calcium was raised beyond 1 mM, a distinct second transition was observed that may correspond to a doubling of the number of oligomer subunits. The collective findings confirm and extend our understanding of the action mode of A54145 and daptomycin.

Exploring the Potent Inhibition of CTP Synthase by Gemcitabine-5'-Triphosphate.

CTP synthase (CTPS) catalyzes the conversion of UTP to CTP and is a target for the development of antiviral, anticancer, antiprotozoal, and immunosuppressive agents. Exposure of cell lines to the antineoplastic cytidine analogue gemcitabine causes depletion of intracellular CTP levels, but the direct inhibition of CTPS by its metabolite gemcitabine-5'-triphosphate (dF-dCTP) has not been demonstrated. We show that dF-dCTP is a potent competitive inhibitor of Escherichia coli CTPS with respect to UTP [Ki =(3.0±0.1) µm], and that its binding affinity exceeds that of CTP ≈75-fold. Site-directed mutagenesis studies indicated that Glu149 is an important binding determinant for both CTP and dF-dCTP. Comparison of the binding affinities of the 5'-triphosphates of 2'-fluoro-2'-deoxycytidine and 2'-fluoro-2'-deoxyarabinocytidine revealed that the 2'-F-arabino group contributes markedly to the strong binding of dF-dCTP. Geminal 2'-F substitution on UTP (dF-dUTP) did not result in an increase in binding affinity with CTPS. Remarkably, CTPS catalyzed the conversion of dF-dUTP into dF-dCTP, thus suggesting that dF-dCTP might be regenerated in vivo from its catabolite dF-dUTP.