We describe the use of iterative in situ click chemistry to design an Akt-specific branched peptide triligand that is a drop-in replacement for monoclonal antibodies in multiple biochemical assays. Each peptide module in the branched structure makes unique contributions to affinity and/or specificity resulting in a 200 nM affinity ligand that efficiently immunoprecipitates Akt from cancer cell lysates and labels Akt in fixed cells. Our use of a small molecule to pre-inhibit Akt prior to screening resulted in low micromolar inhibitory potency and an allosteric mode of inhibition, which is evidenced through a series of competitive enzyme kinetic assays. To demonstrate the efficiency and selectivity of the protein-templated in situ click reaction, we developed a novel QPCR-based methodology that enabled a quantitative assessment of its yield. These results point to the potential for iterative in situ click chemistry to generate potent, synthetically accessible antibody replacements with novel inhibitory properties

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We report on a method to improve in vitro diagnostic assays that detect immune response, with specific application to HIV-1. The inherent polyclonal diversity of the humoral immune response was addressed by using sequential in situ click chemistry to develop a cocktail of peptide-based capture agents, the components of which were raised against different, representative anti-HIV antibodies that bind to a conserved epitope of the HIV-1 envelope protein gp41. The cocktail was used to detect anti-HIV-1 antibodies from a panel of sera collected from HIV-positive patients, with improved signal-to-noise ratio relative to the gold standard commercial recombinant protein antigen. The capture agents were stable when stored as a powder for two months at temperatures close to 60oC.

BackgroundThe complexity of the eukaryotic parasite Trypanosoma (T.) cruzi manifests in its highly dynamic genome, multi-host life cycle, progressive morphologies and immune-evasion mechanisms. Accurate determination of infection or Chagas’ disease activity and prognosis continues to challenge researchers. We hypothesized that a diagnostic platform with higher ligand complexity than previously employed may hold value.MethodologyWe applied the ImmunoSignature Technology (IST) for the detection of T. cruzi-specific antibodies among healthy blood donors. IST is based on capturing the information in an individual’s antibody repertoire by exposing their peripheral blood to a library of >100,000 position-addressable, chemically-diverse peptides.Principal findingsInitially, samples from two Chagas cohorts declared positive or negative by bank testing were studied. With the first cohort, library-peptides displaying differential binding signals between T. cruzi sero-states were used to train an algorithm. A classifier was fixed and tested against the training-independent second cohort to determine assay performance. Next, samples from a mixed cohort of donors declared positive for Chagas, hepatitis B, hepatitis C or West Nile virus were assayed on the same library. Signals were used to train a single algorithm that distinguished all four disease states. As a binary test, the accuracy of predicting T. cruzi seropositivity by IST was similar, perhaps modestly reduced, relative to conventional ELISAs. However, the results indicate that information beyond determination of seropositivity may have been captured. These include the identification of cohort subclasses, the simultaneous detection and discerning of other diseases, and the discovery of putative new antigens.Conclusions & significanceThe central outcome of this study established IST as a reliable approach for specific determination of T. cruzi seropositivity versus disease-free individuals or those with other diseases. Its potential contribution for monitoring and controlling Chagas lies in IST’s delivery of higher resolution immune-state readouts than obtained with currently-used technologies. Despite the complexity of the ligand presentation and large quantitative readouts, performing an IST test is simple, scalable and reproducible.

In our recent JACS article, 1 we presented a study of the exchange dynamics of Xe gas atoms localized near the openings of selfassembled L-alanyl-L-valine (AV) dipeptide nanotubes 2,3 utilizing continuous-flow hyperpolarized two-dimensional NMR exchange spectroscopy (CFHP 2D-EXSY). [4][5][6][7][8][9][10][11][12][13][14] In this method, hyperpolarized gas is transported from the spin-exchange optical pumping cell to the NMR sample space at a constant flow rate, enabling rapid acquisition of 2D spectra with enhanced sensitivity. In deriving the expressions for the cross-peak signals under flow conditions, we noted that the finite residence time of the hyperpolarized gas inside the sample space may under certain conditions suppress the gas-phase diagonal-peak and cross-peak signals associated with exchange between the surface and gas phases. Moreover, we suggested that the undesired signal suppression could be mitigated simply by incorporating a brief pause in the gas flow during the mixing time of the 2D-EXSY pulse sequence. Here we present the dramatic experimental verification of this effect in AV nanotubes.As in ref 1, a 15 mg AV sample was evacuated in-situ to ∼10 -5 Torr at 100°C for 2-3 h prior to experiments. The gas mixture (Spectra Gases) consisted of 2% natural abundance 129 Xe, 2% N 2 , and 96% He at a total pressure of 4600 mbar. The 129 Xe spin polarization is estimated to be ∼20%. Only hyperpolarized 129 Xe yields observable NMR signal, since the thermally polarized gas cannot be detected at this density without signal averaging. A fractional occupancy of θ ) 0.047 in AV nanotubes was inferred from the Xe chemical shift tensor, as described in ref 5. Spectra were acquired at -10°C in a magnetic field of 9.4 T (110.6 MHz 129 Xe Larmor frequency). The isotropic chemical shift difference between the gaseous and adsorbed phases of 129 Xe is about 110 ppm. The continuous-flow hyperpolarized 129 Xe NMR setup is the same as that described in ref 1, except for one modification: to control the flow of hyperpolarized gas, the outlet of the sample space was connected to a two-way solenoid valve (Jefferson Solenoid Valves). An auxiliary transistor-to-transistor logic (TTL) gate on the Bruker Avance NMR spectrometer was used to switch the solenoid valve from the pulse program.Two different 2D-EXSY NMR pulse sequences, adapted for experiments with hyperpolarized gas, are presented in Figure 1. In the continuous flow sequence (Figure 1a), the stream of hyperpolarized gas is circulated through the sample space at a steady rate of 100 mL/min during the experiment, with no interruption.In principle, spectra acquired in CF mode will not be affected by gas flow if the spin relaxation time of the gas is much shorter than the residence time of Xe atoms in the sample space (i.e., T 1g , τ R ). In diamagnetic materials devoid of paramagnetic defects or impurities, 129 Xe spin relaxation times in the gaseous or adsorbed phases are typically much longer than any of the other time-scales relevant to the experiment. In such case...