Search

Proteomic signatures associated with clinical measures of more aggressive cancers could yield molecular clues as to disease drivers. Here, utilizing the Clinical Proteomic Tumor Analysis Consortium (CPTAC) mass-spectrometry-based proteomics datasets, we defined differentially expressed proteins and mRNAs associated with higher grade or higher stage, for each of seven cancer types (breast, colon, lung adenocarcinoma, clear cell renal, ovarian, uterine, and pediatric glioma), representing 794 patients. Widespread differential patterns of total proteins and phosphoproteins involved some common patterns shared between different cancer types. More proteins were associated with higher grade than higher stage. Most proteomic signatures predicted patient survival in independent transcriptomic datasets. The proteomic grade signatures, in particular, involved DNA copy number alterations. Pathways of interest were enriched within the grade-associated proteins across multiple cancer types, including pathways of altered metabolism, Warburg-like effects, and translation factors. Proteomic grade correlations identified protein kinases having functional impact in vitro in uterine endometrial cancer cells, including MAP3K2, MASTL, and TTK. The protein-level grade and stage associations for all proteins profiled—along with corresponding information on phosphorylation, pathways, mRNA expression, and copy alterations—represent a resource for identifying new potential targets. Proteomic analyses are often concordant with corresponding transcriptomic analyses, but with notable exceptions.

DNA-encoded chemical library (DECL) screens are a rapid and economical tool to identify chemical starting points for drug discovery. As a robust transformation for drug discovery, palladium-catalyzed C–N coupling is a valuable synthetic method for the construction of DECL chemical matter; however, currently disclosed methods have only been demonstrated on DNA-attached (hetero)aromatic iodide and bromide electrophiles. We developed conditions utilizing an N-heterocyclic carbene–palladium catalyst that extends this reaction to the coupling of DNA-conjugated (hetero)aromatic chlorides with (hetero)aromatic and select aliphatic amine nucleophiles. In addition, we evaluated steric and electronic effects within this catalyst series, carried out a large substrate scope study on two representative (hetero)aryl bromides, and applied this newly developed method within the construction of a 63 million-membered DECL.

A strategy for DNA-compatible, palladium-catalyzed hydroxycarbonylation of (hetero)aryl halides on DNA–chemical conjugates has been developed. This method generally provided the corresponding carboxylic acids in moderate to very good conversions for (hetero)aryl iodides and bromides, and in poor to moderate conversions for (hetero)aryl chlorides. These conditions were further validated by application within a DNA-encoded chemical library synthesis and subsequent discovery of enriched features from the library in selection experiments against two protein targets.

A hypodiboric acid system for the reduction of nitro groups on DNA–chemical conjugates has been developed. This transformation provided good to excellent yields of the reduced amine product for a variety of functionalized aromatic, heterocyclic, and aliphatic nitro compounds. DNA tolerance to reaction conditions, extension to decigram scale reductions, successful use in a DNA-encoded chemical library synthesis, and subsequent target selection are also described.

We present a method to evaluate the free energies of ligand binding utilizing a Monte Carlo estimation of the configuration integrals concomitant with uncertainty quantification. Ensembles for integration are built through systematically perturbing an initial ligand conformation in a rigid binding pocket, which is optimized separately prior to incorporation of the ligand. We call the procedure producing the ensembles “blurring”, and it is carried out using an in-house developed code. The Boltzmann factor contribution of each pose to the configuration integral is computed and from there the free energy is obtained. Potential function uncertainties are estimated using a fragment-based error propagation method. This method has been applied to a set of small aromatic ligands complexed with T4 Lysozyme L99A mutant. Microstate energies have been determined with the force fields ff99SB and ff94, and the semiempirical method PM6DH2 in conjunction with continuum solvation models including Generalized Born (GB), the Conductor-like Screening Model (COSMO), and SMD. Of the methods studied, PM6DH2-based scoring gave binding free energy estimates, which yielded a good correlation to the experimental binding affinities (R2= 0.7). All methods overestimated the calculated binding affinities. We trace this to insufficient sampling, the single static protein structure, and inaccuracies in the solvent models we have used in this study.

Computational models used in the estimation of thermodynamic quantities of large chemical systems often require approximate energy models that rely on parametrization and cancellation of errors to yield agreement with experimental measurements. In this work, we show how energy function errors propagate when computing statistical mechanics-derived thermodynamic quantities. Assuming that each microstate included in a statistical ensemble has a measurable amount of error in its calculated energy, we derive low-order expressions for the propagation of these errors in free energy, average energy, and entropy. Through gedanken experiments, we show the expected behavior of these error propagation formulas on hypothetical energy surfaces. For very large microstate energy errors, these low-order formulas disagree with estimates from Monte Carlo simulations of error propagation. Hence, such simulations of error propagation may be required when using poor potential energy functions. Propagated systematic errors predicted by these methods can be removed from computed quantities, while propagated random errors yield uncertainty estimates. Importantly, we find that end-point free energy methods maximize random errors and that local sampling of potential energy wells decreases random error significantly. Hence, end-point methods should be avoided in energy computations and should be replaced by methods that incorporate local sampling. The techniques described herein will be used in future work involving the calculation of free energies of biomolecular processes, where error corrections are expected to yield improved agreement with experimental results.

A largely unsolved problem in computational biochemistry is the accurate prediction of binding affinities of small ligands to protein receptors. We present a detailed analysis of the systematic and random errors present in computational methods through the use of error probability density functions, specifically for computed interaction energies between chemical fragments comprising a protein−ligand complex. An HIV−II protease crystal structure with a bound ligand (indinavir) was chosen as a model protein−ligand complex. The complex was decomposed into 21 interacting fragment pairs, which were studied using a number of computational methods. The chemically accurate complete basis set coupled cluster theory (CCSD(T)/CBS) interaction energies were used as reference values to generate our error estimates. In our analysis, we observed significant systematic and random errors in most methods, which was surprising, especially for parametrized classical and semiempirical quantum mechanical calculations. After propagating these fragment-based error estimates over the entire protein−ligand complex, our total error estimates for many methods are large compared to the experimentally determined free energy of binding. Thus, we conclude that statistical error analysis is a necessary addition to any scoring function attempting to produce reliable binding affinity predictions.