Individual and collective human intelligence in drug design: evaluating the search strategy.

In recent years, individual and collective human intelligence, defined as the knowledge, skills, reasoning and intuition of individuals and groups, have been used in combination with computer algorithms to solve complex scientific problems. Such approach was successfully used in different research fields such as: structural biology, comparative genomics, macromolecular crystallography and RNA design. Herein we describe an attempt to use a similar approach in small-molecule drug discovery, specifically to drive search strategies of de novo drug design. This is assessed with a case study that consists of a series of public experiments in which participants had to explore the huge chemical space in silico to find predefined compounds by designing molecules and analyzing the score associate with them. Such a process may be seen as an instantaneous surrogate of the classical design-make-test cycles carried out by medicinal chemists during the drug discovery hit to lead phase but not hindered by long synthesis and testing times. We present first findings on (1) assessing human intelligence in chemical space exploration, (2) comparing individual and collective human intelligence performance in this task and (3) contrasting some human and artificial intelligence achievements in de novo drug design.

Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins.

Addition of a 50 mM mixture of L: -arginine and L: -glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.

Weak oligomerization of low-molecular-weight protein tyrosine phosphatase is conserved from mammals to bacteria.

The well-characterized self-association of a mammalian low-molecular-weight protein tyrosine phosphatase (lmwPTP) produces inactive oligomers that are in equilibrium with active monomers. A role of the inactive oligomers as supramolecular proenzymes has been suggested. The oligomerization equilibrium of YwlE, a lmwPTP from Bacillus subtilis, was studied by NMR. Chemical shift data and NMR relaxation confirm that dimerization takes place through the enzyme's active site, and is fully equivalent to the dimerization previously characterized in a eukaryotic low-molecular-weight phosphatase, with similarly large dissociation constants. The similarity between the oligomerization of prokaryotic and eukaryotic phosphatases extends beyond the dimer and involves higher order oligomers detected by NMR relaxation analysis at high protein concentrations. The conservation across different kingdoms of life suggests a physiological role for lmwPTP oligomerization in spite of the weak association observed in vitro. Structural data suggest that substrate modulation of the oligomerization equilibrium could be a regulatory mechanism leading to the generation of signaling pulses. The presence of a phenylalanine residue in the dimerization site of YwlE, replacing a tyrosine residue conserved in all eukaryotic lmwPTPs, demonstrates that lmwPTP regulation by oligomerization can be independent from tyrosine phosphorylation.

Structural characterization of unphosphorylated STAT5a oligomerization equilibrium in solution by small-angle X-ray scattering.

Signal transducer and activator of transcription (STAT) proteins play a crucial role in the activation of gene transcription in response to extracellular stimuli. The regulation and activity of these proteins require a complex rearrangement of the domains. According to the established models, based on crystallographic data, STATs convert from a basal antiparallel inactive dimer into a parallel active one following phosphorylation. The simultaneous analysis of small-angle X-ray scattering data measured at different concentrations of unphosphorylated human STAT5a core domain unambiguously identifies the simultaneous presence of a monomer and a dimer. The dimer is the minor species but could be structurally characterized by SAXS in the presence of the monomer using appropriate computational tools and shown to correspond to the antiparallel assembly. The equilibrium is governed by a moderate dissociation constant of K(d) approximately 90 microM. Integration of these results with previous knowledge of the N-terminal domain structure and dissociation constants allows the modeling of the full-length protein. A complex network of intermolecular interactions of low or medium affinity is suggested. These contacts can be eventually formed or broken to trigger the dramatic modifications in the dimeric arrangement needed for STAT regulation and activity.

Low-resolution structures of transient protein-protein complexes using small-angle X-ray scattering.

The determination of the three-dimensional structure of a weak protein-protein complex in solution using small-angle X-ray scattering requires the deconvolution of its contribution from those of other components coexisting in equilibrium. Using the oligomerization equilibrium of low molecular weight phosphatase (lmwPTP) as a model system, we show computationally and experimentally that the individual low-resolution structures of monomeric and dimeric lmwPTP can be determined from a small number of SAXS curves using the multivariate curve resolution with alternating least squares (MCR-ALS) algorithm. The dimeric complex represents no more than 15% of the macromolecules in the most concentrated sample. The derived structures are in good agreement with the crystallographic ones and the dissociation constant matches the one measured by NMR. These results demonstrate the power of SAXS, in combination with MCR-ALS, to study transient biomolecular complexes. The limits of the method were explored using a three-species model that describes the oligomerization of lmwPTP at higher concentrations.

Protein tyrosine phosphatase oligomerization studied by a combination of 15N NMR relaxation and 129Xe NMR. Effect of buffer containing arginine and glutamic acid.

15N NMR relaxation and 129Xe NMR chemical shift measurements offer complementary information to study weak protein-protein interactions. They have been applied to study the oligomerization equilibrium of a low-molecular-weight protein tyrosine phosphatase in the presence of 50 mM arginine and 50 mM glutamic acid. These experimental conditions are shown to enhance specific protein-protein interactions while decreasing nonspecific aggregation. In addition, 129Xe NMR chemical shifts become selective reporters of one particular oligomer in the presence of arginine and glutamic acid, indicating that a specific Xe binding site is created in the oligomerization process. It is suggested that the multiple effects of arginine and glutamic acid are related to their effective excluded volume that favors specific protein association and the destabilization of partially unfolded forms that preferentially interact with xenon and are responsible for nonspecific protein aggregation.

Structure-based discovery of new small molecule inhibitors of low molecular weight protein tyrosine phosphatase.

The application of a fully integrated and automated virtual screening method for identifying potential and novel inhibitors of bovine lmwPTP is described. The protocol makes extensive use of our recently introduced LINGO tools, which allow the extraction of the implicit chemical information present in SMILES representations. Nine out of 34 compounds selected from a database of almost 500,000 commercially available compounds were experimentally confirmed to be competitive inhibitors of lmwPTP, two of them showing K(i) values around 10microM. The best inhibitors previously described had K(i) values higher than 1mM. These results constitute an experimental validation of our virtual screening algorithm and provide a basis for the optimization of pharmacologically interesting lmwPTP inhibitors.

An extended winged helix domain in general transcription factor E/IIE alpha.

Initiation of eukaryotic mRNA transcription requires melting of promoter DNA with the help of the general transcription factors TFIIE and TFIIH. Here we define a conserved and functionally essential N-terminal domain in TFE, the archaeal homolog of the large TFIIE subunit alpha. X-ray crystallography shows that this TFE domain adopts a winged helix-turn-helix (winged helix) fold, extended by specific alpha-helices at the N and C termini. Although the winged helix fold is often found in DNA-binding proteins, we show that TFE is not a typical DNA-binding winged helix protein, because its putative DNA-binding face shows a negatively charged groove and an unusually long wing, and because the domain lacks DNA-binding activity in vitro. The groove and a conserved hydrophobic surface patch on the additional N-terminal alpha-helix may, however, allow for interactions with other general transcription factors and RNA polymerase. Homology modeling shows that the TFE domain is conserved in TFIIE alpha, including the potential functional surfaces.