The value of thoroughly understanding the thermodynamics specific to a drug discovery/design study is well known. Over the past decade, the crucial roles of water molecules in protein structure, function, and dynamics have also become increasingly appreciated. This Perspective explores water in the biological environment by adopting its point of view in such phenomena. The prevailing thermodynamic models of the past, where water was seen largely in terms of an entropic gain after its displacement by a ligand, are now known to be much too simplistic. We adopt a set of terminology that describes water molecules as being "hot" and "cold", which we have defined as being easy and difficult to displace, respectively. The basis of these designations, which involve both enthalpic and entropic water contributions, are explored in several classes of biomolecules and structural motifs. The hallmarks for characterizing water molecules are examined, and computational tools for evaluating water-centric thermodynamics are reviewed. This Perspective's summary features guidelines for exploiting water molecules in drug discovery.

The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS), catalyzing L-cysteine formation, is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. 400 MNXXI pentapeptides were generated, docked into OASS active site using GOLD and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4, and to a lesser extent, at position P3, also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptides-OASS complexes were compared with the docked poses. These results, combined with a GRID analysis of the active site, allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.

The SZMAP method computes binding free energies and the corresponding thermodynamic components for water molecules in the binding site of a protein structure [ SZMAP, 1.0.0 ; OpenEye Scientific Software Inc. : Santa Fe, NM, USA , 2011 ]. In this work, the ability of SZMAP to predict water structure and thermodynamic stability is examined for the X-ray crystal structures of a series of protein-ligand complexes. SZMAP results correlate with higher-level replica exchange thermodynamic integration double decoupling calculations of the absolute free energy of bound waters in the test set complexes. In addition, SZMAP calculations show good agreement with experimental data in terms of water conservation (across multiple crystal structures) and B-factors over a subset of the test set. In particular, the SZMAP neutral entropy difference term calculated at crystallographic water positions within each of the complex structures correlates well with whether that crystallographic water is conserved or displaceable. Furthermore, the calculated entropy of the water probe relative to the continuum shows a significant degree of correlation with the B-factors associated with the oxygen atoms of the water molecules. Taken together, these results indicate that SZMAP is capable of quantitatively predicting water positions and their energetics and is potentially a useful tool for determining which waters to attempt to displace, maintain, or build in through water-mediated interactions when evolving a lead series during a drug discovery program.

Previous studies demonstrate that nitric oxide (NO) promotes p53 transcriptional activity by a classical DNA-damage-responsive mechanism involving activation of ATM/ATR and phosphorylation of p53. These studies intentionally used high doses of NO-donors to achieve the maximum DNA-damage. However, lower concentrations of NO donors also stimulate rapid and unequivocal nuclear retention of p53, but apparently do not require ATM/ATR-dependent p53 phosphorylation or total p53 protein accumulation. To identify possible mechanisms for p53 activation at low NO levels, the role of Tyr nitration in p53 activation was evaluated. Low concentrations of the NO donor, DETA NONOate (<200μM), exclusively nitrate Tyr327 within the tetramerization domain promoting p53 oligomerization, nuclear accumulation and increased DNA-binding activity without p53 Ser15 phosphorylation. Molecular modeling indicates that nitration of one Tyr327 stabilizes the dimer by about 2.67 kcal mol−1. Significant quantitative and qualitative differences in the patterns of p53-target gene modulation by low (50μM), non DNA-damaging and high (500μM), DNA-damaging NO donor concentrations was shown. These results demonstrate a new post-translational mechanism for modulating p53 transcriptional activity responsive to low NO concentrations and independent of DNA damage signaling.

O-acetylserine sulfhydrylase (OASS) catalyzes the synthesis of l-cysteine in the last step of the reductive sulfate assimilation pathway in microorganisms. Its activity is inhibited by the interaction with serine acetyltransferase (SAT), the preceding enzyme in the metabolic pathway. Inhibition is exerted by the insertion of SAT C-terminal peptide into the OASS active site. This action is effective only on the A isozyme, the prevalent form in enteric bacteria under aerobic conditions, but not on the B-isozyme, the form expressed under anaerobic conditions. We have investigated the active site determinants that modulate the interaction specificity by comparing the binding affinity of thirteen pentapeptides, derived from the C-terminal sequences of SAT of the closely related species Haemophilus influenzae and Salmonella typhimurium, towards the corresponding OASS-A, and towards S. typhimurium OASS-B. We have found that subtle changes in protein active sites have profound effects on protein-peptide recognition. Furthermore, affinity is strongly dependent on the pentapeptide sequence, signaling the relevance of P3-P4-P5 for the strength of binding, and P1-P2 mainly for specificity. The presence of an aromatic residue at P3 results in high affinity peptides with K(diss) in the micromolar and submicromolar range, regardless of the species. An acidic residue, like aspartate at P4, further strengthens the interaction and results in the higher affinity ligand of S. typhimurium OASS-A described to date. Since OASS knocked-out bacteria exhibit a significantly decreased fitness, this investigation provides key information for the development of selective OASS inhibitors, potentially useful as novel antibiotic agents.