Protein-Ligand Interactions

The human sliding clamp (PCNA) controls access to DNA for many proteins involved in DNA replication and repair.P roteins are recruited to the PCNA surfaceb ym eans of as hort, conserved peptidem otif known as the PCNA-interacting protein box (PIP-box). Inhibitors of these essential protein-protein interactions may be useful as cancer therapeutics by disrupting DNA replication and repair in these highly proliferative cells. PIP-box peptide mimetics have been identified as ap otentially rapid route to potent PCNA inhibitors. Here we describe the rational design and synthesis of the first PCNA peptidomimetic ligands, based on the high affinity PIP-box sequence from the natural PCNA inhibi-tor p21. These mimetics incorporate covalent i,i + 4s idechain/side-chain lactam linkages of differentl engths, designed to constrain the peptides into the 3 10-helicalstructure required for PCNA binding.N MR studies confirmed that while the unmodified p21 peptide had little defined structure in solution, mimetic ACR2 pre-organized into 3 10-helical structure prior to interaction with PCNA. ACR2 displayed higher affinity binding than most known PIP-box peptides, and retains the native PCNA binding mode, as observedi n the co-crystal structure of ACR2 bound to PCNA. This study offers ap romising new strategy for PCNA inhibitor design for use as anti-cancer therapeutics.

The ubiquitin–proteasome system is a master regulator of protein homeostasis, by which proteins are initially targeted for poly-ubiquitination by E3 ligases and then degraded into short peptides by the proteasome. Nature evolved diverse peptidic motifs, termed degrons, to signal substrates for degradation. We discuss degrons of the N-end rule pathway and also degrons characterized by post-translational modifications, including phosphorylation and hydroxylation. In each case we detail the structural basis of E3 ligase:degron recognition and small-molecule mimicry approaches that disrupt those protein– protein interactions. We present as well genetic and chemical technologies that enable targeted degradation of proteins of interest, namely small-molecule dependent inducible degrons and chemical degraders, for example, proteolysis-targeting chimeras (PROTACs).

Background: Enzyme active sites can be connected to the exterior environment by one or more channels passing through the protein. Despite our current knowledge of enzyme structure and function, surprisingly little is known about how often channels are present or about any structural features such channels may have in common.

The peroxisome proliferator-activated receptor gamma (PPARγ) regulates osteoblast and osteoclast differentiation, and is the molecular target of thiazolidinediones (TZDs), insulin sensitizers that enhance glucose utilization and adipocyte differentiation. However, clinical use of TZDs has been limited by side effects including a higher risk of fractures and bone loss. Here we demonstrate that the same post-translational modifications at S112 and S273, which influence PPARγ pro-adipocytic and insulin sensitizing activities, also determine PPARγ osteoblastic (pS112) and osteoclastic (pS273) activities. Treatment of either hyperglycemic or normoglycemic animals with SR10171, an inverse agonist that blocks pS273 but not pS112, increased trabecular and cortical bone while normalizing metabolic parameters. Additionally, SR10171 treatment modulated osteocyte, osteoblast, and osteoclast activities, and decreased marrow adiposity. These data demonstrate that regulation of bone mass and energy metabolism shares similar mechanisms suggesting that one pharmacologic agent could be developed to treat both diabetes and metabolic bone disease.

The penultimate step in the biosynthesis of biotin is the closure of the ureido heterocycle in a reaction requiring a nucleoside triphosphate (NTP). In Mycobacterium tuberculosis this reaction is catalyzed by dethiobiotin synthetase (MtDTBS). MtDTBS is unusual as it can employ multiple (NTPs), with a >100-fold preference for cytidine triphosphate (CTP). Here the molecular basis of NTP binding was investigated using a surface plasmon resonancebased ligand binding assay and X-ray crystallography. The biophysical and structural data revealed two discrete mechanisms by which MtDTBS binds NTPs: (i) A high affinity binding mode employed by CTP (K D 160 nM) that is characterized by a slow dissociation rate between enzyme and ligand (k d 5.3 × 10 −2 s −1) and that is defined by an extended network of specific ligand−protein interactions involving both the cytidine and triphosphate moieties and (ii) a low affinity mode employed by the remaining NTPs (K D > 16.5 μM), that is characterized by weak interactions between protein and ligand. Previously intractable structures of MtDTBS in complex with ATP, GTP, UTP, and ITP were obtained to define the molecular basis of the low affinity ligand binding. Anchoring of the triphosphate moiety into the phosphate binding loop of MtDTBS allows the promiscuous utilization of multiple NTPs. Both high and low binding mechanisms showed conserved hydrogen bonding interactions involving the β-phosphate of NTPs and a high-affinity anion binding site within the phosphate binding loop. This study provides insights into enzymes that can likewise utilize multiple NTPs.

Human serum albumin (HSA) principally tasks as a transport carrier for a vast variety of natural compounds and pharmaceutical drugs. In the present study, two structurally related binuclear Pt (II) complexes containing cis, cis-[Me 2 Pt (μ-NN) (μ-dppm) PtMe 2 ] (1), and cis, cis-[Me 2 Pt(μ-NN)(μ dppm) Pt((CH 2) 4)] (2) in which NN = phthalazine and dppm = bis (diphenylphosphino) methane were used to investigate their interaction with HSA, using UV–Vis absorption spectroscopy, fluorescence, circular dichroism and molecular dynamic analyses. The spectroscopic results suggest that upon binding to HSA, the binuclear Pt (II) complexes could effectively induce structural alteration of this protein. These complexes can bind to HSA with the binding affinities of the following order: complex 2 N complex 1. Moreover, the thermodynamic parameters of binding between these complexes and HSA suggested the existence of entropy-driven spontaneous interaction, which mostly dominated with the hydrophobic forces. The ANS fluorescence results also indicated that two binuclear Pt (II) complexes were competing for the binding to the hydrophobic regions on HSA. In addition, competitive displacement assay and docking simulation study revealed that complexes 1 and 2 bind to the drug binding sites II and I on HSA, respectively. Furthermore, complex 2, with the higher binding affinity for HSA, shows more denaturing effect on this protein. Considering the protein structural damages in the pathway of harmful side effects of platinum drugs, complex 1 with the moderate binding affinity and low denaturing effect might be of high significance.

Current virtual screening tools are fast, but reliable scoring is elusive. Here, we present the 'SQM/COSMO filter', a novel scoring function featuring a quantitative semiempirical quantum mechanical (SQM) description of all types of noncovalent interactions coupled with implicit COSMO solvation. We show unequivocally that it outperforms eight widely used scoring functions. The accuracy and chemical generality of the SQM/COSMO filter make it a perfect tool for late stages of virtual screening. Despite the enormous advances in method development for structure-based in silico drug design, reliable predictions of the structures (docking) and affinities (scoring) of protein–ligand (P–L) complexes still remain an unsolved task. 1 A plethora of scoring functions (SFs) have been devised by utilising experimental data for regression analyses, by constructing knowledge-based potentials, or based on physical laws. 2,3 As none of the SFs is general enough to perform equally strongly for a diverse set of P–L complexes, utilising several SFs at once (consensus scoring) holds promise. 4 Regression analysis and knowledge-based approaches to scoring are trained on a set of P–L complexes and rely on variable master equation terms. Their validity is limited to complexes similar to the training set. In principle, this problem has been overcome in physics-based methods. Because of computational cost, preference has been given to molecular mechanics (MM) methods, such as the combination of MM interaction energies with implicit solvation free energy terms (generalised Born, GB, or Poisson–Boltzmann, PB) to estimate affinities. 2 Additionally, the wide coverage of organic chemical space in the GAFF (general AMBER force field) 5 has made the parameterisation of ligands for MM straightforward. However, an explicit description of quantum mechanical (QM) effects in P–L interactions, such as charge transfer, polarisa-tion, covalent-bond formation or s-hole bonding, was missing. QM methods, which describe these effects qualitatively better than the energy functions used in MM-based SFs, were thus introduced into computational drug design. 6,7 Recent developments in QM methods and algorithms as well as the availability of a powerful computing infrastructure have paved the way to apply them for P–L complexes in numerous setups: linear scaling or efficient parallelisation of semi-empirical QM (SQM) methods, 7–10 QM/MM, 7,8,11,12 DFT-D3 on truncated P–L complexes 13 or various fragmentation methods. 11,14 Specifically, AM1, RM1, PM6 or DF–TB SQM methods have been used 7–9,12,15 as such or with empirical corrections for dispersion, hydrogen-and halogen-bonding 16 to describe the P–L noncovalent interactions. Merz et al. pioneered this area by introducing a QM-based SF (QMScore), a combination of the AM1 SQM method with an empirical dispersion (D) and the PB implicit solvent [eqn (1)]. 17 The method was useful for describing metalloprotein–ligand binding, but further corrections were needed, especially for a quantitative treatment of dispersion and hydrogen bonding. 10 Score = DE int + DDG solv + DG 0w conf À TDS (1) The above equation is a general physics-based SF. The terms are the gas-phase interaction energy (DE int), the change of solvation free energy upon complex formation (DDG solv), the change of conformational 'free' energy (DG 0w conf) and the change of entropy upon ligand binding (ÀTDS). Our approach is systematic. Using accurate calculations in small model systems as a benchmark, we developed corrections for SQM methods that provide reliable and accurate description of a wide range of noncovalent interactions including dispersion , hydrogen-and halogen-bonding. 16 Coupled with the PM6 SQM method, 18 the resulting PM6-D3H4X approach is applicable to a wide chemical space and does not require any

Many enzymes have buried active sites. The properties of the tunnels connecting the active site with bulk solvent affect ligand binding and unbinding and also the catalytic properties. Here, we investigate ligand passage in the haloalkane dehalogenase enzyme LinB and the effect of replacing leucine by a bulky tryptophan at a tunnel-lining position. Transient kinetic experiments show that the mutation significantly slows down the rate of product release. Moreover, the mechanism of bromide ion release is changed from a one-step process in the wild type enzyme to a two-step process in the mutant. The rate constant of bromide ion release corresponds to the overall steady-state turnover rate constant, suggesting that product release became the rate-limiting step of catalysis in the mutant. We explain the experimental findings by investigating the molecular details of the process computationally. Analysis of trajectories from molecular dynamics simulations with a tunnel detection software reveals differences in the tunnels available for ligand egress. Corresponding differences are seen in simulations of product egress using a specialized enhanced sampling technique. The differences in the free energy barriers for egress of a bromide ion obtained using potential of mean force calculations are in good agreement with the differences in rates obtained from the transient kinetic experiments. Interactions of the bromide ion with the introduced tryptophan are shown to affect the free energy barrier for its passage. The study demonstrates how the mechanism of an enzymatic catalytic cycle and reaction kinetics can be engineered by modification of protein tunnels.

Knowledge on neuropeptide receptor systems is integral to understanding animal physiology. Yet, obtaining general insight into neuropeptide signalling in a clade as biodiverse as the insects is problematic. Here we apply fluorescent analogues of three key insect neuropeptides to map renal tissue architecture across systematically chosen representatives of the major insect Orders, to provide an unprecedented overview of insect renal function and control. In endopterygote insects, such as Drosophila, two distinct transporting cell types receive separate neuropeptide signals, whereas in the ancestral exopterygotes, a single, general cell type mediates all signals. Intriguingly, the largest insect Order Coleoptera (beetles) has evolved a unique approach, in which only a small fraction of cells are targets for neuropeptide action. In addition to demonstrating a universal utility of this technology, our results reveal not only a generality of signalling by the evolutionarily ancient neuropeptide families but also a clear functional separation of the types of cells that mediate the signal.