Development of Computational Approaches with a Fragment-Based Drug Design Strategy: In Silico Hsp90 Inhibitors Discovery

A semi-exhaustive approach and a heuristic search algorithm use a fragment-based drug design (FBDD) strategy for designing new inhibitors in an in silico process. A deconstruction reconstruction process uses a set of known Hsp90 ligands for generating new ones. The deconstruction process consists of cutting off a known ligand in fragments. The reconstruction process consists of coupling fragments to develop a new set of ligands. For evaluating the approaches, we compare the binding energy of the new ligands with the known ligands.     

The Binding Mode of an ADP Analogue to a Metallohydrolase Mimics the Likely Transition State

Purple acid phosphatases (PAPs) are members of the large family of metallohydrolases, a group of enzymes that perform a wide range of biological functions, while employing a highly conserved catalytic mechanism. PAPs are found in plants, animals and fungi; in humans they play an important role in bone turnover and are thus of interest for developing treatments for osteoporosis. The majority of metallohydrolases use a metal-bound hydroxide to initiate catalysis, which leads to the formation of a proposed five-coordinate oxyphosphorane species in the transition state. In this work, we crystallized PAP from red kidney beans (rkbPAP) in the presence of both adenosine and vanadate. The in crystallo-formed vanadate analogue of ADP provides detailed insight into the binding mode of a PAP substrate, captured in a structure that mimics the putative fivecoordinate transition state. Our observations not only provide unprecedented insight into the mechanism of metallohydrolases, but might also guide the structure-based design of inhibitors for application in the treatment of several human illnesses.     

Pyrazoloadenine Inhibitors of the RET Lung Cancer Oncoprotein Discovered by a Fragment Optimization Approach

A fragment-based drug-discovery approach was used on a pyrazoloadenine fragment library to uncover new molecules that target the RET (REarranged during Transfection) oncoprotein, which is a driver oncoprotein in ∼2 % of non-small-cell lung cancers. The fragment library was screened against the RET kinase and LC-2/ad (RET-driven), KM-12 (TRKA-driven matched control) and A549 (cytotoxic control) cells to identify selective scaffolds that could inhibit RET-driven growth. An unsubstituted pyrazoloadenine fragment was found to be active on RET in a biochemical assay, but reduced cell viability in non-RET-driven cell lines (EC₅₀=1 and 3 μM, respectively). To increase selectivity for RET, the pyrazoloadenine was modeled in the RET active site, and two domains were identified that were probed with pyrazoloadenine fragment derivatives to improve RET affinity. Scaffolds at each domain were merged to generate a novel lead compound, 8 p , which exhibited improved activity and selectivity for the RET oncoprotein (A549 EC₅₀=5.92 μM, LC-2/ad EC₅₀=0.016 μM, RET IC₅₀=0.000326 μM).     

The Discovery of Potent SHP2 Inhibitors with Anti-Proliferative Activity in Breast Cancer Cell Lines

Despite available treatments, breast cancer is the leading cause of cancer-related death. Knowing that the tyrosine phosphatase SHP2 is a regulator in tumorigenesis, developing inhibitors of SHP2 in breast cells is crucial. Our study investigated the effects of new compounds, purchased from NSC, on the phosphatase activity of SHP2 and the modulation of breast cancer cell lines’ proliferation and viability. A combined ligand-based and structure-based virtual screening protocol was validated, then performed, against SHP2 active site. Top ranked compounds were tested via SHP2 enzymatic assay, followed by measuring IC50 values. Subsequently, hits were tested for their anti-breast cancer viability and proliferative activity. Our experiments identified three compounds 13030, 24198, and 57774 as SHP2 inhibitors, with IC50 values in micromolar levels and considerable selectivity over the analogous enzyme SHP1. Long MD simulations of 500 ns showed a very promising binding mode in the SHP2 catalytic pocket. Furthermore, these compounds significantly reduced MCF-7 breast cancer cells’ proliferation and viability. Interestingly, two of our hits can have acridine or phenoxazine cyclic system known to intercalate in ds DNA. Therefore, our novel approach led to the discovery of SHP2 inhibitors, which could act as a starting point in the future for clinically useful anticancer agents.     

Rapid Discovery of Aspartyl Protease Inhibitors Using an Anchoring Approach

Pharmacophore searches that include anchors, fragments contributing above average to receptor binding, combined with one-step syntheses are a powerful approach for the fast discovery of novel bioactive molecules. Here, we are presenting a pipeline for the rapid and efficient discovery of aspartyl protease inhibitors. First, we hypothesized that hydrazine could be a multi-valent warhead to interact with the active site Asp carboxylic acids. We incorporated the hydrazine anchor in a multicomponent reaction and created a large virtual library of hydrazine derivatives synthetically accessible in one-step. Next, we performed anchor-based pharmacophore screening of the libraries and resynthesized top-ranked compounds. The inhibitory potency of the molecules was finally assessed by an enzyme activity assay and the binding mode confirmed by several soaked crystal structures supporting the validity of the hypothesis and approach. The herein reported pipeline of tools will be of general value for the rapid generation of receptor binders beyond Asp proteases.     

Engineering a Constrained Peptidic Scaffold towards Potent and Selective Furin Inhibitors

We report the engineering of the monocyclic sunflower trypsin inhibitor (SFTI‐1[1,14]) into a potent furin inhibitor. In a rational approach, we converted the native scaffold of this trypsin‐like serine protease inhibitor into a subtilisin‐like one by substitutions in the canonical and, particularly, in the substrate‐binding loop. Although the substrate sequence for furin is Arg‐X‐Arg/Lys‐Arg↓, the most potent inhibitor had a lysine at position P1. C‐terminally truncated versions demonstrated the strongest activity, thus suggesting a lack of interaction between this motif and the surface of furin. This observation was further supported by molecular modeling. With an inhibition constant of 0.49 nm , the engineered peptide H‐KRCKKSIPPICF‐NH₂ is a promising compound for further development of furin inhibitors aimed at controlling the activity of this protease in vitro and in vivo.     

Rational Design and Synthesis of Selective PRMT4 Inhibitors: A New Chemotype for Development of Cancer Therapeutics**

Protein arginine N-methyl transferase 4 (PRMT4) asymmetrically dimethylates the arginine residues of histone H3 and nonhistone proteins. The overexpression of PRMT4 in several cancers has stimulated interest in the discovery of inhibitors as biological tools and, potentially, therapeutics. Although several PRMT4 inhibitors have been reported, most display poor selectivity against other members of the PRMT family of methyl transferases. Herein, we report the structure-based design of a new class of alanine-containing 3-arylindoles as potent and selective PRMT4 inhibitors, and describe key structure–activity relationships for this class of compounds.     

3D-QSAR Study of Potent Inhibitors of Phosphodiesterase-4 Using a CoMFA Approach

Phosphodiesterase-4 (PDE4) plays an important role in treatment of asthma and chronic obstructive pulmonary disease. Thirty-one analogs displaying variable inhibition of PDE4 were selected to develop models for establishing three-dimensional quantitative structure-activity relationships (3D-QSAR). Comparative molecular field analysis (CoMFA) was conducted on the group of analogs to determine the structural requirements for potency in inhibiting PDE4. The resulting model exhibited good q² and r² values up to 0.741 and 0.954 for CoMFA. The contributions from the steric and electrostatic fields were 0.915 and 0.085 respectively. The 3D-QSAR model should be very useful for design of novel PDE 4 inhibitors.     

Rational Computational Design of Fourth-Generation EGFR Inhibitors to Combat Drug-Resistant Non-Small Cell Lung Cancer

Although the inhibitors of singly mutated epidermal growth factor receptor (EGFR) kinase are effective for the treatment of non-small cell lung cancer (NSCLC), their clinical efficacy has been limited due to the emergence of various double and triple EGFR mutants with drug resistance. It has thus become urgent to identify potent and selective inhibitors of triple mutant EGFRs resistant to first-, second-, and third-generation EGFR inhibitors. Herein, we report the discovery of potent and highly selective inhibitors of EGFR exon 19 p.E746_A750del/EGFR exon 20 p.T790M/EGFR exon 20 p.C797S (d746-750/T790M/C797S) mutant, which were derived via two-track virtual screening and de novo design. This two-track approach was performed so as to maximize and minimize the inhibitory activity against the triple mutant and the wild type, respectively. Extensive chemical modifications of the initial hit compounds led to the identification of several low-nanomolar inhibitors of the d746-750/T790M/C797S mutant. Among them, two compounds exhibited more than 10⁴-fold selectivity in the inhibition of EGFR^{d746-750/T790M/C797S} over the wild type. The formations of a hydrogen bond with the mutated residue Ser797 and the van der Waals contact with the mutated residue Met790 were found to be a common feature in the interactions between EGFR^{d746-750/T790M/C797S} and the fourth-generation inhibitors. Such an exceptionally high selectivity could also be attributed to the formation of the hydrophobic contact with a Gly loop residue or the hydrogen bond with Asp855 in the activation loop. The discovery of the potent and selective EGFR^{d746-750/T790M/C797S} inhibitors were actually made possible by virtue of the modified protein–ligand binding free energy function involving a new hydration free energy term with enhanced accuracy. The fourth-generation EGFR inhibitors found in this work are anticipated to serve as a new starting point for the discovery of anti-NSCLC medicines to overcome the problematic drug resistance.     

Rational Design of CYP3A4 Inhibitors: A One-Atom Linker Elongation in Ritonavir-Like Compounds Leads to a Marked Improvement in the Binding Strength

Inhibition of the major human drug-metabolizing cytochrome P450 3A4 (CYP3A4) by pharmaceuticals and other xenobiotics could lead to toxicity, drug–drug interactions and other adverse effects, as well as pharmacoenhancement. Despite serious clinical implications, the structural basis and attributes required for the potent inhibition of CYP3A4 remain to be established. We utilized a rational inhibitor design to investigate the structure–activity relationships in the analogues of ritonavir, the most potent CYP3A4 inhibitor in clinical use. This study elucidated the optimal length of the head-group spacer using eleven (series V) analogues with the R₁/R₂ side-groups as phenyls or R₁–phenyl/R₂–indole/naphthalene in various stereo configurations. Spectral, functional and structural characterization of the inhibitory complexes showed that a one-atom head-group linker elongation, from pyridyl–ethyl to pyridyl–propyl, was beneficial and markedly improved K_s, IC₅₀ and thermostability of CYP3A4. In contrast, a two-atom linker extension led to a multi-fold decrease in the binding and inhibitory strength, possibly due to spatial and/or conformational constraints. The lead compound, 3h, was among the best inhibitors designed so far and overall, the strongest binder (K_s and IC₅₀ of 0.007 and 0.090 µM, respectively). 3h was the fourth structurally simpler inhibitor superior to ritonavir, which further demonstrates the power of our approach.     

Design and Structural Analysis of Aromatic Inhibitors of Type II Dehydroquinase from Mycobacterium tuberculosis

3‐Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3‐dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug‐like aromatic analogues of the most potent inhibitors. A range of carbon–carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high‐micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3‐nitrobenzylgallate‐ and 5‐aminoisophthalate‐based analogues.     

Substrate Selection Influences Molecular Recognition in a Screen for Lymphoid Tyrosine Phosphatase Inhibitors

Assay design is an important variable that influences the outcome of an inhibitor screen. Here, we have investigated the hypothesis that protein tyrosine phosphatase inhibitors with improved biological activity could be identified from a screen by using a biologically relevant peptide substrate, rather than traditional phosphotyrosine mimetic substrates. A 2000‐member library of drugs and drug‐like compounds was screened for inhibitors of lymphoid tyrosine phosphatase (LYP) by using both a peptide substrate (Ac‐ARLIEDNE‐pCAP‐TAREG‐NH₂, peptide 1) and a small‐molecule phosphotyrosine mimetic substrate (difluoromethyl umbelliferyl phosphate, DiFMUP). The results demonstrate that compounds that inhibited enzyme activity on the peptide substrate had greater biological activity than compounds that only inhibited enzyme activity on DiFMUP. Finally, epigallocatechin‐3,5‐digallate was identified as the most potent inhibitor of lymphoid tyrosine phosphatase activity to date, with an IC₅₀ of 50 nM and significant activity in T‐cells. Molecular docking simulations provided a first model for binding of this potent inhibitor to LYP; this will constitute the platform for ongoing lead optimization efforts.     

Critical Assessment of a Structure-Based Screening Campaign for IDO1 Inhibitors: Tips and Pitfalls

Over the last two decades, indoleamine 2,3-dioxygenase 1 (IDO1) has attracted wide interest as a key player in immune regulation, fostering the design and development of small molecule inhibitors to restore immune response in tumor immunity. In this framework, biochemical, structural, and pharmacological studies have unveiled peculiar structural plasticity of IDO1, with different conformations and functional states that are coupled to fine regulation of its catalytic activity and non-enzymic functions. The large plasticity of IDO1 may affect its ligand recognition process, generating bias in structure-based drug design campaigns. In this work, we report a screening campaign of a fragment library of compounds, grounding on the use of three distinct conformations of IDO1 that recapitulate its structural plasticity to some extent. Results are instrumental to discuss tips and pitfalls that, due to the large plasticity of the enzyme, may influence the identification of novel and differentiated chemical scaffolds of IDO1 ligands in structure-based screening campaigns.     

Potent Inhibitors of Plasmodial Serine Hydroxymethyltransferase (SHMT) Featuring a Spirocyclic Scaffold

With the discovery that serine hydroxymethyltransferase (SHMT) is a druggable target for antimalarials, the aim of this study was to design novel inhibitors of this key enzyme in the folate biosynthesis cycle. Herein, 19 novel spirocyclic ligands based on either 2‐indolinone or dihydroindene scaffolds and featuring a pyrazolopyran core are reported. Strong target affinities for Plasmodium falciparum (Pf) SHMT (14–76 nm ) and cellular potencies in the low nanomolar range (165–334 nm ) were measured together with interesting selectivity against human cytosolic SHMT1 (hSHMT1). Four co‐crystal structures with Plasmodium vivax (Pv) SHMT solved at 2.2–2.4 Å resolution revealed the key role of the vinylogous cyanamide for anchoring ligands within the active site. The spirocyclic motif in the molecules enforces the pyrazolopyran core to adopt a substantially more curved conformation than that of previous non‐spirocyclic analogues. Finally, solvation of the spirocyclic lactam ring of the receptor‐bound ligands is discussed.     

Design,Synthesis, and Biological Evaluation of New Urushiol Derivatives as Potent Class I Histone Deacetylase Inhibitors

In the present study, a novel series of 11 urushiol-based hydroxamic acid histone deacetylase (HDAC) inhibitors was designed, synthesized, and biologically evaluated. Compounds 1 – 11 exhibited good to excellent inhibitory activities against HDAC1/2/3 (IC₅₀: 42.09–240.17 nM) and HDAC8 (IC₅₀: 16.11–41.15 nM) in vitro, with negligible activity against HDAC6 (>1409.59 nM). Considering HDAC8, docking experiments revealed some important features contributing to inhibitory activity. According to Western blot analysis, select compounds could notably enhance the acetylation of histone H3 and SMC3 but not-tubulin, indicating their privileged structure is appropriate for targeting class I HDACs. Furthermore, antiproliferation assays revealed that six compounds exerted greater in vitro antiproliferative activity against four human cancer cell lines (A2780, HT-29, MDA-MB-231, and HepG2, with IC₅₀ values ranging from 2.31–5.13 μM) than suberoylanilide hydroxamic acid; administration of these compounds induced marked apoptosis in MDA-MB-231 cells, with cell cycle arrest in the G2/M phase. Collectively, specific synthesized compounds could be further optimized and biologically explored as antitumor agents.     

The role of sulfate as a competitive inhibitor of enzymatically‐mediated heavy metal uptake by Citrobacter sp: implications in the bioremediation of acid mine drainage water using biogenic phosphate precipitant

Heavy metals can be removed from solution via biocrystallization with enzymatically‐liberated inorganic phosphate, according to Michaelis–Menten kinetics, in free whole cells and cells immobilized within polyacrylamide gel in a flow‐through reactor. Sulfate is a competitive inhibitor of phosphate release and a predictive model was developed and shown to describe the effect of sulfate on the efficiency of phosphate release by flow‐through columns. The inhibitory effect was substantially less than anticipated in the case of metal removal by the columns. In the case of lanthanum removal metal removal efficiency was restored by increasing the substrate concentration in accordance with model predictions. In the case of uranyl ion its removal with an equivalent substrate supplement increased the activity by 20% over the initial value at a limiting flow rate. Since the initial loss in activity in the presence of 40 mmol dm⁻³ SO₄²⁻ (approximately twice the K_i value) was only approximately 20% with both metals this was considered to be a minor problem for bioprocess application. In confirmation, calculations made from a published ‘case history’ of application of the system to the bioremediation of acid mine drainage water (AMD) containing 0.22 mmol dm⁻³ of uranyl ion and 35 mmol dm⁻³ of SO showed that the benchscale model is a good representation of performance under actual load conditions. © 1999 Society of Chemical Industry     

Computational Protein Engineering: Bridging the Gap between Rational Design and Laboratory Evolution

Enzymes are tremendously proficient catalysts, which can be used as extracellular catalysts for a whole host of processes, from chemical synthesis to the generation of novel biofuels. For them to be more amenable to the needs of biotechnology, however, it is often necessary to be able to manipulate their physico-chemical properties in an efficient and streamlined manner, and, ideally, to be able to train them to catalyze completely new reactions. Recent years have seen an explosion of interest in different approaches to achieve this, both in the laboratory, and in silico. There remains, however, a gap between current approaches to computational enzyme design, which have primarily focused on the early stages of the design process, and laboratory evolution, which is an extremely powerful tool for enzyme redesign, but will always be limited by the vastness of sequence space combined with the low frequency for desirable mutations. This review discusses different approaches towards computational enzyme design and demonstrates how combining newly developed screening approaches that can rapidly predict potential mutation “hotspots” with approaches that can quantitatively and reliably dissect the catalytic step can bridge the gap that currently exists between computational enzyme design and laboratory evolution studies.     

Noncovalent binding of a reaction intermediate by a designed helix-loop-helix motif-implications for catalyst design

In our search for a catalyst for the transamination reaction of aspartic acid to form oxaloacetate, twenty-five forty-two-residue sequences were designed to fold into helix-loop-helix dimers and form binding sites for the key intermediate along the reaction pathway, the aldimine. This intermediate is formed from aspartic acid and the cofactor pyridoxal phosphate. The design of the binding sites followed a strategy in which exclusively noncovalent forces were used for binding the aldimine. Histidine residues were incorporated to catalyse the rate-limiting 1,3 proton transfer reaction that converts the aldimine into the ketimine, an intermediate that is subsequently hydrolysed to form oxaloacetate and pyridoxamine phosphate. The two most efficient catalysts, T-4 and T-16, selected from the pool of sequences by a simple screening procedure, were shown by CD and NMR spectroscopies to bind the aldimine intermediate with dissociation constants in the millimolar range. The mean residue ellipticity of T-4 in aqueous solution at pH 7.4 and a concentration of 0.75 mM was -18500 deg x cm(2) dmol(-1). Upon addition of 6 mm l-aspartic acid and 1.5 mM pyridoxal phosphate to form the aldimine, the mean residue ellipticity changed to -19900 deg x cm(2) dmol(-1). The corresponding mean residue ellipticities of T-16 were -21200 deg x cm(2) dmol(-1) and -24000 deg x cm(2) dmol(-1). These results show that the helical content increased in the presence of the aldimine, and that the folded polypeptides bound the aldimine. The (1)H NMR relaxation time of the imine CH proton of the aldimine was affected by the presence of T-4 as was the (31)P NMR resonance linewidth. The catalytic efficiencies of T-4 and T-16 were compared to that of imidazole and found to be more than three orders of magnitude larger. The designed binding sites were thus shown to be capable of binding the aldimine in close proximity to His residues, by noncovalent forces, into conformations that proved to be catalytically active. The results show for the first time the design of well-defined catalytic sites that bind a reaction intermediate with enzyme-like affinities under equilibrium conditions and represent an important advance in de novo catalyst design.