Computer graphic modelling in rug design: conformational analysis of dihydrofolate reductase inhibitors

Lipophilic 2,4-diaminopyrimidines with a 5-adamantyl substituent are effective inhibitors of mammalian dihydrofolate reductase (DHFR) and produce an additional 1000-fold increase in their cytotoxic activity when the substituent in position six is changed from hydrogen to ethyl, but drops at propyl. The results of X-ray crystal structure analysis of these antifolates show that the pyrimidine ring and its substituents become more distorted from coplanarity as the size of the 6-substituent increases. Computer graphic modelling of the binding of these antifolates in the active site of the chicken liver DHFR-NADPH binary complex indicates that both the adamantyl group and 6-substituent occupy hydrophobic pockets. Exploration of the size and character of the protein environment about the 6-position suggests that neither the ethyl nor the propyl group make optimal contacts with the functional groups surrounding this pocket. From these studies the design of alternative 6-substituent antifolates are suggested which could make specific contacts with the residues in this region of the protein.     

Non-classical antifolates. Part 2: Synthesis,biological evaluation,and molecular modeling study of some new 2,6-substituted-quinazolin-4-ones

A new series of 2,6-substituted-quinazolin-4-ones was designed, synthesized, and evaluated for their in vitro DHFR inhibition, antimicrobial, and antitumor activities. Compounds 22, 33–37, 39–43, and 45 proved to be active DHFR inhibitors with IC₅₀ range of 0.4–1.0 μM. Compound 18 showed broad-spectrum antimicrobial activity comparable to the known antibiotic gentamicin. Compounds 34 and 36 showed antitumor activity at GI₅₀ (MG-MID) concentrations of 11.2, and 24.2 μM, respectively. Molecular modeling study including flexible alignment; electrostatic, hydrophobic mappings; and pharmacophore prediction were performed. A main featured pharmacophore model was developed which justifies the importance of the main pharmacophoric groups as well as of their relative distances. The substitution pattern and spatial considerations of the π-systems in regard to the quinazoline nucleus proved critical for biological activity.     

Descriptor-free molecular discovery in large libraries by adaptive substituent reordering

Molecular discovery often involves identification of the best functional groups (substituents) on a scaffold. When multiple substitution sites are present, the number of possible substituent combinations can be very large. This article introduces a strategy for efficiently optimizing the substituent combinations by iterative rounds of compound sampling, substituent reordering to produce the most regular property landscape, and property estimation over the landscape. Application of this approach to a large pharmaceutical compound library demonstrates its ability to find active compounds with a threefold reduction in synthetic and assaying effort, even without knowing the molecular identity of any compound.     

Experimental screening of dihydrofolate reductase yields a "test set" of 50,000 small molecules for a computational data-mining and docking competition

High-throughput screening (HTS) generates an abundance of data that are a valuable resource to be mined. Dockers and data miners can use "real-world" HTS data to test and further develop their tools. A screen of 50,000 diverse small molecules was carried out against Escherichia coli dihydrofolate reductase (DHFR) and compared with a previous screen of 50,000 compounds against the same target. Identical assays and conditions were maintained for both studies. Prior to the completion of the second screen, the original screening data were publicly released for use as a "training set", and computational chemists and data analysts were challenged to predict the activity of compounds in this second "test set". Upon completion, the primary screen of the test set generated no potent inhibitors of DHFR activity.     

Kinetics of the inhibition of bovine liver dihydrofolate reductase by tea catechins: origin of slow-binding inhibition and pH studies

Dihydrofolate reductase (DHFR) is the subject of intensive investigation since it appears to be the primary target enzyme for "antifolate" drugs, such as methotrexate and trimethoprim. Fluorescence quenching and stopped-flow fluorimetry show that the ester bond-containing tea polyphenols (-)-epigallocatechin gallate (EGCG) and (-)-epicatechin gallate (ECG) are potent and specific inhibitors of DHFR with inhibition constants (K(I)) of 120 and 82 nM, respectively. Both tea compounds showed the characteristics of slow-binding inhibitors of bovine liver DHFR. In this work, we have determined a complete kinetic scheme to explain the slow-binding inhibition and the pH effects observed during the inhibition of bovine liver DHFR by these tea polyphenols. Experimental data, based on fluorimetric titrations, and transient phase and steady-state kinetic studies confirm that EGCG and ECG are competitive inhibitors with respect to 7,8-dihydrofolate, which bind preferentially to the free form of the enzyme. The origin of their slow-binding inhibition is proposed to be the formation of a slow dissociation ternary complex by the reaction of NADPH with the enzyme-inhibitor complex. The pH controls both the ionization of critical catalytic residues of the enzyme and the protonation state of the inhibitors. At acidic pH, EGCG and ECG are mainly present as protonated species, whereas near neutrality, they evolve toward deprotonated species due to ionization of the ester-bonded gallate moiety (pK = 7.8). Although DHFR exhibits different affinities for the protonated and deprotonated forms of EGCG and ECG, it appears that the ionization state of Glu-30 in DHFR is critical for its inhibition. The physiological implications of these pH dependencies are also discussed.     

Anti-idiotypic antibodies elicited by pterin recognize active site epitopes in dihydrofolate reductases and dihydropteridine reductase

Monoclonal antibodies (mAbs) against antipterin immunoglobulin and dihydropteridine reductase (DHPR) and also polyclonal antibodies against human dihydrofolate reductase (DHFR) were obtained. The anti-idiotypic mAbs and anti-DHPR mAbs bind specifically to human DHFR, Escherichia coli DHFR, soybean seedling DHFR, and human DHPR in solid-phase immunoassays. Further, the mAbs bind to the native but not to the denatured forms of DHFRs. The monoclonal antibodies also inhibit the enzymatic activity of human DHFR but not that of human DHPR. Competitive solid-phase immunoassays show stoichiometric inhibition by methotrexate and partial inhibition by NADPH of mAb binding to human DHFR. Cyanogen bromide fragments derived from human DHFR (residues 15-52 and 53-111), containing several active site residues, bind partially to some of the monoclonal antibodies. Accordingly, polyclonal antibodies to peptide 53-111 of human DHFR cross-react to some extent with human DHPR. Data from competitive immunoassays in which the binding of the various mAbs was tested singly and in combination with other mAbs suggest that these antibodies bind to a common region on human DHFR. The results also indicate that the mAbs display some heterogeneity with respect to specific epitopes. These data suggest that despite the absence of significant amino acid sequence homologies among the various DHFRs and DHPR, they have a fundamentally similar topography at the site of binding of the pterin moiety that is recognized by the anti-idiotypic mAbs generated by pterin. In the relatively simple structure of the pterin ring system there are different substituent groups at positions C4 and C6 in methotrexate, 7,8-dihydrofolate, and 7,8-dihydrobiopterin, suggesting that these antibodies are specific for regions on various proteins that interact with the remainder of the pterin moiety. These mAbs and similar mAbs specified by substituent groups on pterin may thus be used as specific probes or inhibitors of various folate-dependent enzymes and transport proteins. They should also provide insights into some of the general features of antibody recognition of protein antigens.     

Design,synthesis, biological evaluation and computational investigation of novel inhibitors of dihydrofolate reductase of opportunistic pathogens

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted ΔG_bind were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC₅₀ values and MM-GBSA predicted ΔG_bind. Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity.     

Alpha-ketoamides and alpha-ketocarbonyls: conformational analysis and development of all-atom OPLS force field

The molecular structures and barriers for the internal rotation around the OC-CO single bond in four alpha-ketoamides and eight alpha-ketocarbonyls have been determined from the MP3/aug-cc-pVDZ and MP2/aug-cc-pVDZ calculations. Alpha-ketocarbonyls with non-bulky substituents adopt planar conformations with two carbonyl oxygens in s-trans arrangement. The s-cis conformation is significantly less stable due to the electrostatic repulsion between the two carbonyl groups. Primary and secondary alpha-ketoamides are planar when the substituent at the carbonyl carbon is hydrogen or methyl group but tertiary alpha-ketoamides adopt a conformation where the OC-CO unit is significantly bent. Based on current ab initio structural data, a set of OPLS-AA force field parameters has been derived. These parameters can be used for the modeling of a variety of alpha-ketoamide or alpha-ketocarbonyl containing drugs such as novel protease inhibitors or neuroregenerative polyketides.     

Molecular docking studies on DMDP derivatives as human DHFR inhibitors

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r² = 0.499) for the 73 compounds between docking score and IC₅₀ values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.     

Pyrrolo[2,3-d]pyrimidines containing diverse N-7 substituents as potent inhibitors of Lck

A series of pyrrolo[2,3-d]pyrimidines was synthesized and evaluated as inhibitors of Lck. Lck accommodates a diverse set of substituents at N-7. Altering the substituent at N-7 provided compound 13, an orally available lck inhibitor which inhibited TCR mediated IL-2 production after oral dosing.     

Determination of the syn-anti equilibrium of some purine 3':5'-nucleotides by nuclear-magnetic-relaxation perturbation in the presence of a lanthanide-ion probe.

The aqueous solution conformation of four purine 3':5'-nucleotides varying in their substituents at C-6 and C-8 has been studied using gadolinium(III) to perturb the proton relaxation times. The ribose conformations are inferred. All the nucleotides are best described as being in a dynamic equilibrium between syn and anti conformations and the position of this equilibrium is not dramatically affected by changing the substituent at C-6. These nucleotides in their neutral base form slightly favour an anti conformation. In the presence of a bulky methylthio group at C-8 the equilibrium is shifted towards a dominance from the syn conformation due to steric repulsion factors.     

Conformational and NMR study of some furan derivatives by DFT methods

4′-substituted neutral/protonated furfurylidenanilines and trans-styrylfurans are able to exist in two different conformations related to the rotation around the furan ring-bridge double bond. In this work, the equilibrium geometry and the corresponding rotational barrier of the benzene ring for each furan derivative conformation were calculated by DFT methods. The trend and shape of the rotational barrier are rationalized within natural bond orbitals as well as atoms-in-molecules approach. For the corresponding equilibrium geometries, ¹H and ¹³C substituent induced shifts (SIS) were calculated and compared with experimental values. Calculated shielding constants are shown to be sensitive to the substituent effect through a linear fit with substituent’s Hammett constants. An alternative approach was followed for assessing the effect of substituents over SIS through comparing the differences in isotropic shielding constants with NBO charges as well as with ¹H and ¹³C experimental chemical shifts.     

Crystal structure of a novel trimethoprim-resistant dihydrofolate reductase specified in Escherichia coli by R-plasmid R67

Crystalline R67 dihydrofolate reductase (DHFR) is a dimeric molecule with two identical 78 amino acid subunits, each folded into a beta-barrel conformation. The outer surfaces of the three longest beta strands in each protomer together form a third beta barrel having six strands at the subunit interface. A unique feature of the enzyme structure is that while the intersubunit beta barrel is quite regular over most of its surface, an 8-A "gap" runs the full length of the barrel, disrupting potential hydrogen bonds between beta-strand D in subunit I and the adjacent corresponding strand of subunit II. It is proposed that this deep groove is the NADPH binding site and that the association between protein and cofactor is modulated by hydrogen-bonding interactions along one face of this antiparallel beta-barrel structure. A hypothetical model is proposed for the R67 DHFR-NADPH-folate ternary complex that is consistent with both the known reaction stereoselectivity and the weak binding of 2,4-diamino inhibitors to the plasmid-specified reductase. Geometrical comparison of this model with an experimentally determined structure for chicken DHFR suggests that chromosomal and type II R-plasmid specified enzymes may have independently evolved similar catalytic machinery for substrate reduction.     

Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.     

Studies of the thermotropic phase behavior of phosphatidylcholines containing 2-alkyl substituted fatty acyl chains: a new class of phosphatidylcholines forming inverted nonlamellar phases.

We have synthesized a number of 1,2-diacyl phosphatidylcholines with hydrophobic substituents adjacent to the carbonyl group of the fatty acyl chain and studied their thermotropic phase behavior by differential scanning calorimetry, 31P-nuclear magnetic resonance spectroscopy, and x-ray diffraction. Our results indicate that the hydrocarbon chain-melting phase transition temperatures of these lipids are lower than those of the n-saturated diacylphosphatidylcholines of similar chain length. In the gel phase, the 2-alkyl substituents on the fatty acyl chains seem to inhibit the formation of tightly packed, partially dehydrated, quasi-crystalline bilayers (Lc phases), although possibly promoting the formation of chain-interdigitated bilayers. In the liquid-crystalline state, however, these 2-alkyl substituents destabilize the lamellar phase with respect to one or more inverted nonlamellar structures. In general, increases in the length, bulk, or rigidity of the alkyl substituent result in an increased destabilization of the lamellar gel and liquid-crystalline phases and a greater tendency to form inverted nonlamellar phases, the nature of which depends upon the size of the 2-alkyl substituent. Unlike normal non-lamella-forming lipids such as the phosphatidylethanolamines, increases in the length of the main acyl chain stabilize the lamellar phases and reduce the tendency to form nonlamellar structures. Our results establish that with a judicious choice of a 2-alkyl substituent and hydrocarbon chain length, phosphatidylcholines (and probably most other so-called "bilayer-preferring" lipids) can be induced to form a range of inverted nonlamellar structures at relatively low temperatures. The ability to vary the lamellar/nonlamellar phase preference of such lipids should be useful in studies of bilayer/nonbilayer phase transitions and of the molecular organization of various nonlamellar phases. Moreover, because the nonlamellar phases can easily be induced at physiologically relevant temperatures and hydration levels while avoiding changes in polar headgroup composition, this new class of 2-alkyl-substituted phosphatidylcholines should prove valuable in studies of the physiological role of non-lamella-forming lipids in reconstituted lipid-protein model membranes.     

Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.     

Designing potential HDAC3 inhibitors to improve memory and learning

The work presented here explores the structural and physicochemical features important for benzamide-based HDAC3 inhibitors to get an idea about the design aspect of potential inhibitors. A number of molecular modeling studies (3D-QSAR CoMFA and CoMSIA, Bayesian classification modeling) were performed on 113 diverse set of benzamide-based HDAC3 inhibitors. All these models developed are statistically reliable and correlate the SAR observations. Electron withdrawing substitution is favorable but the bulky hydrophobic group at the cap region reduces HDAC3 inhibition. Hydrophobicity and steric feature of the aryl linker function favor the activity. Aryl group substituted benzamide functionality is not favorable for HDAC3 inhibition. The amide function of the benzamide moiety is essential for Zn²⁺ chelation and the carboxylic acid function may serve as a hydrogen bond acceptor (HBA) feature. Moreover, electron withdrawing substituent at the benzamide moiety influences activity whereas steric and hydrophobic substituents reduce HDAC3 inhibition. Overall, this study may provide a valuable insight on the design of better active HDAC3 inhibitors in future.

Communicated by Ramaswamy H. Sarma