Shared structural mechanisms of general anaesthetics and benzodiazepines.

Most general anaesthetics and classical benzodiazepine drugs act through positive modulation of γ-aminobutyric acid type A (GABAA) receptors to dampen neuronal activity in the brain1-5. However, direct structural information on the mechanisms of general anaesthetics at their physiological receptor sites is lacking. Here we present cryo-electron microscopy structures of GABAA receptors bound to intravenous anaesthetics, benzodiazepines and inhibitory modulators. These structures were solved in a lipidic environment and are complemented by electrophysiology and molecular dynamics simulations. Structures of GABAA receptors in complex with the anaesthetics phenobarbital, etomidate and propofol reveal both distinct and common transmembrane binding sites, which are shared in part by the benzodiazepine drug diazepam. Structures in which GABAA receptors are bound by benzodiazepine-site ligands identify an additional membrane binding site for diazepam and suggest an allosteric mechanism for anaesthetic reversal by flumazenil. This study provides a foundation for understanding how pharmacologically diverse and clinically essential drugs act through overlapping and distinct mechanisms to potentiate inhibitory signalling in the brain.

Sesquiterpenes and sesquiterpenoids harbor modulatory allosteric potential and affect inhibitory GABAA receptor function in vitro.

Naturally occurring compounds such as sesquiterpenes and sesquiterpenoids (SQTs) have been shown to modulate GABAA receptors (GABAA Rs). In this study, the modulatory potential of 11 SQTs at GABAA Rs was analyzed to characterize their potential neurotropic activity. Transfected HEK293 cells and primary hippocampal neurons were functionally investigated using electrophysiological whole-cell recordings. Significantly different effects of ß-caryophyllene and α-humulene, as well as their respective derivatives ß-caryolanol and humulol, were observed in the HEK293 cell system. In neurons, the concomitant presence of phasic and tonic GABAA R configurations accounts for differences in receptor modulation by SQTs. The in vivo presence of the γ2 and δ subunits is important for SQT modulation. While phasic GABAA receptors in hippocampal neurons exhibited significantly altered GABA-evoked current amplitudes in the presence of humulol and guaiol, negative allosteric potential at recombinantly expressed α1 ß2 γ2 receptors was only verified for humolol. Modeling and docking studies provided support for the binding of SQTs to the neurosteroid-binding site of the GABAA R localized between transmembrane segments 1 and 3 at the (+ α)-(- α) interface. In sum, differences in the modulation of GABAA R isoforms between SQTs were identified. Another finding is that our results provide an indication that nutritional digestion affects the neurotropic potential of natural compounds.

Synthetic, Mechanistic, and Biological Interrogation of Ginkgo biloba Chemical Space En Route to (-)-Bilobalide.

Here we interrogate the structurally dense (1.64 mcbits/Å3) GABAA receptor antagonist bilobalide, intermediates en route to its synthesis, and related mechanistic questions. 13C isotope labeling identifies an unexpected bromine migration en route to an α-selective, catalytic asymmetric Reformatsky reaction, ruling out an asymmetric allylation pathway. Experiment and computation converge on the driving forces behind two surprising observations. First, an oxetane acetal persists in concentrated mineral acid (1.5 M DCl in THF-d8/D2O); its longevity is correlated to destabilizing steric clash between substituents upon ring-opening. Second, a regioselective oxidation of des-hydroxybilobalide is found to rely on lactone acidification through lone-pair delocalization, which leads to extremely rapid intermolecular enolate equilibration. We also establish equivalent effects of (-)-bilobalide and the nonconvulsive sesquiterpene (-)-jiadifenolide on action potential-independent inhibitory currents at GABAergic synapses, using (+)-bilobalide as a negative control. The high information density of bilobalide distinguishes it from other scaffolds and may characterize natural product (NP) space more generally. Therefore, we also include a Python script to quickly (ca. 132 000 molecules/min) calculate information content (Böttcher scores), which may prove helpful to identify important features of NP space.

Novel fused 1,2,3-triazolo-benzodiazepine derivatives as potent anticonvulsant agents: design, synthesis, in vivo, and in silico evaluations.

A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a-o has been synthesized and evaluated in vivo for their anticonvulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. The synthetic approach started with diazotizing 2-aminobenzoic acids 1 to produce 2-azidobenzoic acids 2. Next, reaction of the latter compounds with propargylamine 3, benzaldehyde 4, and isocyanides 5 led to the formation of the title compounds 6a-o, in good yields. All the synthesized compounds exhibited high anticonvulsant activity in the PTZ test, comparable to or better than the standard drug diazepam. Among the tested compounds, N-(tert-butyl)-2-(9-chloro-6-oxo-4H-[1,2,3]triazolo[1,5-a][1,4]benzodiazepin-5(6H)-yl)-2-(3-bromophenyl)acetamide 6h was the most potent compound in this assay. Moreover, compounds 6i and 6k showed excellent activity in MES test. Loss of the anticonvulsant effect of compound 6h in the presence of flumazenil in the PTZ test and appropriate interaction of this compound in the active site of benzodiazepine (BZD)-binding site of GABAA receptor confirm involvement of BZD receptors in the anticonvulsant activity of compound 6h. A novel series of 1,2,3-triazolo-benzodiazepine derivatives 6a-o have been synthesized and evaluated in vivo for their anticonvulsant activities using by pentylenetetrazole (PTZ)- and maximal electroshock (MES)-induced seizures in mice. All the synthesized compounds exhibited high anticonvulsant activity, comparable to or better than the standard drug diazepam in the PTZ test and compounds 6i and 6k showed excellent activity in MES test. Flumazenil test and in silico docking study confirm involvement of benzodiazepine receptors in the anticonvulsant activity of these compounds.

What is the form of muscimol from fly agaric mushroom (Amanita muscaria) in water? An insight from NMR experiment supported by molecular modeling.

The biologically active alkaloid muscimol is present in fly agaric mushroom (Amanita muscaria), and its structure and action is related to human neurotransmitter γ -aminobutyric acid (GABA). The current study reports on determination of muscimol form present in water solution using multinuclear 1 H and 13 C nuclear magnetic resonance (NMR) experiments supported by density functional theory molecular modeling. The structures of three forms of free muscimol molecule both in the gas phase and in the presence of water solvent, modeled by polarized continuous model, and nuclear magnetic isotropic shieldings, the corresponding chemical shifts, and indirect spin-spin coupling constants were calculated. Several J-couplings observed in proton and carbon NMR spectra, not available before, are reported. The obtained experimental spectra, supported by theoretical calculations, favor the zwitterion form of muscimol in water. This structure differs from NH isomer, previously determined in dimethyl sulfoxide (DMSO) solution. In addition, positions of signals C3 and C5 are reversed in both solvents.

GABAA Receptor Ligands Often Interact with Binding Sites in the Transmembrane Domain and in the Extracellular Domain-Can the Promiscuity Code Be Cracked?

Many allosteric binding sites that modulate gamma aminobutyric acid (GABA) effects have been described in heteropentameric GABA type A (GABAA) receptors, among them sites for benzodiazepines, pyrazoloquinolinones and etomidate. Diazepam not only binds at the high affinity extracellular "canonical" site, but also at sites in the transmembrane domain. Many ligands of the benzodiazepine binding site interact also with homologous sites in the extracellular domain, among them the pyrazoloquinolinones that exert modulation at extracellular α+/ß- sites. Additional interaction of this chemotype with the sites for etomidate has also been described. We have recently described a new indole-based scaffold with pharmacophore features highly similar to pyrazoloquinolinones as a novel class of GABAA receptor modulators. Contrary to what the pharmacophore overlap suggests, the ligand presented here behaves very differently from the identically substituted pyrazoloquinolinone. Structural evidence demonstrates that small changes in pharmacophore features can induce radical changes in ligand binding properties. Analysis of published data reveals that many chemotypes display a strong tendency to interact promiscuously with binding sites in the transmembrane domain and others in the extracellular domain of the same receptor. Further structural investigations of this phenomenon should enable a more targeted path to less promiscuous ligands, potentially reducing side effect liabilities.

Synthesis of New GABAA Receptor Modulator with Pyrazolo[1,5-a]quinazoline (PQ) Scaffold.

We previously published a series of 8-methoxypirazolo[1,5-a]quinazolines (PQs) and their 4,5-dihydro derivatives (4,5(H)PQ) bearing the (hetero)arylalkylester group at position 3 as ligands at the γ-aminobutyric type A (GABAA) subtype receptor. Continuing the study in this field, we report here the design and synthesis of 3-(hetero)arylpyrazolo[1,5-a]quinazoline and 3-(hetero)aroylpyrazolo[1,5-a]quinazoline 8-methoxy substituted as interesting analogs of the above (hetero)arylalkylester, in which the shortening or the removal of the linker between the 3-(hetero)aryl ring and the PQ was performed. Only compounds that are able to inhibit radioligand binding by more than 80% at 10 µM have been selected for electrophysiological studies on recombinant α1ß2γ2L GABAA receptors. Some compounds show a promising profile. For example, compounds 6a and 6b are able to modulate the GABAAR in an opposite manner, since 6b enhances and 6a reduces the variation of the chlorine current, suggesting that they act as a partial agonist and an inverse partial agonist, respectively. The most potent derivative was 3-(4-methoxyphenylcarbonyl)-8-methoxy-4,5-dihydropyrazolo[1,5-a] quinazoline 11d, which reaches a maximal activity at 1 µM (+54%), and it enhances the chlorine current at ≥0.01 µM. Finally, compound 6g, acting as a null modulator at α1ß2γ2L, shows the ability to antagonize the full agonist diazepam and the potentiation of CGS 9895 on the new α+/ß- 'non-traditional' benzodiazepine site.

Design of a Highly Bistable Photoswitchable Tethered Ligand for Rapid and Sustained Manipulation of Neurotransmission.

Photoswitchable neurotransmitter receptors are powerful tools for precise manipulation of neural signaling. However, their applications for slow or long-lasting biological events are constrained by fast thermal relaxation of cis-azobenzene. We address this issue by modifying the ortho positions of azobenzene used in the tethered ligand. In cultured cells and intact brain tissue, conjugating inhibitory neurotransmitter receptors with one of the derivatives, dMPC1, allows bidirectional receptor control with 380 and 500 nm light. Moreover, the receptors can be locked in either an active or an inactive state in darkness after a brief pulse of light. This strategy thus enables both rapid and sustained manipulation of neurotransmission, allowing optogenetic interrogation of neural functions over a broad range of time scales.

3ß-Methyl-Neurosteroid Analogs Are Preferential Positive Allosteric Modulators and Direct Activators of Extrasynaptic δ-Subunit γ-Aminobutyric Acid Type A Receptors in the Hippocampus Dentate Gyrus Subfield.

Neurosteroids are powerful modulators of γ-aminobutyric acid (GABA)-A receptors. Ganaxolone (3α-hydroxy-3ß-methyl-5α-pregnan-20-one, GX) and synthetic analogs of the neurosteroid allopregnanolone (AP) are designed to treat epilepsy and related conditions. However, their precise mechanism of action in native neurons remains unclear. Here, we sought to determine the mode of action of GX and its analogs at GABA-A receptors in native hippocampal neurons by analyzing extrasynaptic receptor-mediated tonic currents and synaptic receptor-mediated phasic currents. Concentration-response profiles of GX were determined in two cell types: δ-containing dentate gyrus granule cells (DGGCs) and γ2-containing CA1 pyramidal cells (CA1PCs). GX produced significantly greater potentiation of the GABA-A receptor-activated chloride currents in DGGCs (500%) than CA1PCs (200%). In the absence of GABA, GX evoked 2-fold greater inward currents in DGGCs than CA1PCs, which were 2-fold greater than AP within DGGCs. In hippocampus slices, GX potentiated and directly activated tonic currents in DGGCs. These responses were significantly diminished in DGGCs from δ-subunit knockout (δKO) mice, confirming GX's selectivity for δGABA-A receptors. Like AP, GX potentiation of tonic currents was prevented by protein kinase C inhibition. Furthermore, GX's protection against hippocampus-kindled seizures was significantly diminished in δKO mice. GX analogs exhibited greater potency and efficacy than GX on δGABA-A receptor-mediated tonic inhibition. In summary, these results provide strong evidence that GX and its analogs are preferential allosteric modulators and direct activators of extrasynaptic δGABA-A receptors regulating network inhibition and seizures in the dentate gyrus. Therefore, these findings provide a mechanistic rationale for the clinical use of synthetic neurosteroids in epilepsy and seizure disorders.

Structural changes at the myrtenol backbone reverse its positive allosteric potential into inhibitory GABAA receptor modulation.

GABAA receptors are ligand-gated anion channels that form pentameric arrangements of various subunits. Positive allosteric modulators of GABAA receptors have been reported as being isolated either from plants or synthesized analogs of known GABAA receptor targeting drugs. Recently, we identified monoterpenes, e.g. myrtenol as a positive allosteric modulator at α1ß2 GABAA receptors. Here, along with pharmacophore-based virtual screening studies, we demonstrate that scaffold modifications of myrtenol resulted in the loss of modulatory activity. Two independent approaches, fluorescence-based compound analysis and electrophysiological recordings in whole-cell configurations were used for analysis of transfected cells. C-atoms 1 and 2 of the myrtenol backbone were identified as crucial to preserve positive allosteric potential. A modification at C-atom 2 and lack of the hydroxyl group at C-atom 1 exhibited significantly reduced GABAergic currents at α1ß2, α1ß2γ, α2ß3, α2ß3γ and α4ß3δ receptors. This effect was independent of the γ2 subunit. A sub-screen with side chain length and volume differences at the C-atom 1 identified two compounds that inhibited GABAergic responses but without receptor subtype specificity. Our combined approach of pharmacophore-based virtual screening and functional readouts reveals that side chain modifications of the bridged six-membered ring structure of myrtenol are crucial for its modulatory potential at GABAA receptors.

Synthesis of (-)-11-O-Debenzoyltashironin: Neurotrophic Sesquiterpenes Cause Hyperexcitation.

11-O-Debenzoyltashironin (1) is a member of the neurotrophic sesquiterpenes, trace plant metabolites that enhance neurite outgrowth in cultured neurons. We report its synthesis in six steps from a butenolide heterodimer via its likely biosynthetic precursor, 3,6-dideoxy-10-hydroxypseudoanisatin, here identified as the chain tautomer of 1. Access to the tashironin chemotype fills a gap in a comparison set of convulsive and neurotrophic sesquiterpenes, which we hypothesized to share a common target. Here we show that both classes mutually hyperexcite rat cortical neurons, consistent with antagonism of inhibitory channels and a mechanism of depolarization-induced neurite outgrowth.

GABAA receptor target of tetramethylenedisulfotetramine.

Use of the highly toxic and easily prepared rodenticide tetramethylenedisulfotetramine (TETS) was banned after thousands of accidental or intentional human poisonings, but it is of continued concern as a chemical threat agent. TETS is a noncompetitive blocker of the GABA type A receptor (GABAAR), but its molecular interaction has not been directly established for lack of a suitable radioligand to localize the binding site. We synthesized [(14)C]TETS (14 mCi/mmol, radiochemical purity >99%) by reacting sulfamide with H(14)CHO and s-trioxane then completion of the sequential cyclization with excess HCHO. The outstanding radiocarbon sensitivity of accelerator mass spectrometry (AMS) allowed the use of [(14)C]TETS in neuroreceptor binding studies with rat brain membranes in comparison with the standard GABAAR radioligand 4'-ethynyl-4-n-[(3)H]propylbicycloorthobenzoate ([(3)H]EBOB) (46 Ci/mmol), illustrating the use of AMS for characterizing the binding sites of high-affinity (14)C radioligands. Fourteen noncompetitive antagonists of widely diverse chemotypes assayed at 1 or 10 µM inhibited [(14)C]TETS and [(3)H]EBOB binding to a similar extent (r(2) = 0.71). Molecular dynamics simulations of these 14 toxicants in the pore region of the α1ß2γ2 GABAAR predict unique and significant polar interactions for TETS with α1T1' and γ2S2', which are not observed for EBOB or the GABAergic insecticides. Several GABAAR modulators similarly inhibited [(14)C]TETS and [(3)H]EBOB binding, including midazolam, flurazepam, avermectin Ba1, baclofen, isoguvacine, and propofol, at 1 or 10 µM, providing an in vitro system for recognizing candidate antidotes.

Cloaked Caged Compounds: Chemical Probes for Two-Photon Optoneurobiology.

Caged neurotransmitters, in combination with focused light beams, enable precise interrogation of neuronal function, even at the level of single synapses. However, most caged transmitters are, surprisingly, severe antagonists of ionotropic gamma-aminobutyric acid (GABA) receptors. By conjugation of a large, neutral dendrimer to a caged GABA probe we introduce a "cloaking" technology that effectively reduces such antagonism to very low levels. Such cloaked caged compounds will enable the study of the signaling of the inhibitory neurotransmitter GABA in its natural state using two-photon uncaging microscopy for the first time.

Development of Anionically Decorated Caged Neurotransmitters: In Vitro Comparison of 7-Nitroindolinyl- and 2-(p-Phenyl-o-nitrophenyl)propyl-Based Photochemical Probes.

Neurotransmitter uncaging, especially that of glutamate, has been used to study synaptic function for over 30 years. One limitation of caged glutamate probes is the blockade of γ-aminobutyric acid (GABA)-A receptor function. This problem comes to the fore when the probes are applied at the high concentrations required for effective two-photon photolysis. To mitigate such problems one could improve the photochemical properties of caging chromophores and/or remove receptor blockade. We show that addition of a dicarboxylate unit to the widely used 4-methoxy-7-nitroindolinyl-Glu (MNI-Glu) system reduced the off-target effects by about 50-70 %. When the same strategy was applied to an electron-rich 2-(p-Phenyl-o-nitrophenyl)propyl (PNPP) caging group, the pharmacological improvements were not as significant as in the MNI case. Finally, we used very extensive biological testing of the PNPP-caged Glu (more than 250 uncaging currents at single dendritic spines) to show that nitro-biphenyl caging chromophores have two-photon uncaging efficacies similar to that of MNI-Glu.

Synthesis and evaluation of anticonvulsant properties of new N-Mannich bases derived from 3-(1-phenylethyl)- and 3-benzyl-pyrrolidine-2,5-dione.

Two series of new derivatives of pyrrolidine-2,5-dione were synthesized and evaluated for their anticonvulsant properties. Initial screening for their anticonvulsant properties was performed in mice after intraperitoneal administration, using the maximal electroshock (MES), subcutaneous pentylenetetrazole (scPTZ) and 6-Hz seizure tests. Quantitative pharmacological research revealed that the highest level of protection was demonstrated by compound N-[{4-methylpiperazin-1-yl}-methyl]-3-(1-phenylethyl)-pyrrolidine-2,5-dione monohydrochloride (22) which was effective both in the scPTZ test (ED50=39 mg/kg) and in the 6-Hz test (ED50=36 mg/kg). This molecule showed higher potency than reference antiepileptic drugs such as ethosuximide, lacosamide and valproic acid. With the aim of explaining the possible mechanism of action of the selected molecule, its influence on sodium and calcium channels as well as NMDA and GABAA receptors binding properties were evaluated in vitro.

Azogabazine; a photochromic antagonist of the GABAA receptor.

The design and synthesis of azogabazine is described, which represents a highly potent (IC50 = 23 nM) photoswitchable antagonist of the GABAA receptor. An azologization strategy is adopted, in which a benzyl phenyl ether in a high affinity gabazine analogue is replaced by an azobenzene, with resultant retention of antagonist potency. We show that cycling from blue to UV light, switching between trans and cis isomeric forms, leads to photochemically controlled antagonism of the GABA ion channel.

The GABAA antagonist DPP-4-PIOL selectively antagonises tonic over phasic GABAergic currents in dentate gyrus granule cells.

GABAA receptors mediate two different types of inhibitory currents: phasic inhibitory currents when rapid and brief presynaptic GABA release activates postsynaptic GABAA receptors and tonic inhibitory currents generated by low extrasynaptic GABA levels, persistently activating extrasynaptic GABAA receptors. The two inhibitory current types are mediated by different subpopulations of GABAA receptors with diverse pharmacological profiles. Selective antagonism of tonic currents is of special interest as excessive tonic inhibition post-stroke has severe pathological consequences. Here we demonstrate that phasic and tonic GABAA receptor currents can be selectively inhibited by the antagonists SR 95531 and the 4-PIOL derivative, 4-(3,3-diphenylpropyl)-5-(4-piperidyl)-3-isoxazolol hydrobromide (DPP-4-PIOL), respectively. In dentate gyrus granule cells, SR 95531 was found approximately 4 times as potent inhibiting phasic currents compared to tonic currents (IC50 values: 101 vs. 427 nM). Conversely, DPP-4-PIOL was estimated to be more than 20 times as potent inhibiting tonic current compared to phasic current (IC50 values: 0.87 vs. 21.3 nM). Consequently, we were able to impose a pronounced reduction in tonic GABA mediated current (>70 %) by concentrations of DPP-4-PIOL, at which no significant effect on the phasic current was seen. Our findings demonstrate that selective inhibition of GABA mediated tonic current is possible, when targeting a subpopulation of GABAA receptors located extrasynaptically using the antagonist, DPP-4-PIOL.

Seeking potential anticonvulsant agents that target GABAA receptors using experimental and theoretical procedures.

The aim of this study was to identify compounds that possess anticonvulsant activity by using a pentylenetetrazol (PTZ)-induced seizure model. Theoretical studies of a set of ligands, explored the binding affinities of the ligands for the GABA(A) receptor (GABA(A)R), including some benzodiazepines. The ligands satisfy the Lipinski rules and contain a pharmacophore core that has been previously reported to be a GABA(A)R activator. To select the ligands with the best physicochemical properties, all of the compounds were analyzed by quantum mechanics and the energies of the highest occupied molecular orbital and lowest unoccupied molecular orbital were determined. Docking calculations between the ligands and the GABA(A)R were used to identify the complexes with the highest Gibbs binding energies. The identified compound D1 (dibenzo(b,f)(1,4)diazocine-6,11(5H,12H)-dione) was synthesized, experimentally tested, and the GABA(A)R-D1 complex was submitted to 12-ns-long molecular dynamics (MD) simulations to corroborate the binding conformation obtained by docking techniques. MD simulations were also used to analyze the decomposition of the Gibbs binding energy of the residues involved in the stabilization of the complex. To validate our theoretical results, molecular docking and MD simulations were also performed for three reference compounds that are currently in commercial use: clonazepam (CLZ), zolpidem and eszopiclone. The theoretical results show that the GABA(A)R-D1, and GABA(A)R-CLZ complexes bind to the benzodiazepine binding site, share a similar map of binding residues, and have similar Gibbs binding energies and entropic components. Experimental studies using a PTZ-induced seizure model showed that D1 possesses similar activity to CLZ, which corroborates the predicted binding free energy identified by theoretical calculations.

Inhibitory effects of carvone isomers on the GABAA receptor in primary cultures of rat cortical neurons.

Carvone is a natural terpene which can be purified as R-(-) or S-(+) enantiomers. There are many reports about its antibacterial, antifungal, and insecticide activities, and also of some effects on the nervous system, where both enantiomers showed different potencies. Considering that the GABA(A) receptor is a major insecticide target, we studied the pharmacological activity of both carvone enantiomers, and of thujone as a reference compound acting on the receptor, on native GABA(A) by determining their effects on benzodiazepine recognition sites using primary neuronal cultures. Both isomers were able to inhibit the GABA-induced stimulation of [(3)H]flunitrazepam binding, suggesting their interaction with the GABA(A) receptor as negative allosteric modulators. Their activity was comparable to that described for thujone in the present article, with the R-(-)-carvone being the more similar and potent stereoisomer. The different configuration of the isopropenyl group in position 5 thus seems to be significant for receptor interaction and the bicycle structure not to be critical for receptor recognition. The concentrations necessary to induce negative modulation of the receptor were not cytotoxic in a murine neuron culture system. These results confirm that, at least partially, the reported insecticidal activity of carvones may be explained by their interaction with the GABA(A) receptor at its noncompetitive blocker site.

Exploring QSARs of the interaction of flavonoids with GABA (A) receptor using MLR, ANN and SVM techniques.

Quantitative Structure-Activity Relationship (QSAR) models for binding affinity constants (log Ki) of 78 flavonoid ligands towards the benzodiazepine site of GABA (A) receptor complex were calculated using the machine learning methods: artificial neural network (ANN) and support vector machine (SVM) techniques. The models obtained were compared with those obtained using multiple linear regression (MLR) analysis. The descriptor selection and model building were performed with 10-fold cross-validation using the training data set. The SVM and MLR coefficient of determination values are 0.944 and 0.879, respectively, for the training set and are higher than those of ANN models. Though the SVM model shows improvement of training set fitting, the ANN model was superior to SVM and MLR in predicting the test set. Randomization test is employed to check the suitability of the models.