A selective small-molecule inhibitor of macrophage migration inhibitory factor-2 (MIF-2), a MIF cytokine superfamily member, inhibits MIF-2 biological activity.

Cytokine macrophage migration inhibitory factor-2 (MIF-2 or D-dopachrome tautomerase) is a recently characterized second member of the MIF cytokine superfamily in mammalian genomes. MIF-2 shares pro-inflammatory and tumorigenic properties with the clinical target MIF (MIF-1), but the precise contribution of MIF-2 to immune physiology or pathology is unclear. Like MIF-1, MIF-2 has intrinsic keto-enol tautomerase activity and mediates biological functions by engaging the cognate, common MIF family receptor CD74. Evidence that the catalytic site of MIF family cytokines has a structural role in receptor binding has prompted exploration of tautomerase inhibitors as potential biological antagonists and therapeutic agents, although few catalytic inhibitors inhibit receptor activation. Here we describe the discovery and biochemical characterization of a selective small-molecule inhibitor of MIF-2. An in silico screen of 1.6 million compounds targeting the MIF-2 tautomerase site yielded several hits for potential catalytic inhibitors of MIF-2 and identified 4-(3-carboxyphenyl)-2,5-pyridinedicarboxylic acid (4-CPPC) as the most functionally potent compound. We found that 4-CPPC has an enzymatic IC50 of 27 µm and 17-fold selectivity for MIF-2 versus MIF-1. An in vitro binding assay for MIF-1/MIF-2 to the CD74 ectodomain (sCD74) indicated that 4-CPPC inhibits MIF-2-CD74 binding in a dose-dependent manner (0.01-10 µm) without influencing MIF-1-CD74 binding. Notably, 4-CPPC inhibited MIF-2-mediated activation of CD74 and reduced CD74-dependent signal transduction. These results open opportunities for development of more potent and pharmacologically auspicious MIF-2 inhibitors to investigate the distinct functions of this MIF family member in vivo.

Novel l-prolyl-l-leucylglycinamide (PLG) tripeptidomimetics based on a 2-azanorbornane scaffold as positive allosteric modulators of the D2R.

An efficient and straightforward orthogonal methodology was successfully developed to achieve constrained l-prolyl-l-leucylglycinamide (PLG) analogues starting from two proline mimetics based on a 2-azanorbornane scaffold. A preliminary dopamine D2 receptor radiolabeled binding assay with [3H]-N-propylnorapomorphine shows that enantiopurity of PLG peptidomimetics based on 2-azanorbornane is a requirement to achieve statistically significant positive modulators of the D2 receptor. This is the first documented active peptidomimetic of PLG whose bioactivity is not correlated with the C-terminal carboxamide pharmacophore and which cannot adopt the hypothesized type II ß-turn conformation.

Discovery of New Potent Positive Allosteric Modulators of Dopamine D2 Receptors: Insights into the Bioisosteric Replacement of Proline to 3-Furoic Acid in the Melanostatin Neuropeptide.

The control of Parkinson's disease (PD) is challenged by the motor and non-motor fluctuations as well as dyskinesias associated with levodopa long-term therapy. As such, pharmacological alternatives to reduce the reliance on this drug are needed. Melanostatin (MIF-1), a positive allosteric modulator (PAM) of D2 receptors (D2R), is being explored as a novel pharmacological approach focused on D2R potentiation. In this work, 3-furoic acid (3-Fu) was successfully employed as an l-proline (Pro) surrogate, affording two potent MIF-1 analogues, methyl 3-furoyl-l-leucylglycinate (4a) and 3-furoyl-l-leucylglycinamide (6a). In this series, the C-terminal carboxamide moiety was found crucial to enhancing the potency and toxicological profile, yet it is not considered a requisite for the PAM activity. Conformational analysis excludes 4a from adopting the claimed type II ß-turn. The discovery and validation of 6a as a lead compound open a new avenue for the development of a novel class of anti-Parkinson therapeutics targeting the D2R.

Perturbation Theory/Machine Learning Model of ChEMBL Data for Dopamine Targets: Docking, Synthesis, and Assay of New l-Prolyl-l-leucyl-glycinamide Peptidomimetics.

Predicting drug-protein interactions (DPIs) for target proteins involved in dopamine pathways is a very important goal in medicinal chemistry. We can tackle this problem using Molecular Docking or Machine Learning (ML) models for one specific protein. Unfortunately, these models fail to account for large and complex big data sets of preclinical assays reported in public databases. This includes multiple conditions of assays, such as different experimental parameters, biological assays, target proteins, cell lines, organism of the target, or organism of assay. On the other hand, perturbation theory (PT) models allow us to predict the properties of a query compound or molecular system in experimental assays with multiple boundary conditions based on a previously known case of reference. In this work, we report the first PTML (PT + ML) study of a large ChEMBL data set of preclinical assays of compounds targeting dopamine pathway proteins. The best PTML model found predicts 50000 cases with accuracy of 70-91% in training and external validation series. We also compared the linear PTML model with alternative PTML models trained with multiple nonlinear methods (artificial neural network (ANN), Random Forest, Deep Learning, etc.). Some of the nonlinear methods outperform the linear model but at the cost of a notable increment of the complexity of the model. We illustrated the practical use of the new model with a proof-of-concept theoretical-experimental study. We reported for the first time the organic synthesis, chemical characterization, and pharmacological assay of a new series of l-prolyl-l-leucyl-glycinamide (PLG) peptidomimetic compounds. In addition, we performed a molecular docking study for some of these compounds with the software Vina AutoDock. The work ends with a PTML model predictive study of the outcomes of the new compounds in a large number of assays. Therefore, this study offers a new computational methodology for predicting the outcome for any compound in new assays. This PTML method focuses on the prediction with a simple linear model of multiple pharmacological parameters (IC50, EC50, Ki, etc.) for compounds in assays involving different cell lines used, organisms of the protein target, or organism of assay for proteins in the dopamine pathway.

Allosteric modulation of the dopamine receptor by conformationally constrained type VI beta-turn peptidomimetics of Pro-Leu-Gly-NH2.

A peptidomimetic of Pro-Leu-Pro-NH2, 7, possessing an indolizidinone type VI beta-turn mimic was synthesized via improved high-yielding protocols for the preparation and Cbz protection of alpha-allylproline. Bicyclic peptidomimetic 7 and spirobicylic peptidomimetic 8 enhanced the binding of [3H] N-propylnorapomorphine to dopamine receptors indicating that a type VI beta-turn is a possible bioactive conformation of the homochiral Pro-Leu-Pro-NH2 and Pro-Pro-Pro-NH 2 analogues of Pro-Leu-Gly-NH2 at the dopamine receptor allosteric regulatory site.

MIF-1 and its peptidomimetic analogs attenuate haloperidol-induced vacuous chewing movements and modulate apomorphine-induced rotational behavior in 6-hydroxydopamine-lesioned rats.

Two melanocyte-stimulating hormone release inhibiting factor-1 (MIF-1) also known as L-prolyl-L-leucyl-glycinamide (PLG) peptidomimetic analogs, 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]-amino]-3-(butyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (A) and 3(R)-[[[2(S)-pyrrolidinyl]carbonyl]amino]-3-(benzyl)-2-oxo-1-pyrrolidineacetamide trifluoroacetate (B), were evaluated for their ability to modulate dopaminergic activity by measuring apomorphine-induced rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats, and haloperidol (HP)-induced vacuous chewing movements (VCMs) in rats; animal models of Parkinson's disease (PD) and human tardive dyskinesia (TD), respectively. In the 6-OHDA model, both analogs were found to potentiate the contralateral rotational behavior induced by apomorphine dose-dependently and with approximately the same potency. Furthermore, each analog was able to significantly attenuate HP-induced VCMs with almost equal efficacy. The potency and efficacy of these analogs were significantly greater than their parent compound, PLG. These results suggest that both analogs can modulate dopaminergic activity in vivo, likely by the same mechanisms recruited by PLG previously reported.

An extraordinary relationship involving MIF-1 and other peptides.

In commemoration of Abba J. Kastin's exceptional service as the founding editor for the international journal Peptides, I review our collaborative work on how neuropeptides are involved in depression and other neuropsychiatric behavior. A special focus is on MIF-1 (prolyl-leucyl-glycinamide) that was discovered in the Kastin laboratory and shown effective to treat human depression with greater efficacy and faster onset of action than traditional antidepressants at the time of clinical trial. My personal reflection of the evolving changes of translational research on neuropeptides will hopefully provide some insight to young investigators.

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.

Design, synthesis and evaluation of a PLG tripeptidomimetic based on a pyridine scaffold.

A 2,3,4-substituted pyridine derivative has been identified as a potential tripeptidomimetic scaffold. The design of the scaffold was based on conformational and electrostatic comparisons with a natural tripeptide. The scaffold has been used in the synthesis of a Pro-Leu-Gly-NH2 (PLG) mimetic. The different substituents in the 2-, 3-, and 4-positions of the pyridine ring were introduced via an aromatic nucleophilic substitution reaction, a "halogen-dancing" reaction, and a Grignard coupling of a Boc-protected amino aldehyde, respectively. The synthetic route involves eight steps and provides the mimetic in 20% overall yield. The pyridine based PLG-mimetic was evaluated for its ability to enhance the maximum response of the dopamine agonist N-propylapomorphine (NPA) at human D2 receptors using a cell based assay (the R-SAT assay). The dose-response curve of the mimetic was found to exhibit a down-turn phase, similar to that of PLG. In addition, the mimetic was more potent than PLG to enhance the NPA response; the maximum response was found to be 146% at 10 nM concentration, as compared to 115% for PLG at the same concentration. Interestingly, conformational analysis by molecular modeling showed that the pyridine mimetic cannot adopt a type II beta-turn conformation that previously has been suggested to be the bioactive conformation of PLG.

Synthesis and dopamine receptor modulating activity of 3-substituted gamma-lactam peptidomimetics of L-prolyl-L-leucyl-glycinamide.

gamma-Lactam peptidomimetic 2 of Pro-Leu-Gly-NH(2) (PLG) was substituted at the 3-position with isobutyl, butyl, and benzyl moieties to give the PLG peptidomimetics 3-5, respectively. These compounds were synthesized to test the hypothesis that attaching a hydrophobic moiety to the lactam ring to mimic the isobutyl side chain of the leucyl residue of PLG would increase the dopamine receptor modulating activity of such peptidomimetics. These peptidomimetics were tested for their ability to enhance the binding of [(3)H]-N-propylnorapomorphine to dopamine receptors isolated from bovine striatal membranes. The rank order of effectiveness of the 3-substituent was benzyl > n-butyl > isobutyl > H.

Synthesis and dopamine receptor modulating activity of novel peptidomimetics of L-prolyl-L-leucyl-glycinamide featuring alpha,alpha-disubstituted amino acids.

In the present study, L-prolyl-L-leucyl-glycinamide (1) peptidomimetics 3a-3d and 4a-4d were synthesized utilizing alpha, alpha-disubstituted amino acids. These analogues were designed to explore the conformational effects of constraints at the phi3 and psi3 torsion angles. Constrained conformations were verified by the use of X-ray crystallography and circular dichroism. The effects of Pro-Leu-Gly-NH2 analogues 3a-3d and 4a-4d on enhancing rotational behavior induced by apomorphine in the 6-hydroxydopamine-lesioned animal models of Parkinson's disease were studied. The ability of these peptidomimetics to increase the binding of agonist N-propylnorapomorphine (NPA) to the dopamine D2 receptor was also examined. Extended analogue Pro-Leu-Deg-NH2 was the most active compound of this series. It was 10 times more potent and almost 2 times more effective than 1 in increasing apomorphine-induced rotations (56 +/- 15% at 1.0 mg/kg ip) and in enhancing [3H]NPA specific binding (40%).

Design, synthesis, and dopamine receptor modulating activity of spiro bicyclic peptidomimetics of L-prolyl-L-leucyl-glycinamide.

In the present study, the synthesis of the 5.5.6. and 5.6.5. spiro bicyclic lactam PLG peptidomimetics, compounds 3 and 4, respectively, was undertaken. These peptidomimetics were designed to examine the following: (1) the effect that changing the size of the thiazolidine and lactam ring systems would have on the ability of these systems to mimic the type-II beta-turn and (2) the effect that these structural perturbations would have on the ability of the peptidomimetics to modulate dopamine receptors. Through the use of the [3H]spiroperidol/N-propylnorapomorphine (NPA) dopamine D2 receptor competitive binding assay, 3 and 4, at a concentration of 100 nM, decreased the dissociation constant of the high-affinity state of the dopamine receptor for the agonist. These effects were observed when either Gpp(NH)p was absent or present and they were comparable to those produced by PLG at a concentration of 1 microM. Peptidomimetics 3 and 4 also increased the percentage of D2 receptors that existed in the high-affinity state. Even with Gpp(NH)p present, 3 and 4 were able to return the RH/RL ratios to values observed in the respective controls where Gpp(NH)p was absent. Furthermore, both peptidomimetics were able to attenuate the Gpp(NH)p-induced shift to the low-affinity state to a greater extent than PLG. Peptidomimetics 3 and 4 were evaluated in vivo as modulators of apomorphine-induced rotational behavior in the 6-hydroxydopamine-lesioned rat model of hemiparkinsonism, and each affected the rotational behavior in a bell-shaped dose-response relationship producing increases of 95 +/- 31% (0.01 mg/kg, ip) and 88 +/- 14% (0.001 mg/kg, ip), respectively. In comparison, the previously reported 5.5.5. spiro bicyclic lactam 2 increased rotational behavior by 25 +/- 11% (0.01 mg/kg, ip).

Synthesis and dopamine receptor modulating activity of substituted bicyclic thiazolidine lactam peptidomimetics of L-prolyl-L-leucyl-glycinamide.

6-Substituted bicyclic thiazolidine lactam peptidomimetics of Pro-Leu-Gly-NH(2) (1) were synthesized to test the hypothesis that incorporation of a hydrophobic side chain into the bicyclic thiazolidine lactam scaffold would further enhance the dopamine receptor modulating activity of such peptidomimetics. The substituents employed were the isobutyl, butyl, and benzyl groups to give peptidomimetics 3-5, respectively. These peptidomimetics were evaluated in vivo as modulators of apomorphine-induced rotational behavior in the 6-hydroxydopamine-lesioned rat model of hemiparkinsonism and were compared with the unsubstituted bicyclic thiazolidine lactam Pro-Leu-Gly-NH(2) peptidomimetic 2. Peptidomimetics 3-5 each affected rotational behavior in a bell-shaped dose-response relationship producing maximal increases of 44% (1 microgram/kg,ip), 56% (0.1 microgram/kg,ip), and 30% (1 microgram/kg, ip), respectively. In comparison, unsubstituted peptidomimetic 2 increased rotational behavior by only 23% at a dose of 0.1 microgram/kg, ip.

Comparison of active conformations of the insectatachykinin/tachykinin and insect kinin/Tyr-W-MIF-1 neuropeptide family pairs.

A comparison of solution conformations of active, restricted-conformation analogues of two sequence-similar insect/vertebrate neuropeptide family pairs shed light on the potential existence of molecular evolutionary relationships. Analogues of the locustatachykinins and the mammalian tachykinin substance P, containing a sterically hindered Aib-NMePhe/Tyr residue block, share similar low-energy turn conformations incorporating a cis peptide bond. Conversely, restricted conformation analogues of the insect kinins and the mammalian opiate peptide Tyr-W-MIF-1, with near identical C-terminal tetrapeptide sequences, adopt different conformations. The insect kinins adopt a cisPro 1-4 beta-turn, in which the Phe1 is critical for bioactivity. Tyr-W-MIF-1 prefers a transPro 2-5 turn, and an additional N-terminal Phe severely inhibits mu-opiate receptor binding. Comparisons of the chemical/conformational requirements for receptor interaction are consistent with a distant evolutionary relationship between the insectatachykinins and tachykinins, but not between the insect kinins and Tyr-W-MIF-1. Therefore, analogues of the insect kinins with pest control potential can be readily designed to avoid mammalian interactions.

Modulatory effects of PLG and its peptidomimetics on haloperidol-induced catalepsy in rats.

A behavioral model of dopaminergic function in the rat was used to examine the anticataleptic effects of L-prolyl-L-leucyl-glycinamide (PLG) and peptidomimetic analogs of PLG. Administration of 1 mg/kg PLG intraperitoneally significantly attenuated haloperidol (1 mg/kg)-induced catalepsy (as measured by the standard horizontal bar test), whereas doses of 0.1 and 10 mg/kg PLG did not. Eight synthetic PLG peptidomimetics (Calpha, alpha-dialkylated glycyl residues with lactam bridge constraint [1-4] and without [5-8]) were tested in the same manner (at a dose of 1 microg/kg) and categorized according to their activity, i.e. very active (5), moderately active (2, 3, 4, and 6), and inactive (1, 7, and 8). The catalepsy-reversal action of the diethylglycine-substituted peptidomimetic 5 was examined further and found to exhibit a U-shaped dose-response effect with an optimal dose of 1 microg/kg. The similarity between the effects of PLG and the synthetic peptidomimetics suggests a common mechanism of action. Finally, the synthetic peptidomimetics examined here, particularly peptidomimetic 5, were more effective than PLG in attenuating haloperidol-induced catalepsy.

Chronic treatment with MIF-1 prevents the painful stimuli threshold elevation induced by neonatal handling in mice.

Chronic postnatal stressful handling results in a hyposensitivity to thermal nociceptive stimuli. This phenomenon is strongly affected by manipulations of the opioid system. In the present experiment, we report that chronic treatment with MIF-1 during the neonatal period prevents the behavioral alterations induced by handling while it is completely ineffective if injected acutely before antinociceptive testing by the tail flick test at 45 days of life.

Opioid binding profile of morphiceptin, Tyr-MIF-1 and dynorphin-related peptides in rat brain membranes.

Opioid properties of several morphiceptin- (Tyr-Pro-Phe-Pro-NH2), Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) and dynorphin-derivatives were characterized in rat brain in vitro receptor binding assay and in electrically stimulated longitudinal muscle strip preparation of guinea pig ileum. In the case of morphiceptin-related peptides, an excellent correlation was found between the [3H]-naloxone binding displacement data and the agonist potencies determined in the bioassay. The "turning point' was the C-terminal amidation in the tri- and tetrapeptide pairs in both series. Tyr-MIF-1 derivatives showed weak affinity in the opioid receptor binding assay and none of them had any remarkable effect in the bioassay either as agonist or antagonist. The dynorphin A(1-10)-peptides modified at positions 5 and 8 retained their affinity with Pro5-, Pro8-, and Ala8-substituents, whereas some loss of affinity was observed in the case of Gly8-Dyn A(1-10).

Melanostatin, a new melanin synthesis inhibitor. Production, isolation, chemical properties, structure and biological activity.

Melanostatin, a new antibiotic with melanin synthesis inhibitor activity, was isolated from the fermentation broth of Streptomyces clavifer No. N924-2. Its structure was determined by spectral analysis and degradation experiments. Melanostatin strongly inhibited melanin formation in Streptomyces bikiniensis NRRL B-1049 and B16 melanoma cells.

Synthesis, stereochemistry, and biological properties of the depigmenting agents, melanostatin, feldamycin and analogs.

Syntheses of melanostatin and feldamycin have been completed from L-serine and L-threonine, respectively, and the configuration of unknown asymmetric carbons determined. Feldamycin analogs have also been prepared and the L-tryptophyl analog was the most potent in the depigmentation of Streptomyces bikiniensis and B16 melanoma cells.

Antiopioid properties of the TYR-MIF-1 family.

The Tyr-MIF-1 family of peptides includes MIF-1, Tyr-MIF-1, Tyr-W-MIF-1 and Tyr-K-MIF-1, which have been isolated from bovine hypothalamus and human brain cortex. All these peptides interact with opioid receptors and in addition bind to non-opiate sites specific for each of the peptides. Data in the literature suggest that peptides of the Tyr-MIF-1 family (Tyr-MIF-1s) have antiopioid and opioid- like effects. It is known that some anti-opioid peptides (AOP) could reverse morphine-induced analgesia in rodents and men and able to inhibit the expression of some forms of stress-induced analgesia (SIA) in various species. We examined the effects of the Tyr-MIF-1 peptides (all in dose 1 mg/kg i.p.) in the male Wistar rats on morphine-induced analgesia in acute pain using the paw-pressure (PP) and the tail-flick (TF) tests and on immobilization stress-induced antinociception using the PP test. Our results showed that the Tyr-MIF-1 peptides significantly decreased the analgesic effect of morphine (1 mg/kg i.p.) in both tests used. Immobilization of the rats increased the pain threshold for at least 1 h. The Tyr-MIF-1 peptides reduced stress-induced antinociception in PP test. In conclusion, our findings indicate that Tyr-MIF-1s modulate the analgesic effects of morphine and SIA, which corresponds with the hypothesis about AOP mentioned above.