Human
macrophage migration inhibitory factor (MIF) is both a keto–enol
tautomerase and a cytokine associated with numerous inflammatory diseases
and cancer. Consistent with observed correlations between inhibition
of the enzymatic and biological activities, discovery of MIF inhibitors
has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates.
The accuracy of these assays is compromised by several issues including
substrate instability, spectral interference, and short linear periods
for product formation. In this work, we report the syntheses of fluorescently
labeled MIF inhibitors and their use in the first fluorescence polarization-based
assay to measure the direct binding of inhibitors to the active site.
The assay allows the accurate and efficient identification of competitive,
noncompetitive, and covalent inhibitors of MIF in a manner that can
be scaled for high-throughput screening. The results for 22 compounds
show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and
the other is a compound from the patent literature. X-ray crystal
structures for two of the most potent compounds bound to MIF are also
reported here. Striking combinations of protein–ligand hydrogen
bonding, aryl–aryl, and cation−π interactions
are responsible for the high affinities. A new chemical series was
then designed using this knowledge to yield two more strong MIF inhibitors/binders.
Inhibitors of human macrophage migration inhibitory factor (MIF) previously reported in the literature have been reexamined by synthesis, assaying for tautomerase activity, and protein crystallography. Substantial inconsistencies between prior and current assay results are noted. They appear to arise from difficulties with the tautomerase substrates, solubility issues, and especially covalent inhibition. Incubation time variation shows that 3, 4, 6, and 9 are covalent or slow-binding inhibitors. Two protein crystal structures are provided; one confirms that the twice-discovered 3 is a covalent inhibitor.
We report the synthesis of new compounds 4− 35 based on structural modifications of different moieties of previously described lead UCM-2550. The new nonpiperazine derivatives, representing second-generation agonists, were assessed for binding affinity, selectivity, and functional activity at the 5-HT 1A receptor (5-HT 1A R). Computational β 2 -based homology models of the ligand−receptor complexes were used to explain the observed structure−affinity relationships. Selected candidates were also evaluated for their potential in vitro and in vivo neuroprotective properties. Interestingly, compound 26 (2-{6-[(3,4-dihydro-2H-chromen-2-ylmethyl)amino]hexyl}tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione) has been characterized as a high-affinity and potent 5-HT 1A R agonist (K i = 5.9 nM, EC 50 = 21.8 nM) and exhibits neuroprotective effect in neurotoxicity assays in primary cell cultures from rat hippocampus and in the MCAO model of focal cerebral ischemia in rats.
We report the synthesis of new compounds 4-35 based on two different openings (A and B) of the chromane ring present in the previously identified 5-HT1A receptor (5-HT1AR) ligand 3. The synthesized compounds were assessed for binding affinity, selectivity, and functional activity at the 5-HT1AR. Selected candidates resulting from B opening were also evaluated for their potential antinociceptive effect in vivo and pharmacokinetic properties in vitro. Analogue 19 [2-(4-{[2-(2-ethoxyphenoxy)ethyl]amino}butyl)tetrahydro-1H-pyrrolo[1,2-c]imidazole-1,3(2H)-dione] has been characterized as a high-affinity and potent 5-HT1AR agonist (Ki = 2.3 nM; EC50 = 19 nM). Pharmacokinetic studies indicated that compound 19 displays a good metabolic stability in human liver microsomes (t1/2 ∼ 3 h and CLint = 3.5 mL/min/kg, at 5 μM), and a low level of protein binding (25%, at 5 μM). Interestingly, 19 (3 mg/kg, ip, and 30 mg/kg, po) caused significant attenuation of formalin-induced behavior in early and late phases of the mouse intradermal formalin test of pain, and this in vivo effect was reversed by the selective 5-HT1AR antagonist WAY-100635. Thus, the new 5-HT1AR agonist identified in this work, 19, exhibits oral analgesic activity, and the results herein represent a step toward identifying new therapeutics for the control of pain.
Covalent inhibitors of wild-type HIV-1 reverse transcriptase (CRTIs) are reported. Three compounds derived from catechol diether nonnucleoside inhibitors (NNRTIs) with addition of a fluorosulfate warhead are demonstrated to covalently modify Tyr181 of HIV-RT. X-ray crystal structures for complexes of the CRTIs with the enzyme are provided, which fully demonstrate the covalent attachment, and confirmation is provided by appropriate mass shifts in ESI-TOF mass spectra. The three CRTIs and six noncovalent analogues are found to be potent inhibitors with both IC 50 values for in vitro inhibition of WT RT and EC 50 values for cytopathic protection of HIV-1-infected human T-cells in the 5−320 nM range.
Protozoans of the genus Cryptosporidium are the causative agent of the gastrointestinal disease, cryptosporidiosis, which can be fatal in immunocompromised individuals. Cryptosporidium hominis (C. hominis) bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in the folate biosynthesis pathway and a molecular target for inhibitor design. Previous studies have demonstrated the importance of the ChTS-DHFR linker region "crossover helix" to the enzymatic activity and stability of the ChDHFR domain. We conducted a virtual screen of a novel non-active site pocket located at the interface of the ChDHFR domain and crossover helix. From this screen we have identified and characterized a noncompetitive inhibitor, compound 15, a substituted diphenyl thiourea. Through subsequent structure activity relationship studies, we have identified a time-dependent inhibitor lead, compound 15D17, a thiol-substituted 2-hydroxy-N-phenylbenzamide, which is selective for ChTS-DHFR, and whose effects appear to be mediated by covalent bond formation with a non-catalytic cysteine residue adjacent to the nonactive site pocket.
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