Patrick Chaltin

Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication

Lens epithelium-derived growth factor (LEDGF/p75) is a cellular cofactor of HIV-1 integrase that promotes viral integration by tethering the preintegration complex to the chromatin. By virtue of its crucial role in the early steps of HIV replication, the interaction between LEDGF/p75 and integrase represents an attractive target for antiviral therapy. We have rationally designed a series of 2-(quinolin-3-yl)acetic acid derivatives (LEDGINs) that act as potent inhibitors of the LEDGF/p75-integrase interaction and HIV-1 replication at submicromolar concentration by blocking the integration step. A 1.84-A resolution crystal structure corroborates the binding of the inhibitor in the LEDGF/p75-binding pocket of integrase. Together with the lack of cross-resistance with two clinical integrase inhibitors, these findings define the 2-(quinolin-3-yl)acetic acid derivatives as the first genuine allosteric HIV-1 integrase inhibitors. Our work demonstrates the feasibility of rational design of small molecules inhibiting the protein-protein interaction between a viral protein and a cellular host factor.

Small-Molecule Inhibitors of the LEDGF/p75 Binding Site of Integrase Block HIV Replication and Modulate Integrase Multimerization

ABSTRACT Targeting the HIV integrase (HIV IN) is a clinically validated approach for designing novel anti-HIV therapies. We have previously described the discovery of a novel class of integration inhibitors, 2-(quinolin-3-yl)acetic acid derivatives, blocking HIV replication at a low micromolar concentration through binding in the LEDGF/p75 binding pocket of HIV integrase, hence referred to as LEDGINs. Here we report the detailed characterization of their mode of action. The design of novel and more potent analogues with nanomolar activity enabled full virological evaluation and a profound mechanistic study. As allosteric inhibitors, LEDGINs bind to the LEDGF/p75 binding pocket in integrase, thereby blocking the interaction with LEDGF/p75 and interfering indirectly with the catalytic activity of integrase. Detailed mechanism-of-action studies reveal that the allosteric mode of inhibition is likely caused by an effect on HIV-1 integrase oligomerization. The multimodal inhibition by LEDGINs results in a block in HIV integration and in a replication deficiency of progeny virus. The allosteric nature of LEDGINs leads to synergy in combination with the clinically approved active site HIV IN strand transfer inhibitor (INSTI) raltegravir, and cross-resistance profiling proves the distinct mode of action of LEDGINs and INSTIs. The allosteric nature of inhibition and compatibility with INSTIs underline an interest in further (clinical) development of LEDGINs.

Repurposing as a means to increase the activity of amphotericin B and caspofungin against Candida albicans biofilms

OBJECTIVES Biofilms of Candida species, often formed on medical devices, are generally resistant to currently available antifungal drugs. The aim of this study was to identify compounds that increase the activity of amphotericin B and caspofungin, commonly used antifungal agents, against Candida biofilms. METHODS A library containing off-patent drugs was screened for compounds, termed enhancers, that increase the in vitro activity of amphotericin B against Candida albicans biofilms. Biofilms were grown in 96-well plates and growth was determined by the cell titre blue assay. Synergy between identified enhancers and antifungal agents was further characterized in vitro using fractional inhibitory concentration index (FICI) values and in vivo using a worm biofilm infection model. In light of the application of these enhancers onto implants, their possible effect on the growth potential of MG63 osteoblast-like cells was assessed. RESULTS Pre-incubation of C. albicans biofilms with subinhibitory concentrations of the enhancers drospirenone, perhexiline maleate or toremifene citrate significantly increased the activity of amphotericin B or caspofungin (FICI  < 0.5) against C. albicans and Candida glabrata biofilms. Moreover, these enhancers did not affect the growth potential of osteoblasts. Interestingly, toremifene citrate also enhanced the in vitro activity of caspofungin in a mixed biofilm consisting of C. albicans and Staphylococcus epidermidis. Furthermore, we demonstrate synergy between toremifene citrate and caspofungin in an in vivo worm C. albicans biofilm infection model. CONCLUSIONS Our data demonstrate an in vitro and in vivo enhancement of the antibiofilm activity of caspofungin by toremifene citrate. Furthermore, our results pave the way for implant-related applications of the identified enhancers.

LEDGINs, non-catalytic site inhibitors of HIV-1 integrase: a patent review (2006 – 2014)

Introduction: Integration of the viral genome into the host cell chromatin is a central step in the replication cycle of the HIV. Blocking the viral integrase (IN) enzyme therefore provides an attractive therapeutic strategy, as evidenced by the recent clinical approval of three IN strand transfer inhibitors. Viral resistance and cross-resistance among these inhibitors, however, warrant the search for compounds targeting HIV integration through alternative mechanisms of action. Areas covered: The most potent class of allosteric IN inhibitors was independently identified at the University of Leuven, Belgium, and at Boehringer Ingelheim, Canada. These compounds, coined LEDGINs (after the lens epithelium-derived growth factor/p75 cofactor binding pocket on IN) or non-catalytic site IN inhibitors (NCINIs) by the respective groups, have shown remarkable antiviral activity. This review provides a brief introduction to the compound class and discusses the recent patent literature (2006 to the present). Expert opinion: LEDGINs are still early in development. Trials with clinical candidate BI-224436 were put on hold despite promising results. Literature, however, reveals that almost all major pharmaceutical companies active in the treatment of HIV/AIDS have taken a significant interest in this class. As a result, several of these inhibitors may soon enter clinical trials.

Activation of TRPM3 by a potent synthetic ligand reveals a role in peptide release

Significance The cation channel TRPM3 is highly expressed in the sensory system, where it plays a key role in the detection of noxious heat and the development of inflammatory heat hypersensitivity. Our understanding of the physiological role of TRPM3 in the sensory system and other tissues is hampered by the lack of potent pharmacologic tools, however. This study describes CIM0216, a small-molecule TRPM3 agonist. Our results indicate that CIM0216 is much more potent than established TRPM3 agonists, particularly owing to its ability to open two distinct cation-permeable pores in TRPM3. Using CIM0216 as a pharmacologic tool, we reveal that activation of TRPM3 evokes the release of calcitonin gene-related peptide from sensory nerve terminals and of insulin from pancreatic islets. Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation.

Synthesis and pharmacological evaluation of carboxycoumarins as a new antitumor treatment targeting lactate transport in cancer cells.

Under hypoxia, cancer cells consume glucose and release lactate at a high rate. Lactate was recently documented to be recaptured by oxygenated cancer cells to fuel the TCA cycle and thereby to support tumor growth. Monocarboxylate transporters (MCT) are the main lactate carriers and therefore represent potential therapeutic targets to limit cancer progression. In this study, we have developed and implemented a stepwise in vitro screening procedure on human cancer cells to identify new potent MCT inhibitors. Various 7-substituted carboxycoumarins and quinolinone derivatives were synthesized and pharmacologically evaluated. Most active compounds were obtained using a palladium-catalyzed Buchwald-Hartwig type coupling reaction, which proved to be a quick and efficient method to obtain aminocarboxycoumarin derivatives. Inhibition of lactate flux revealed that the most active compound 19 (IC50 11 nM) was three log orders more active than the CHC reference compound. Comparison with warfarin, a conventional anticoagulant coumarin, further showed that compound 19 did not influence the prothrombin time which, together with a good in vitro ADME profile, supports the potential of this new family of compounds to act as anticancer drugs through inhibition of lactate flux.

Degradation profile of [His7]-corazonin in the hemolymph of the desert locust Schistocerca gregaria.

Degradation of the neuropeptide [His7]-corazonin, a key hormone in phase transition in locusts was studied using [3H][His7]-corazonin, RP-HPLC and mass spectrometry. After 4h incubation, 50 and 75% of [His7]-corazonin could still be found in hemolymph of gregarious and solitarious Schistocerca gregaria, respectively. Under in vivo conditions the half-life was 30 min. These results are in contrast to many other neuropeptides that usually have half lives of a few minutes. The peptide is cleaved first by an endopeptidase, either just before or after the Tyr residue at position 5. Next, the C-terminal degradation fragments are further degraded by a dipeptidyl-peptidase, whereas the N-terminal fragments are further broken down one amino acid at a time. In addition, [Dopa5][His7]-corazonin was detected. Upon synthesis, this unexpected molecular modification turned out to be biologically active in bringing about cuticular melanization.

New dsDNA Binding Unnatural Oligopeptides with Pyrimidine Selectivity

Solid phase peptide library screening followed by extension of a lead recognition element for binding to a dsDNA sequence (NF binding site of IL6) using solution phase screening, delivered a new DNA binding peptide, Ac-Arg-Ual-Sar-Chi-Chi-Tal-Arg-CONH2. In the present research, the contribution of the different amino acid side chains to the binding strength of the peptide to dsDNA was investigated using an ethidium bromide displacement test. Based on these results, the lead structure was optimized by deconvolution. Eight new unnatural amino acids were evaluated at two positions of the heptapeptide replacing the Ual-Sar fragment. The strongest dsDNA binding was observed using ([(3-chlorophenyl)methyl]amino)acetic acid (Cbg) and beta-cyclohexyl-l-alanine (Cha) respectively, at those two positions. A 10-fold increase in affinity compared to the Ual-Sar sequence was obtained. Further enhancement of dsDNA binding was obtained with hybrid molecules linking the newly developed peptide fragment to an acridine derivative with a flexible spacer. This resulted in ligands with affinities in the microM range for the dsDNA target (K(d) of 2.1 x 10(-6) M). DNase I footprinting with the newly developed oligopeptide motifs showed the presence of a pronounced pyrimidine specificity, while conjugation to an intercalator seems to redirect the interaction to mixed sequences. This way, new unnatural oligopeptide motifs and hybrid molecules have been developed endowed with different sequence selectivities. The results demonstrate that the unnatural peptide library approach combined with subsequent modification of selected amino acid positions, is very suited for the discovery of novel sequence-specific dsDNA binding ligands.

Elucidation of the Mode of Action of a New Antibacterial Compound Active against Staphylococcus aureus and Pseudomonas aeruginosa

Nosocomial and community-acquired infections caused by multidrug resistant bacteria represent a major human health problem. Thus, there is an urgent need for the development of antibiotics with new modes of action. In this study, we investigated the antibacterial characteristics and mode of action of a new antimicrobial compound, SPI031 (N-alkylated 3, 6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol), which was previously identified in our group. This compound exhibits broad-spectrum antibacterial activity, including activity against the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa. We found that SPI031 has rapid bactericidal activity (7-log reduction within 30 min at 4x MIC) and that the frequency of resistance development against SPI031 is low. To elucidate the mode of action of SPI031, we performed a macromolecular synthesis assay, which showed that SPI031 causes non-specific inhibition of macromolecular biosynthesis pathways. Liposome leakage and membrane permeability studies revealed that SPI031 rapidly exerts membrane damage, which is likely the primary cause of its antibacterial activity. These findings were supported by a mutational analysis of SPI031-resistant mutants, a transcriptome analysis and the identification of transposon mutants with altered sensitivity to the compound. In conclusion, our results show that SPI031 exerts its antimicrobial activity by causing membrane damage, making it an interesting starting point for the development of new antibacterial therapies.

Synthetic strategy and antiviral evaluation of diamide containing heterocycles targeting dengue and yellow fever virus

High-throughput screening of a subset of the CD3 chemical library (Centre for Drug Design and Discovery; KU Leuven) provided us with a lead compound 1, displaying low micromolar potency against dengue virus and yellow fever virus. Within a project aimed at discovering new inhibitors of flaviviruses, substitution of its central imidazole ring led to synthesis of variably substituted pyrazine dicarboxylamides and phthalic diamides, which were evaluated in cell-based assays for cytotoxicity and antiviral activity against the dengue virus (DENV) and yellow fever virus (YFV). Fourteen compounds inhibited DENV replication (EC50 ranging between 0.5 and 3.4 μM), with compounds 6b and 6d being the most potent inhibitors (EC50 0.5 μM) with selectivity indices (SI) > 235. Compound 7a likewise exhibited anti-DENV activity with an EC50 of 0.5 μM and an SI of >235. In addition, good antiviral activity of seven compounds in the series was also noted against the YFV with EC50 values ranging between 0.4 and 3.3 μM, with compound 6n being the most potent for this series with an EC50 0.4 μM and a selectivity index of >34. Finally, reversal of one of the central amide bonds as in series 13 proved deleterious to the inhibitory activity.