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1.  Structural Basis for Rational Design of Inhibitors Targeting Trypanosoma cruzi Sterol 14α-Demethylase: Two Regions of the Enzyme Molecule Potentiate Its Inhibition 
Journal of Medicinal Chemistry  2014;57(15):6704-6717.
Chagas disease, which was once thought to be confined to endemic regions of Latin America, has now gone global, becoming a new worldwide challenge with no cure available. The disease is caused by the protozoan parasite Trypanosoma cruzi, which depends on the production of endogenous sterols, and therefore can be blocked by sterol 14α-demethylase (CYP51) inhibitors. Here we explore the spectral binding parameters, inhibitory effects on T. cruzi CYP51 activity, and antiparasitic potencies of a new set of β-phenyl imidazoles. Comparative structural characterization of the T. cruzi CYP51 complexes with the three most potent inhibitors reveals two opposite binding modes of the compounds ((R)-6, EC50 = 1.2 nM, vs (S)-2/(S)-3, EC50 = 1.0/5.5 nM) and suggests the entrance into the CYP51 substrate access channel and the heme propionate-supporting ceiling of the binding cavity as two distinct areas of the protein that enhance molecular recognition and therefore could be used for the development of more effective antiparasitic drugs.
PMCID: PMC4136671  PMID: 25033013
2.  Identification of Highly Conserved Residues Involved in Inhibition of HIV-1 RNase H Function by Diketo Acid Derivatives 
Antimicrobial Agents and Chemotherapy  2014;58(10):6101-6110.
HIV-1 reverse transcriptase (RT)-associated RNase H activity is an essential function in viral genome retrotranscription. RNase H is a promising drug target for which no inhibitor is available for therapy. Diketo acid (DKA) derivatives are active site Mg2+-binding inhibitors of both HIV-1 RNase H and integrase (IN) activities. To investigate the DKA binding site of RNase H and the mechanism of action, six couples of ester and acid DKAs, derived from 6-[1-(4-fluorophenyl)methyl-1H-pyrrol-2-yl)]-2,4-dioxo-5-hexenoic acid ethyl ester (RDS1643), were synthesized and tested on both RNase H and IN functions. Most of the ester derivatives showed selectivity for HIV-1 RNase H versus IN, while acids inhibited both functions. Molecular modeling and site-directed mutagenesis studies on the RNase H domain demonstrated different binding poses for ester and acid DKAs and proved that DKAs interact with residues (R448, N474, Q475, Y501, and R557) involved not in the catalytic motif but in highly conserved portions of the RNase H primer grip motif. The ester derivative RDS1759 selectively inhibited RNase H activity and viral replication in the low micromolar range, making contacts with residues Q475, N474, and Y501. Quantitative PCR studies and fluorescence-activated cell sorting (FACS) analyses showed that RDS1759 selectively inhibited reverse transcription in cell-based assays. Overall, we provide the first demonstration that RNase H inhibition by DKAs is due not only to their chelating properties but also to specific interactions with highly conserved amino acid residues in the RNase H domain, leading to effective targeting of HIV retrotranscription in cells and hence offering important insights for the rational design of RNase H inhibitors.
PMCID: PMC4187941  PMID: 25092689
3.  Basic Quinolinonyl Diketo Acid Derivatives as Inhibitors of HIV Integrase and their Activity against RNase H Function of Reverse Transcriptase 
Journal of Medicinal Chemistry  2014;57(8):3223-3234.
A series of antiviral basic quinolinonyl diketo acid derivatives were developed as inhibitors of HIV-1 IN. Compounds 12d,f,i inhibited HIV-1 IN with IC50 values below 100 nM for strand transfer and showed a 2 order of magnitude selectivity over 3′-processing. These strand transfer selective inhibitors also inhibited HIV-1 RNase H with low micromolar potencies. Molecular modeling studies based on both the HIV-1 IN and RNase H catalytic core domains provided new structural insights for the future development of these compounds as dual HIV-1 IN and RNase H inhibitors.
PMCID: PMC4203401  PMID: 24684270
4.  Phenylpyrazolo[1,5-a]quinazolin-5(4H)-one: a suitable scaffold for the development of non-camptothecin Topoisomerase I (Top1) inhibitors 
Journal of medicinal chemistry  2013;56(18):7458-7462.
In search for a novel chemotype to develop Topoisomerase I (Top1) inhibitors, the pyrazolo[1,5–a]quinazoline nucleus, structurally related to the indenoisoquinoline system precursor of well-known Top1 poisons, was variously decorated (i.e. a substituted phenyl ring at 2– or 3–position, a protonable side chain at 4– or 5–position) affording a number of Top1 inhibitors with cleavage patterns common to CPT and MJ–III–65. SARs data were rationalized by means of an advanced docking protocol.
PMCID: PMC3982121  PMID: 23987476
Topoisomerase I; pyrazoloquinazoline derivatives; structure–activity relationships; medicinal chemistry
6.  New Promising Compounds with in Vitro Nanomolar Activity against Trypanosoma cruzi 
ACS Medicinal Chemistry Letters  2013;4(6):538-541.
The antiparasitic activity of azole and new 4-aminopyridine derivatives has been investigated. The imidazoles 1 and 3–5 showed a potent in vitro antichagasic activity with IC50 values in the low nanomolar concentration range. The (S)-1, (S)-3, and (S)-5 enantiomers showed (up to) a thousand-fold higher activity than the reference drug benznidazole and furthermore low cytotoxicity on rat myogenic L6 cells.
PMCID: PMC4027138  PMID: 24900706
Trypanosoma cruzi; CYP51; antiparasitic activity; Chagas disease; azole; aminopyridine
7.  Efficient Electrochemical N-Alkylation of N-Boc-Protected 4-Aminopyridines: Towards New Biologically Active Compounds 
ISRN organic chemistry  2014;2014:621592.
The use of electrogenerated acetonitrile anion allows the alkylation of N-Boc-4-aminopyridine in very high yields, under mild conditions and without by-products. The high reactivity of this base is due to its large tetraethylammonium counterion, which leaves the acetonitrile anion “naked.” The deprotection of the obtained compounds led to high yields in N-alkylated 4-aminopyridines. Nonsymmetrically dialkylated 4-aminopyridines were obtained by subsequent reaction of monoalkylated ones with t-BuOK and alkyl halides, while symmetrically dialkylated 4-aminopyridines were obtained by direct reaction of 4-aminopyridine with an excess of t-BuOK and alkyl halides. Some mono- and dialkyl-4-aminopyridines were selected to evaluate antifungal and antiprotozoal activity; the dialkylated 4-aminopyridines 3ac, 3ae and 3ff showed antifungal towards Cryptococcus neoformans; whereas 3cc, 3ee and 3ff showed antiprotozoal activity towards Leishmania infantum and Plasmodium falciparum.
PMCID: PMC4041018  PMID: 24955255
8.  Inhibiting the HIV Integration Process: Past, Present, and the Future 
Journal of Medicinal Chemistry  2013;57(3):539-566.
HIV integrase (IN) catalyzes the insertion into the genome of the infected human cell of viral DNA produced by the retrotranscription process. The discovery of raltegravir validated the existence of the IN, which is a new target in the field of anti-HIV drug research. The mechanism of catalysis of IN is depicted, and the characteristics of the inhibitors of the catalytic site of this viral enzyme are reported. The role played by the resistance is elucidated, as well as the possibility of bypassing this problem. New approaches to block the integration process are depicted as future perspectives, such as development of allosteric IN inhibitors, dual inhibitors targeting both IN and other enzymes, inhibitors of enzymes that activate IN, activators of IN activity, as well as a gene therapy approach.
PMCID: PMC3926363  PMID: 24025027
9.  Novel 3,5-Bis(bromohydroxybenzylidene)piperidin-4-ones as Coactivator-associated Arginine Methyltransferase 1 Inhibitors: Enzyme Selectivity and Cellular Activity 
Journal of medicinal chemistry  2011;54(13):4928-4932.
Coactivator-associated arginine methyltransferase 1 (CARM1) represents a valuable target for hormone-dependent tumors such as prostate and breast cancers. Here we report the enzyme and cellular characterization of the 1-benzyl-3,5-bis(3-bromo-4-hydroxybenzylidene) piperidin-4-one (7g) and its analogues 8a-l. Among them, 7g, 8e, and 8l displayed high and selective CARM1 inhibition, with lower or no activity against a panel of different PRMTs or HKMTs. In human LNCaP cells, 7g showed a significant dose-dependent reduction of the PSA promoter activity.
PMCID: PMC3487391  PMID: 21612300
10.  Novel Quinolinonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, and Biological Activities 
Journal of Medicinal Chemistry  2008;51(15):4744-4750.
Novel quinolinonyl diketo acids were designed to obtain integrase (IN) inhibitors selectively active against the strand transfer (ST) step of the HIV integration process. Those new compounds are characterized by a single aryl diketo acid (DKA) chain in comparison to 4, a bifunctional diketo acid reported by our group as an anti-IN agent highly potent against both the 3′-processing and ST steps. Compound 6d was the most potent derivative in IN enzyme assays, while 6i showed the highest potency against HIV-1 in acutely infected cells. The selective inhibition of ST suggested the newly designed monofunctional DKAs bind the IN−DNA acceptor site without affecting the DNA donor site.
PMCID: PMC2646871  PMID: 18646746
11.  Human Immunodeficiency Virus Type 1 (HIV-1) Integration: a Potential Target for Microbicides To Prevent Cell-Free or Cell-Associated HIV-1 Infection ▿  
Conceptually, blocking human immunodeficiency virus type 1 (HIV-1) integration is the last possibility for preventing irreversible cellular infection. Using cocultures of monocyte-derived dendritic cells and CD4+ T cells, which represent primary targets in sexual transmission, we demonstrated that blocking integration with integrase strand transfer inhibitors (InSTIs), particularly L-870812, could consistently block cell-free and cell-associated HIV-1 infection. In a pretreatment setting in which the compound was present before and during infection and was afterwards gradually diluted during the culture period, the naphthyridine carboxamide L-870812 blocked infection with the cell-free and cell-associated HIV-1 Ba-L strain at concentrations of, respectively, 1,000 and 10,000 nM. The potency of L-870812 was similar to that of the nucleotide reverse transcriptase inhibitor R-9-(2-phosphonylmethoxypropyl) adenine (PMPA) but one or two orders of magnitude lower than those of the nonnucleoside reverse transcriptase inhibitors UC781 and TMC120. In contrast, the diketo acid RDS derivative InSTIs showed clear-cut but weaker antiviral activity than L-870812. Moreover, L-870812 completely blocked subtype C and CRFO2_AG primary isolates, which are prevalent in the African heterosexual epidemic. Furthermore, the addition of micromolar concentrations of L-870812 even 24 h after infection could still block both cell-free and cell-associated Ba-L, opening the prospect of postexposure prophylaxis. Finally, an evaluation of the combined activity of L-870812 with either T20, zidovudine, PMPA, UC781, or TMC120 against replication-deficient HIV-1 Ba-L (env) pseudovirus suggested synergistic activity for all combinations. Importantly, compounds selected for the study by using the coculture model were devoid of acute or delayed cytotoxic effects at HIV-blocking concentrations. Therefore, these findings provide evidence supporting consideration of HIV-1 integration as a target for microbicide development.
PMCID: PMC2443891  PMID: 18474579
12.  Novel Bifunctional Quinolonyl Diketo Acid Derivatives as HIV-1 Integrase Inhibitors: Design, Synthesis, Biological Activities and Mechanism of Action 
Journal of medicinal chemistry  2006;49(6):1939-1945.
The virally encoded integrase protein is an essential enzyme in the life cycle of the HIV-1 virus and represents an attractive and validated target in the development of therapeutics against HIV infection. Drugs that selectively inhibit this enzyme, when used in combination with inhibitors of reverse transcriptase and protease, are believed to be highly effective in suppressing the viral replication. Among the HIV-1 integrase inhibitors, the β-diketo acids (DKAs) represent a major lead for anti-HIV-1drug development. In this study, novel bifunctional quinolonyl diketo acid derivatives were designed, synthesized and tested for their inhibitory ability against HIV-1 integrase. The compounds are potent inhibitors of integrase activity. Particularly, derivative 8 is a potent IN inhibitor for both steps of the reaction (3′-processing and strand transfer) and exhibits both high antiviral activity against HIV-1 infected cells and low cytotoxicity. Molecular modeling studies provide a plausible mechanism of action, which is consistent with ligand SARs and enzyme photo-crosslinking experiments.
PMCID: PMC2602756  PMID: 16539381
13.  HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein 
Retrovirology  2007;4:17.
The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN) (IN inhibitors, IINs) has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp) thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA) derivatives active on the HIV-1 IN strand transfer (ST) step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR) reversing ability.
The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells.
To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.
PMCID: PMC1828167  PMID: 17343726
14.  Development of a Human Immunodeficiency Virus Vector-Based, Single-Cycle Assay for Evaluation of Anti-Integrase Compounds 
Antimicrobial Agents and Chemotherapy  2006;50(10):3407-3417.
Therapeutic strategies aimed at inhibiting human immunodeficiency virus type 1 (HIV-1) replication employ a combination of drugs targeted to two viral enzymes (reverse transcriptase and protease) and to the viral entry/fusion step. However, the high propensity of HIV-1 to develop resistance makes the development of novel compounds targeting different steps of the HIV-1 life cycle essential. Among these, integrase (IN) inhibitors have successfully passed the early phases of clinical development. By preventing integration, IN inhibitors preclude viral replication while allowing production of extrachromosomal forms of viral DNA (E-DNA). Here, we describe an improved and standardized assay aimed at evaluating IN inhibitors by taking advantage of the transcriptional activity of E-DNA produced by HIV-derived vectors in the absence of replication-competent virus. In this context, the use of the firefly luciferase gene as a reporter gene provides a rapid and quantitative measure of viral-vector infectivity, thus making it a safe and cost-effective assay for evaluating novel IN inhibitors.
PMCID: PMC1610086  PMID: 17005823

Results 1-14 (14)