Arnout Voet

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.

The Saccharomyces cerevisiae EHT1 and EEB1 Genes Encode Novel Enzymes with Medium-chain Fatty Acid Ethyl Ester Synthesis and Hydrolysis Capacity

Fatty acid ethyl esters are secondary metabolites produced by Saccharomyces cerevisiae and many other fungi. Their natural physiological role is not known but in fermentations of alcoholic beverages and other food products they play a key role as flavor compounds. Information about the metabolic pathways and enzymology of fatty acid ethyl ester biosynthesis, however, is very limited. In this work, we have investigated the role of a three-member S. cerevisiae gene family with moderately divergent sequences (YBR177c/EHT1, YPL095c/EEB1, and YMR210w). We demonstrate that two family members encode an acyl-coenzymeA:ethanol O-acyltransferase, an enzyme required for the synthesis of medium-chain fatty acid ethyl esters. Deletion of either one or both of these genes resulted in severely reduced medium-chain fatty acid ethyl ester production. Purified glutathione S-transferase-tagged Eht1 and Eeb1 proteins both exhibited acyl-coenzymeA:ethanol O-acyltransferase activity in vitro, as well as esterase activity. Overexpression of Eht1 and Eeb1 did not enhance medium-chain fatty acid ethyl ester content, which is probably due to the bifunctional synthesis and hydrolysis activity. Molecular modeling of Eht1 and Eeb1 revealed the presence of a α/β-hydrolase fold, which is generally present in the substrate-binding site of esterase enzymes. Hence, our results identify Eht1 and Eeb1 as novel acyl-coenzymeA:ethanol O-acyltransferases/esterases, whereas the third family member, Ymr210w, does not seem to play an important role in medium-chain fatty acid ethyl ester formation.

Virus Evolution Reveals an Exclusive Role for LEDGF/p75 in Chromosomal Tethering of HIV

Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75–integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase–LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.

Reconstruction of Ancestral Metabolic Enzymes Reveals Molecular Mechanisms Underlying Evolutionary Innovation through Gene Duplication

Resurrection of ancient fungal maltase enzymes uncovers the molecular details of how repeated gene duplications allow the evolution of protein variants with different functions.

Fragment Based Drug Design: From Experimental to Computational Approaches

Fragment based drug design has emerged as an effective alternative to high throughput screening for the identification of lead compounds in drug discovery in the past fifteen years. Fragment based screening and optimization methods have achieved credible success in many drug discovery projects with one approved drug and many more compounds in clinical trials. The fragment based drug design starts with the identification of fragments or low molecular weight compounds that generally bind with weak affinity to the target of interest. The fragments that form high quality interactions are then optimized to lead compounds with high affinity and selectivity. The weak affinity of fragments for their target requires the use of biophysical techniques such as nuclear magnetic resonance, X-ray crystallography or surface plasmon resonance to identify hits. These techniques are very sensitive and some of them provide detailed protein fragment interaction information that is important for fragment to lead optimization. Despite the huge advances in technology in the past years, experimental methods of fragment screening suffer several challenges such as low throughput, high cost of instruments and experiments, high protein and fragment concentration requirements. To address challenges posed by experimental screening approaches, computational methods were developed that play an important role in fragment library design, fragment screening and optimization of initial fragment hits. The computational approaches of fragment screening and optimization are most useful when they are used in combination with experimental approaches. The use of virtual fragment based screening in combination with experimental methods has fostered the application of fragment based drug design to important biological targets including protein-protein interactions and membrane proteins such as GPCRs. This review provides an overview of experimental and computational screening approaches used in fragment based drug discovery with an emphasis on recent successes achieved in discovering potent lead molecules using these approaches.

Computational design of a self-assembling symmetrical β-propeller protein

Significance In this study, we have designed and experimentally validated, to our knowledge, the first perfectly symmetrical β-propeller protein. Our results provide insight not only into protein evolution through duplication events, but also into methods for creating designer proteins that self-assemble according to simple arithmetical rules. Such proteins may have very wide uses in bionanotechnology. Furthermore our design approach is both rapid and applicable to many different protein templates. Our novel propeller protein consists of six identical domains known as “blades.” Using a variety of biophysical techniques, we show it to be highly stable and report several high-resolution crystal structures of different forms of the protein. Domain swapping allows us to generate related oligomeric forms with fixed numbers of blades per complex. The modular structure of many protein families, such as β-propeller proteins, strongly implies that duplication played an important role in their evolution, leading to highly symmetrical intermediate forms. Previous attempts to create perfectly symmetrical propeller proteins have failed, however. We have therefore developed a new and rapid computational approach to design such proteins. As a test case, we have created a sixfold symmetrical β-propeller protein and experimentally validated the structure using X-ray crystallography. Each blade consists of 42 residues. Proteins carrying 2–10 identical blades were also expressed and purified. Two or three tandem blades assemble to recreate the highly stable sixfold symmetrical architecture, consistent with the duplication and fusion theory. The other proteins produce different monodisperse complexes, up to 42 blades (180 kDa) in size, which self-assemble according to simple symmetry rules. Our procedure is suitable for creating nano-building blocks from different protein templates of desired symmetry.

The transcriptional co-activator LEDGF/p75 displays a dynamic scan-and-lock mechanism for chromatin tethering

Nearly all cellular and disease related functions of the transcriptional co-activator lens epithelium-derived growth factor (LEDGF/p75) involve tethering of interaction partners to chromatin via its conserved integrase binding domain (IBD), but little is known about the mechanism of in vivo chromatin binding and tethering. In this work we studied LEDGF/p75 in real-time in living HeLa cells combining different quantitative fluorescence techniques: spot fluorescence recovery after photobleaching (sFRAP) and half-nucleus fluorescence recovery after photobleaching (hnFRAP), continuous photobleaching, fluorescence correlation spectroscopy (FCS) and an improved FCS method to study diffusion dependence of chromatin binding, tunable focus FCS. LEDGF/p75 moves about in nuclei of living cells in a chromatin hopping/scanning mode typical for transcription factors. The PWWP domain of LEDGF/p75 is necessary, but not sufficient for in vivo chromatin binding. After interaction with HIV-1 integrase via its IBD, a general protein–protein interaction motif, kinetics of LEDGF/p75 shift to 75-fold larger affinity for chromatin. The PWWP is crucial for locking the complex on chromatin. We propose a scan-and-lock model for LEDGF/p75, unifying paradoxical notions of transcriptional co-activation and lentiviral integration targeting.

Guards of the great wall: bacterial lysozyme inhibitors

Peptidoglycan is the major structural component of the bacterial cell wall. It provides resistance against turgor and its cleavage by hydrolases such as lysozymes results in bacteriolysis. Most, if not all, animals produce lysozymes as key effectors of their innate immune system. Recently, highly specific bacterial proteinaceous lysozyme inhibitors against the three major animal lysozyme families have been discovered in bacteria, and these may represent a bacterial answer to animal lysozymes. Here, we will review their properties and phylogenetic distribution, present their structure and molecular interaction mechanism with lysozyme, and discuss their possible biological functions and potential applications.

Mutations in Human Immunodeficiency Virus Type 1 Integrase Confer Resistance to the Naphthyridine L-870,810 and Cross-Resistance to the Clinical Trial Drug GS-9137

ABSTRACT To gain further insight into the understanding of the antiviral resistance patterns and mechanisms of the integrase strand transfer inhibitor L-870,810, the prototypical naphthyridine analogue, we passaged the human immunodeficiency virus type 1 strain HIV-1(IIIB) in cell culture in the presence of increasing concentrations of L-870,810 (IIIB/L-870,810). The mutations L74M, E92Q, and S230N were successively selected in the integrase. The L74M and E92Q mutations have both been associated in the past with resistance against the diketo acid (DKA) analogues L-708,906 and S-1360 and the clinical trial drugs MK-0518 and GS-9137. After 20, 40, and 60 passages in the presence of L-870,810, IIIB/L-870,810 displayed 22-, 34-, and 110-fold reduced susceptibility to L-870,810, respectively. Phenotypic cross-resistance against the DKA analogue CHI-1043 and MK-0518 was modest but that against GS-9137 was pronounced. Recombination of the mutant integrase genes into the wild-type background reproduced the resistance profile of the resistant IIIB/L-870,810 strains. In addition, resistance against L-870,810 was accompanied by reduced viral replication kinetics and reduced enzymatic activity of integrase. In conclusion, the accumulation of L74M, E92Q, and S230N mutations in the integrase causes resistance to the naphthyridine L-870,810 and cross-resistance to GS-9137. These data may have implications for cross-resistance of different integrase inhibitors in the clinic.

Androgen receptor antagonists for prostate cancer therapy

Androgen deprivation is the mainstay therapy for metastatic prostate cancer (PCa). Another way of suppressing androgen receptor (AR) signaling is via AR antagonists or antiandrogens. Despite being frequently prescribed in clinical practice, there is conflicting evidence concerning the role of AR antagonists in the management of PCa. In the castration-resistant settings of PCa, docetaxel has been the only treatment option for decades. With recent evidence that castration-resistant PCa is far from AR-independent, there has been an increasing interest in developing new AR antagonists. This review gives a concise overview of the clinically available antiandrogens and the experimental AR antagonists that tackle androgen action with a different approach.