Structure-assisted Drug Design Research Articles

Revealing protonation states and tracking substrate in serine hydroxymethyltransferase with room-temperature X-ray and neutron crystallography

Pyridoxal 5’-phosphate (PLP)-dependent enzymes utilize a vitamin B6-derived cofactor to perform a myriad of chemical transformations on amino acids and other small molecules. Some PLP-dependent enzymes, such as serine hydroxymethyltransferase (SHMT), are promising drug targets for the design of small-molecule antimicrobials and anticancer therapeutics, while others have been used to synthesize pharmaceutical building blocks. Understanding PLP-dependent catalysis and the reaction specificity is crucial to advance structure-assisted drug design and enzyme engineering. Here we report the direct determination of the protonation states in the active site of Thermus thermophilus SHMT (TthSHMT) in the internal aldimine state using room-temperature joint X-ray/neutron crystallography. Conserved active site architecture of the model enzyme TthSHMT and of human mitochondrial SHMT (hSHMT2) were compared by obtaining a room-temperature X-ray structure of hSHMT2, suggesting identical protonation states in the human enzyme. The amino acid substrate serine pathway through the TthSHMT active site cavity was tracked, revealing the peripheral and cationic binding sites that correspond to the pre-Michaelis and pseudo-Michaelis complexes, respectively. At the peripheral binding site, the substrate is bound in the zwitterionic form. By analyzing the observed protonation states, Glu53, but not His residues, is proposed as the general base catalyst, orchestrating the retro-aldol transformation of L-serine into glycine.

Identification of potent compounds against SARs-CoV-2: An in-silico based drug searching against Mpro

The worldwide pandemic of coronavirus disease 2019 (COVID-19) along with the various newly discovered major SARS-CoV-2 variants, including B.1.1.7, B.1.351, and B.1.1.28, constitute the Variant of Concerns (VOC). It's difficult to keep these variants from spreading over the planet. As a result of these VOCs, the fifth wave has already begun in several countries. The rapid spread of VOCs is posing a serious threat to human civilization. There is currently no specific medicine available for the treatment of COVID-19. Here, we present the findings of methods that used a combination of structure-assisted drug design, virtual screening, and high-throughput screening to swiftly generate lead compounds against Mpro protein of SARs-CoV-2. Therapeutics, in addition to vaccinations, are an essential element of the healthcare response to COVID-19's persistent threat. In the current study, we designed the efficient compounds that may combat all emerging variants of SARs-CoV-2 by targeting the common Mpro protein. The present study was aimed to discover new compounds that may be proposed as new therapeutic agents to treat COVID-19 infection without any adverse effects. For this purpose, a computational-based virtual screening of 352 in-house synthesized compounds library was performed through molecular docking and Molecular Dynamics (MD) simulation approach. As a result, four novel potent compounds were successfully shortlisted by implementing certain pharmacological, physiological, and ADMET criteria i.e., compounds 3, 4, 21, and 22. Furthermore, MD simulations were performed to evaluate the stability and dynamic behavior of these compounds with Mpro complex for about 30 ns. Eventually, compound 22 was found to be highly potent against Mpro protein and was further evaluated by applying 100 ns simulations. Our findings showed that these shortlisted compounds may have potency to treat the COVID-19 infection for which further experimental validation is proposed as part of a follow-up investigation.

High-Throughput Screening and Molecular Dynamics Simulation of Natural Products for the Identification of Anticancer Agents against MCM7 Protein.

Minichromosome maintenance complex component 7 (MCM7) belongs to the minichromosome maintenance family that is necessary for the initiation of eukaryotic DNA replication. Overexpression of the MCM7 protein is linked to cellular proliferation and is accountable for critical malignancy in many cancers. Mechanistically, the suppression of MCM7 greatly lowers the cellular proliferation associated with cancer. Advances in immunotherapy have revolutionized treatments for many types of cancer. To date, no effective small molecular candidate has been found that can stop the advancement of cancer produced by the MCM7 protein. Here, we present the findings of methods that used a combination of structure-assisted drug design, high-throughput virtual screening, and simulations studies to swiftly generate lead compounds against MCM7 protein. In the current study, we designed efficient compounds that may combat all emerging cancer targeting the common MCM7 protein. For this objective, a molecular docking and molecular dynamics (MD) simulation-based virtual screening of 29,000 NPASS library was carried out. As a consequence of using specific pharmacological, physiological, and ADMET criteria, four new prevailing compounds, NPA000018, NPA000111, NPA00305, and NPA014826, were successfully selected. The MD simulations were also used for a time period of 50 ns to evaluate for stability and dynamics behavior of the compounds. Eventually, compounds NPA000111 and NPA014826 were found to be highly potent against MCM7 protein. According to our results, the selected compounds may be effective in treating certain cancer subtypes, for which additional follow-up experimental validation is recommended.

Evolving cryo-EM structural approaches for GPCR drug discovery

G protein-coupled receptors (GPCRs) are the largest class of cell surface drug targets. Advances in stabilization of GPCR:transducer complexes, together with improvements in cryoelectron microscopy (cryo-EM) have recently been applied to structure-assisted drug design for GPCR agonists. Nonetheless, limitations in the commercial application of these approaches, including the use of nanobody 35 (Nb35) to aid complex stabilization and the high cost of 300 kV imaging, have restricted broad application of cryo-EM in drug discovery. Here, using the PF 06882961-bound GLP-1R as exemplar, we validated the formation of stable complexes with a modified Gs protein in the absence of Nb35. In parallel, we compare 200 versus 300 kV image acquisition using a Falcon 4 or K3 direct electron detector. Moreover, the 200 kV Glacios-Falcon 4 yielded a 3.2 Å map with clear density for bound drug and multiple structurally ordered waters. Our work paves the way for broader commercial application of cryo-EM for GPCR drug discovery.

Re-Profiling of US-FDA Approved Drugs for COVID-19 by In-silico Studies

Covid-19 is an infectious disease that has rattled the world. Although several new vaccines are being administered to prevent infections, there is a large unmet need for antiviral drugs to ameliorate disease progression. In this study, structures of endogenous proteins were modelled using the genome browser and screened for molecular docking of drugs with spike protein of SARS COV-2 binding to ACE2 receptor using bioinformatics approaches such as structure-assisted drug design (SBDD), virtual drug screening (VS), and high-throughput screening (HTS). We have screened 150 drugs that were shortlisted from extensive literature data, 110 of these have been approved by the US-FDA for various diseases. We have developed a molecule assessment process from which we have identified fifteen drugs that have the potential for repurposing towards blocking SARS-COV2 proteins with endogenous proteins. Using these approaches, we have identified asthma drugs such as Cromolyn sodium, Ciclesonide and Zafirlukast.

Science, Medicine, and the Anesthesiologist

Science, Medicine, and the Anesthesiologist

Structure of Mpro from SARS-CoV-2 and discovery of its inhibitors.

A new coronavirus, known as severe acute respiratory syndrome coronavirus2 (SARS-CoV-2), is the aetiological agent responsible for the 2019-2020 viral pneumonia outbreak of coronavirus disease2019 (COVID-19)1-4. Currently, there are no targeted therapeutic agents for the treatment of this disease, and effective treatment options remain very limited. Here we describe the results of a programme that aimed to rapidly discover lead compounds for clinical use, by combining structure-assisted drug design, virtual drug screening and high-throughput screening. This programme focused on identifying drug leads that target main protease (Mpro) of SARS-CoV-2: Mpro is a key enzyme of coronaviruses and has a pivotal role in mediating viral replication and transcription, making it an attractive drug target for SARS-CoV-25,6. We identified a mechanism-based inhibitor (N3) by computer-aided drug design, and then determined the crystal structure of Mpro of SARS-CoV-2 in complex with this compound. Through a combination of structure-based virtual and high-throughput screening, we assayed more than 10,000compounds-including approved drugs, drug candidates in clinical trials and other pharmacologically active compounds-as inhibitors of Mpro. Six of these compounds inhibited Mpro, showing half-maximal inhibitory concentration values that ranged from 0.67 to 21.4μM. One of these compounds (ebselen) also exhibited promising antiviral activity in cell-based assays. Our results demonstrate the efficacy of our screening strategy, which can lead to the rapid discovery of drug leads with clinical potential in response to new infectious diseases for which no specific drugs or vaccines are available.

Cryo-EM structure of a 40\xa0kDa SAM-IV riboswitch RNA at 3.7\u2009\xc5 resolution

Specimens below 50 kDa have generally been considered too small to be analyzed by single-particle cryo-electron microscopy (cryo-EM). The high flexibility of pure RNAs makes it difficult to obtain high-resolution structures by cryo-EM. In bacteria, riboswitches regulate sulfur metabolism through binding to the S-adenosylmethionine (SAM) ligand and offer compelling targets for new antibiotics. SAM-I, SAM-I/IV, and SAM-IV are the three most commonly found SAM riboswitches, but the structure of SAM-IV is still unknown. Here, we report the structures of apo and SAM-bound SAM-IV riboswitches (119-nt, ~40 kDa) to 3.7 Å and 4.1 Å resolution, respectively, using cryo-EM. The structures illustrate homologies in the ligand-binding core but distinct peripheral tertiary contacts in SAM-IV compared to SAM-I and SAM-I/IV. Our results demonstrate the feasibility of resolving small RNAs with enough detail to enable detection of their ligand-binding pockets and suggest that cryo-EM could play a role in structure-assisted drug design for RNA.

Structural Insights into the Atomistic Mechanisms of Action of Small Molecule Inhibitors Targeting the KCa3.1 Channel Pore.

The intermediate-conductance Ca2+-activated K+ channel (KCa3.1) constitutes an attractive pharmacological target for immunosuppression, fibroproliferative disorders, atherosclerosis, and stroke. However, there currently is no available crystal structure of this medically relevant channel that could be used for structure-assisted drug design. Using the Rosetta molecular modeling suite we generated a molecular model of the KCa3.1 pore and tested the model by first confirming previously mapped binding sites and visualizing the mechanism of TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole), senicapoc (2,2-bis-(4-fluorophenyl)-2-phenylacetamide), and NS6180 (4-[[3-(trifluoromethyl)phenyl]methyl]-2H-1,4-benzothiazin-3(4H)-one) inhibition at the atomistic level. All three compounds block ion conduction directly by fully or partially occupying the site that would normally be occupied by K+ before it enters the selectivity filter. We then challenged the model to predict the receptor sites and mechanisms of action of the dihydropyridine nifedipine and an isosteric 4-phenyl-pyran. Rosetta predicted receptor sites for nifedipine in the fenestration region and for the 4-phenyl-pyran in the pore lumen, which could both be confirmed by site-directed mutagenesis and electrophysiology. While nifedipine is thus not a pore blocker and might be stabilizing the channel in a nonconducting conformation or interfere with gating, the 4-phenyl-pyran was found to be a classical pore blocker that directly inhibits ion conduction similar to the triarylmethanes TRAM-34 and senicapoc. The Rosetta KCa3.1 pore model explains the mechanism of action of several KCa3.1 blockers at the molecular level and could be used for structure-assisted drug design.

Structure-function insights reveal the human ribosome as a cancer target for antibiotics.

Many antibiotics in clinical use target the bacterial ribosome by interfering with the protein synthesis machinery. However, targeting the human ribosome in the case of protein synthesis deregulations such as in highly proliferating cancer cells has not been investigated at the molecular level up to now. Here we report the structure of the human 80S ribosome with a eukaryote-specific antibiotic and show its anti-proliferative effect on several cancer cell lines. The structure provides insights into the detailed interactions in a ligand-binding pocket of the human ribosome that are required for structure-assisted drug design. Furthermore, anti-proliferative dose response in leukaemic cells and interference with synthesis of c-myc and mcl-1 short-lived protein markers reveals specificity of a series of eukaryote-specific antibiotics towards cytosolic rather than mitochondrial ribosomes, uncovering the human ribosome as a promising cancer target.

Structures of lactate dehydrogenase A (LDHA) in apo, ternary and inhibitor-bound forms.

Lactate dehydrogenase (LDH) is an essential metabolic enzyme that catalyzes the interconversion of pyruvate and lactate using NADH/NAD(+) as a co-substrate. Many cancer cells exhibit a glycolytic phenotype known as the Warburg effect, in which elevated LDH levels enhance the conversion of glucose to lactate, making LDH an attractive therapeutic target for oncology. Two known inhibitors of the human muscle LDH isoform, LDHA, designated 1 and 2, were selected, and their IC50 values were determined to be 14.4 ± 3.77 and 2.20 ± 0.15 µM, respectively. The X-ray crystal structures of LDHA in complex with each inhibitor were determined; both inhibitors bind to a site overlapping with the NADH-binding site. Further, an apo LDHA crystal structure solved in a new space group is reported, as well as a complex with both NADH and the substrate analogue oxalate bound in seven of the eight molecules and an oxalate only bound in the eighth molecule in the asymmetric unit. In this latter structure, a kanamycin molecule is located in the inhibitor-binding site, thereby blocking NADH binding. These structures provide insights into LDHA enzyme mechanism and inhibition and a framework for structure-assisted drug design that may contribute to new cancer therapies.

Validation of KCa3.1 Channel Nifedipine Interaction Site Predicted by Rosetta Modeling Method

Calcium-activated potassium channels, including the intermediate-conductance KCa3.1 channel and the related small-conductance KCa2 channels, monitor and regulate intracellular calcium and are ideal pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, the development of drugs specifically for these medically relevant channels faces serious challenges because there is no available crystal structure that could be used for structure-assisted drug design. We used the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a template to generate a homology model of the KCa3.1 channel transmembrane region using the Rosetta modeling method. Docking of small molecules that are known to block KCa3.1 channel currents using Rosetta generated structural models of channel - drug complexes that can be validated using experimental approaches. Here we report the validation of the binding sites for the dihydropyridine nifedipine predicted by Rosetta's lowest energy stimulation of the KCa3.1 channel complex. We used a combination of site-directed mutagenesis and electrophysiological patch-clamp recording to test the identified amino acid residues located between the pore lining S5 and S6 segment. Blocking of the KCa3.1 channel by nifedipine was reduced by altering the side chains at positions L209, T212 and V278 to either alanine or valine, with T212 the most affected residue. Bulkier side-chain change to phenylalanine confirmed T212 as the main interacting site for nifedipine without compromising the channel's affinity for TRAM-34. Thus, by correctly identified the interaction of the dihydropyridine nifedipine with the KCa3.1 channel complex, Rosetta models can be used to understand the molecular mechanism of action of KCa channel blockers and gating modulators and assist with drug design efforts.

Structural Modeling of KCa3.1 Channel Interaction with Small Molecules

The intermediate-conductance KCa3.1 channel and the related small-conductance KCa2 channels constitute potential pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, there currently is no available crystal structure or molecular model of these medically relevant targets that could be used for structure-assisted drug design. We used the Kv1.2-Kv2.1 channel structure (pdb id: 2R9R) as a template to generate a homology model of the KCa3.1 channel transmembrane region with the Rosetta modeling method. Docking of small molecules that are known to block KCa3.1 channel currents using Rosetta Dock generated structural channel - drug complexes that can be validated using experimental approaches. The lowest energy confirmation of the KCa3.1 channel complex with TRAM-34 identified T250 and V275 as interaction sites, two residues which had been previously demonstrated to completely abolish triarylmethane sensitivity when mutated to the corresponding residues in KCa2.3 channel. The model further correctly identified the interaction of the benzothiazinone-type KCa3.1 channel blocker NS6180 with T250 and V275 in KCa3.1 channel and modeled binding of the negative KCa2 channel gating modulator NS8593 to a KCa3.1-T250S- V275A mutant channel, which is blocked more potently by NS8593 than the WT-KCa2.3 channel. For the dihydropyridine nifedipine, which blocks KCa3.1 channels with low micromolar activity, the Rosetta model suggested a receptor site located between the pore lining S5 and S6 segment in agreement with available experimental data showing that nifedipine does not interact with T250 in the selectivity filter. The Rosetta KCa2/3 channel models can be used to understand the molecular mechanism of action of KCa channel blockers and gating modulators and assist with drug design efforts. Supported by RO1 GM076063 (to H.W.) and UC Davis startup (to V.Y-Y.).

Pyridomycin bridges the NADH- and substrate-binding pockets of the enoyl reductase InhA

Pyridomycin, a natural product with potent antituberculosis activity, inhibits a major drug target, the InhA enoyl reductase. Here, we unveil the co-crystal structure and unique ability of pyridomycin to block both the NADH cofactor- and lipid substrate-binding pockets of InhA. This is to our knowledge a first-of-a-kind binding mode that discloses a new means of InhA inhibition. Proof-of-principle studies show how structure-assisted drug design can improve the activity of new pyridomycin derivatives.

Rosetta modeling of the inner KCa3.1 pore, a hotspot for small molecule modulation

The intermediate‐conductance KCa3.1 and the related small‐conductance KCa2 channels constitute potential pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, there currently is no available crystal structure or molecular model of these medically relevant channels that could be used for structure‐assisted drug design. Using the Rosetta modeling method we constructed a model of the KCa3.1 pore and energy minimized it in the presence of the KCa3.1 blocker TRAM‐34. The lowest energy confirmation of the KCa3.1 complex with TRAM‐34 identified Thr250 and Val275 as interaction sites, two residues which had been previously demonstrated to completely abolish triarylmethane sensitivity when mutated to the corresponding residues in KCa2.3. The model further correctly identified the interaction of the benzothiazinone‐type KCa3.1 blocker NS6180 with Thr250 and Val275 in KCa3.1 and modeled binding of the negative KCa2 channel gating modulator NS8593 to a KCa3.1‐Thr250Ser‐ Val275Ala mutant channel, which is blocked more potently by NS8593 than the WT‐KCa2.3 channel. The Rosetta KCa3.1 model can be used to understand the molecular mechanism of action of KCa2/3 channel modulators and assist with drug design of blockers and gating modulators.Supported by RO1 GM076063 (to H.W.) and UC Davis startup (to V.Y.).

Exploring Wild-Type and Mutant E. coli Strains for the Synthesis of Site-Specific Labels to Study RNA Structure and Dynamics by NMR

Ribonucleic acids are involved in many biological processes including catalysis, transfer and translation of genetic material, and regulation of gene expression. This unique ability to perform a variety of functions, traditionally associated with proteins is largely due to their capacity to adopt three-dimensional structural folds. Studying the 3D architecture of RNAs is critical for not only understanding the molecular basis of RNA function, but will eventually help with structure-assisted drug design, discovery and delivery. Heteronuclear NMR has become a powerful tool for studying the structure and dynamics of RNAs. To date, several RNAs have been well characterized by this method. However, overcrowding of chemical shifts and rapid signal loss in larger RNAs renders current NMR methods ineffectual. To study the structure and dynamics of larger RNAs, the use of site-selectively 13C-labeled nucleotides promises to be very helpful. To synthesize these labels, it was hypothesized that the metabolic pathways of various Escherichia coli wild type and mutant strains are capable of producing specifically labeled nucleotides necessary for making RNA. To test this hypothesis, we evaluated the growth of mutant strains K10-1516 (deficient in glucose-6-phosphate dehydrogenase of the pentose phosphate pathway) and DL323 (deficient in the Krebs cycle) on optimal LeMaster Richards's minimal media supplemented with carbon source 2-13C-glycerol alone or in combination with 13C-formate to enhance labeling at carbon positions bearing protons that are useful for NMR studies. Experimental results showed that the strains produced specific ribose and nucleotide labels that were readily predicted. These labels will enable us to study the structure, function and dynamics of higher size RNA molecules using NMR that would have been otherwise difficult if the commercially available uniformly labeled or unlabeled ribonucleotides were utilized.

Structural studies of series HIV-1 nonnucleoside reverse transcriptase inhibitors 1-(2,6-difluorobenzyl)-2-(2,6-difluorophenyl)-benzimidazoles with different 4-substituents

Over the past 10 years, several anti-viral drugs have become available to fight the HIV infection. Antiretroviral treatment reduces the mortality of AIDS. Nonnucleoside inhibitors of HIV-1 reverse transcriptase are specific and potentially nontoxic drugs against AIDS. The crystal structures of five nonnucleoside inhibitors of HIV-1 reverse transcriptase are presented here. The structural parameters, especially those describing the angular orientation of the π-electron systems and influencing biological activity, were determined for all of the investigated inhibitors. The chemical character and orientation of the substituent at C4 position of the benzimidazole moiety substantially influences the anti-viral activity. The structural data of the investigated inhibitors is a good basis for modeling enzyme–inhibitor interactions for structure-assisted drug design.

Structural Determinants for Signal Transducer and Activator of Transcription (STAT) 3 Recruitment and Activation by the Granulocyte Colony-Stimulating Factor Receptor (G-CSFR) at Phosphotyrosine Ligands 704 and 744.

Four tyrosine (Y) residues within the cytoplasmic domain of the G-CSFR (Y704, Y228, Y744 and Y764 in the human receptor; Y703, Y227, Y743 and Y763 in the murine receptor) become phosphorylated by Jak kinases upon ligand binding leading to recruitment of Src homology (SH) 2 domain-containing proteins that link to programs for myeloid cell survival and differentiation (Stat3 recruitment to Y704 and Y744) and proliferation (SHP-2 and PI3K recruitment to Y04; Grb2, Shc and SHP-2 recruitment to Y764). While the preference of SH2 domain binding to specific phospho (p) Y peptide ligands was shown to map to the three residues immediately C-terminal to the pY (+1, +2, +3 residues), the structural basis for these preferences is poorly understood but could be exploited to specifically target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation, which has been demonstrated in a subset of acute myelogenous leukemia (AML) patients whose cells contain Flt3 internal tandem duplications and who suffer relapse following initial chemotherapy. To establish the structural basis for Stat3 recruitment and activation by the G-CSFR at Y704 and Y744, we generated purified recombinant full-length Stat3 and phosphododecapeptides based on the sequence surrounding each Y within the G-CSFR. In peptide pull-down assays, recombinant Stat3 bound only to Y704 and Y744 phosphododecapeptide, which contain core pY motifs consisting of pYVLQ and pYLRC, respectively. In mirror resonance affinity assays employed to obtain quantitative binding information, Stat3 bound to each phosphododecapeptide with similar kinetics (e.g. KDs = 0.703 and 0.95 μM, respectively). We tested three models for Stat3 SH2-pY ligand binding proposed by us and others using wild type and mutant recombinant Stat3 proteins in peptide pull-down and mirror resonance affinity assays along with computer modeling of this interaction using the known structures of Statβ SH2 and EGFR pY ligand (EpY1068INQ). Our results revealed loss of binding of Stat3 to Y704 and Y744 phosphododecapeptides only in Stat3 mutated within the SH2 domain at K591 or R609, whose side chains interacted with the pY phosphate group, and in Stat3 mutated within the SH2 domain at E638, whose amide hydrogen bonded with oxygen within the +3 Q side chain (or with sulfur within the +3 C side chain) when the pY ligand assumes a β turn. G-CSF stimulation of cells co-expressing full-length G-CSFR and either wild type or mutant Stat3 constructs confirmed the requirements for the side chain of R609 and the amide hydrogen of E638 within the Stat3 SH2 domain for binding to the G-CSFR and subsequent phosphorylation of Stat3 on Y705. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of their interaction at Y704 and Y744 i.e. a β turn within the receptor pY motif and a key hydrogen bond formed between the polar side chain of the +3 residue and the amide hydrogen of E638 within the Stat3 SH2 domain. These features explain the preference of the Stat3 SH2 domain for pY peptide ligands with +3 Q or C as well as +3 T (pY705LKT within Stat3) and +3 S (pY743IRS within the murine G-CSFR) and can be exploited using a structure-assisted drug design strategy to develop new therapies for a subset of AML patients with poor prognosis whose cells demonstrate aberrant activation of Stat3.

Atomic structure of PDE4: insights into phosphodiesterase mechanism and specificity.

Cyclic nucleotides are second messengers that are essential in vision, muscle contraction, neurotransmission, exocytosis, cell growth, and differentiation. These molecules are degraded by a family of enzymes known as phosphodiesterases, which serve a critical function by regulating the intracellular concentration of cyclic nucleotides. We have determined the three-dimensional structure of the catalytic domain of phosphodiesterase 4B2B to 1.77 angstrom resolution. The active site has been identified and contains a cluster of two metal atoms. The structure suggests the mechanism of action and basis for specificity and will provide a framework for structure-assisted drug design for members of the phosphodiesterase family.

Inhibitors of HIV-1 protease: a major success of structure-assisted drug design.

Retroviral protease (PR) from the human immunodeficiency virus type 1 (HIV-1) was identified over a decade ago as a potential target for structure-based drug design. This effort was very successful. Four drugs are already approved, and others are undergoing clinical trials. The techniques utilized in this remarkable example of structure-assisted drug design included crystallography, NMR, computational studies, and advanced chemical synthesis. The development of these drugs is discussed in detail. Other approaches to designing HIV-1 PR inhibitors, based on the concepts of symmetry and on the replacement of a water molecule that had been found tetrahedrally coordinated between the enzyme and the inhibitors, are also discussed. The emergence of drug-induced mutations of HIV-1 PR leads to rapid loss of potency of the existing drugs and to the need to continue the development process. The structural basis of drug resistance and the ways of overcoming this phenomenon are mentioned.