Reshaping Echinocandin Antifungal Drugs To Circumvent Glucan Synthase Point-Mutation-Mediated Resistance.

Echinocandins are a class of antifungal drugs that inhibit the activity of the ß-(1,3)-glucan synthase complex, which synthesizes fungal cell wall ß-(1,3)-glucan. Echinocandin resistance is linked to mutations in the FKS gene, which encodes the catalytic subunit of the glucan synthase complex. We present a molecular-docking-based model that provides insight into how echinocandins interact with the target Fks protein: echinocandins form a ternary complex with both Fks and membrane lipids. We used reductive dehydration of alcohols to generate dehydroxylated echinocandin derivatives and evaluated their potency against a panel of Candida pathogens constructed by introducing resistance-conferring mutations in the FKS gene. We found that removing the hemiaminal alcohol, which drives significant conformational alterations in the modified echinocandins, reduced their efficacy. Conversely, eliminating the benzylic alcohol of echinocandins enhanced potency by up to two orders of magnitude, in a manner dependent upon the resistance-conferring mutation. Strains that have developed resistance to either rezafungin, the most recently clinically approved echinocandin, or its dehydroxylated derivative RZF-1, exhibit high resistance to rezafungin while demonstrating moderate resistance to RZF-1. These findings provide valuable insight for combating echinocandin resistance through chemical modifications.

Identification and characterization of the key enzyme in the biosynthesis of the neurotoxin ß-ODAP in grass pea.

Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, ß-N-oxalyl-L-α,ß-diaminopropionic acid (ß-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with ß-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-ß-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for ß-ODAP production in vivo. The identification of BOS may pave the way toward engineering ß-ODAP-free grass pea cultivars, which are safe for human and animal consumption.

Phylogeny and abiotic conditions shape the diel floral emission patterns of desert Brassicaceae species.

A key facet of floral scent is diel fluctuations in emission, often studied in the context of plant-pollinator interactions, while contributions of environment and phylogeny remain overlooked. Here, we ask if these factors are involved in shaping temporal variations in scent emission. To that end, we coupled light/dark floral emission measurements of 17 desert Brassicaceae species with environmental and phylogenetic data to explore the individual/combined impacts of these predictors on diel emission patterns. We further investigated these patterns by conducting high-resolution emission measurements in a subset of genetically distant species with contrasting temporal dynamics. While diel shifts in magnitude and richness of emission were strongly affected by genetic relatedness, they also reflect the environmental conditions under which the species grow. Specifically, light/dark emission ratios were negatively affected by an increase in winter temperatures, known to impact both plant physiology and insect locomotion, and sandy soil fractions, previously shown to exert stress that tempers with diel metabolic rhythms. Additionally, the biosynthetic origins of the compounds were associated with their corresponding production patterns, possibly to maximize emission efficacy. Using a multidisciplinary chemical/ecological approach, we uncover and differentiate the main factors shaping floral scent diel fluctuations, highlighting their consequences under changing global climate.

Inhibition of Cysteine Proteases by 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea.

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.

Pre-equilibrium competitive library screening for tuning inhibitor association rate and specificity toward serine proteases.

High structural and sequence similarity within protein families can pose significant challenges to the development of selective inhibitors, especially toward proteolytic enzymes. Such enzymes usually belong to large families of closely similar proteases and may also hydrolyze, with different rates, protein- or peptide-based inhibitors. To address this challenge, we employed a combinatorial yeast surface display library approach complemented with a novel pre-equilibrium, competitive screening strategy for facile assessment of the effects of multiple mutations on inhibitor association rates and binding specificity. As a proof of principle for this combined approach, we utilized this strategy to alter inhibitor/protease association rates and to tailor the selectivity of the amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI) for inhibition of the oncogenic protease mesotrypsin, in the presence of three competing serine proteases, anionic trypsin, cationic trypsin and kallikrein-6. We generated a variant, designated APPIP13W/M17G/I18F/F34V, with up to 30-fold greater specificity relative to the parental APPIM17G/I18F/F34V protein, and 6500- to 230 000-fold improved specificity relative to the wild-type APPI protein in the presence of the other proteases tested. A series of molecular docking simulations suggested a mechanism of interaction that supported the biochemical results. These simulations predicted that the selectivity and specificity are affected by the interaction of the mutated APPI residues with nonconserved enzyme residues located in or near the binding site. Our strategy will facilitate a better understanding of the binding landscape of multispecific proteins and will pave the way for design of new drugs and diagnostic tools targeting proteases and other proteins.

A computational combinatorial approach identifies a protein inhibitor of superoxide dismutase 1 misfolding, aggregation, and cytotoxicity.

Molecular agents that specifically bind and neutralize misfolded and toxic superoxide dismutase 1 (SOD1) mutant proteins may find application in attenuating the disease progression of familial amyotrophic lateral sclerosis. However, high structural similarities between the wild-type and mutant SOD1 proteins limit the utility of this approach. Here we addressed this challenge by converting a promiscuous natural human IgG-binding domain, the hyperthermophilic variant of protein G (HTB1), into a highly specific aggregation inhibitor (designated HTB1M) of two familial amyotrophic lateral sclerosis-linked SOD1 mutants, SOD1G93A and SOD1G85R We utilized a computational algorithm for mapping protein surfaces predisposed to HTB1 intermolecular interactions to construct a focused HTB1 library, complemented with an experimental platform based on yeast surface display for affinity and specificity screening. HTB1M displayed high binding specificity toward SOD1 mutants, inhibited their amyloid aggregation in vitro, prevented the accumulation of misfolded proteins in living cells, and reduced the cytotoxicity of SOD1G93A expressed in motor neuron-like cells. Competition assays and molecular docking simulations suggested that HTB1M binds to SOD1 via both its α-helical and ß-sheet domains at the native dimer interface that becomes exposed upon mutated SOD1 misfolding and monomerization. Our results demonstrate the utility of computational mapping of the protein-protein interaction potential for designing focused protein libraries to be used in directed evolution. They also provide new insight into the mechanism of conversion of broad-spectrum immunoglobulin-binding proteins, such as HTB1, into target-specific proteins, thereby paving the way for the development of new selective drugs targeting the amyloidogenic proteins implicated in a variety of human diseases.

Digoxin derivatives with selectivity for the α2ß3 isoform of Na,K-ATPase potently reduce intraocular pressure.

The ciliary epithelium in the eye consists of pigmented epithelial cells that express the α1ß1 isoform of Na,K-ATPase and nonpigmented epithelial cells that express mainly the α2ß3 isoform. In principle, a Na,K-ATPase inhibitor with selectivity for α2ß3 that penetrates the cornea could effectively reduce intraocular pressure, with minimal systemic or local toxicity. We have recently synthesized perhydro-1,4-oxazepine derivatives of digoxin by NaIO4 oxidation of the third digitoxose and reductive amination with various R-NH2 substituents and identified derivatives with significant selectivity for human α2ß1 over α1ß1 (up to 7.5-fold). When applied topically, the most α2-selective derivatives effectively prevented or reversed pharmacologically raised intraocular pressure in rabbits. A recent structure of Na,K-ATPase, with bound digoxin, shows the third digitoxose approaching one residue in the ß1 subunit, Gln84, suggesting a role for ß in digoxin binding. Gln84 in ß1 is replaced by Val88 in ß3. Assuming that alkyl substituents might interact with ß3Val88, we synthesized perhydro-1,4-oxazepine derivatives of digoxin with diverse alkyl substituents. The methylcyclopropyl and cyclobutyl derivatives are strongly selective for α2ß3 over α1ß1 (22-33-fold respectively), as determined either with purified human isoform proteins or intact bovine nonpigmented epithelium cells. When applied topically on rabbit eyes, these derivatives potently reduce both pharmacologically raised and basal intraocular pressure. The cyclobutyl derivative is more efficient than Latanoprost, the most widely used glaucoma drug. Thus, the conclusion is that α2ß3-selective digoxin derivatives effectively penetrate the cornea and inhibit the Na,K-ATPase, hence reducing aqueous humor production. The new digoxin derivatives may have potential for glaucoma drug therapy.

Selective Assembly of Na,K-ATPase α2ß2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The ß subunit differentially regulates maturation, trafficking, and activity of α-ß heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three ß isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with ß2 in T-tubules of cardiac myocytes, forming α2ß2 heterodimers. We have expressed human α1ß1, α2ß1, α2ß2, and α2ß3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2ß2 and α2ß3 differ significantly from both α2ß1 and α1ß1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2ß2 and α2ß3 over α1ß1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2ß2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2ß2 for cardiac muscle contractility. The high sensitivity of α2ß2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2ß2-selective digoxin derivatives for reducing cardiotoxicity.

Prediction of homoprotein and heteroprotein complexes by protein docking and template-based modeling: A CASP-CAPRI experiment.

We present the results for CAPRI Round 30, the first joint CASP-CAPRI experiment, which brought together experts from the protein structure prediction and protein-protein docking communities. The Round comprised 25 targets from amongst those submitted for the CASP11 prediction experiment of 2014. The targets included mostly homodimers, a few homotetramers, and two heterodimers, and comprised protein chains that could readily be modeled using templates from the Protein Data Bank. On average 24 CAPRI groups and 7 CASP groups submitted docking predictions for each target, and 12 CAPRI groups per target participated in the CAPRI scoring experiment. In total more than 9500 models were assessed against the 3D structures of the corresponding target complexes. Results show that the prediction of homodimer assemblies by homology modeling techniques and docking calculations is quite successful for targets featuring large enough subunit interfaces to represent stable associations. Targets with ambiguous or inaccurate oligomeric state assignments, often featuring crystal contact-sized interfaces, represented a confounding factor. For those, a much poorer prediction performance was achieved, while nonetheless often providing helpful clues on the correct oligomeric state of the protein. The prediction performance was very poor for genuine tetrameric targets, where the inaccuracy of the homology-built subunit models and the smaller pair-wise interfaces severely limited the ability to derive the correct assembly mode. Our analysis also shows that docking procedures tend to perform better than standard homology modeling techniques and that highly accurate models of the protein components are not always required to identify their association modes with acceptable accuracy. Proteins 2016; 84(Suppl 1):323-348. © 2016 Wiley Periodicals, Inc.

Chiral Fluorescent Antifungal Azole Probes Detect Resistance, Uptake Dynamics, and Subcellular Distribution in Candida Species.

Azoles are essential for fungal infection treatment, yet the increasing resistance highlights the need for innovative diagnostic tools and strategies to revitalize this class of antifungals. We developed two enantiomers of a fluorescent antifungal azole probe (1 S and 1 R ), analyzing 60 Candida strains via live-cell microscopy. A database of azole distribution images in strains of Candida albicans, Candida glabrata, and Candida parapsilosis, among the most important pathogenic Candida species, was established and analyzed. This analysis revealed distinct populations of yeast cells based on the correlation between fluorescent probe uptake and cell diameter. Varied uptake levels and subcellular distribution patterns were observed in C. albicans, C. glabrata, and C. parapsilosis, with the latter displaying increased localization to lipid droplets. Comparison of the more potent fluorescent antifungal azole probe enantiomer 1 S with the moderately potent enantiomer 1 R highlighted time-dependent differences in the uptake profiles. The former displayed a marked elevation in uptake after approximately 150 min, indicating the time required for significant cell permeabilization to occur and its association with the azole's antifungal activity potency. Divergent uptake levels between susceptible and high efflux-based azole-resistant strains were detected, offering a rapid diagnostic approach for identifying azole resistance. This study highlights unique insights achievable through fluorescent antifungal azole probes, unraveling the complexities of azole resistance, subcellular dynamics, and uptake within fungal pathogens.

1-(sulfonyl)-5-(arylsulfonyl)indoline as activators of the tumor cell specific M2 isoform of pyruvate kinase.

Cancer cells preferentially use glycolysis rather than oxidative phosphorylation for their rapid growth. They consume large amount of glucose to produce lactate even when oxygen is abundant, a phenomenon known as the Warburg effect. This metabolic change originates from a shift in the expression of alternative spliced isoforms of the glycolytic enzyme pyruvate kinase (PK), from PKM1 to PKM2. While PKM1 is constitutively active, PKM2 is switched from an inactive dimer form to an active tetramer form by small molecule activators. The prevalence of PKM2 in cancer cells relative to the prevalence of PKM1 in many normal cells, suggests a therapeutic strategy whereby activation of PKM2 may counter the abnormal cellular metabolism in cancer cells, and consequently decreased cellular proliferation. Herein we describe the discovery and optimization of a series of PKM2 activators derived from the 2-((2,3-dihydrobenzo[b][1,4] dioxin-6-yl)thio)-1-(2-methyl-1-(methylsulfonyl)indolin-5-yl) ethanone scaffold. The synthesis, SAR analysis, enzyme active site docking, enzymatic reaction kinetics, selectivity and pharmaceutical properties are discussed.

Small molecule correctors of F508del-CFTR discovered by structure-based virtual screening.

Folding correctors of F508del-CFTR were discovered by in silico structure-based screening utilizing homology models of CFTR. The intracellular segment of CFTR was modeled and three cavities were identified at inter-domain interfaces: (1) Interface between the two Nucleotide Binding Domains (NBDs); (2) Interface between NBD1 and Intracellular Loop (ICL) 4, in the region of the F508 deletion; (3) multi-domain interface between NBD1:2:ICL1:2:4. We hypothesized that compounds binding at these interfaces may improve the stability of the protein, potentially affecting the folding yield or surface stability. In silico structure-based screening was performed at the putative binding-sites and a total of 496 candidate compounds from all three sites were tested in functional assays. A total of 15 compounds, representing diverse chemotypes, were identified as F508del folding correctors. This corresponds to a 3% hit rate, ~tenfold higher than hit rates obtained in corresponding high-throughput screening campaigns. The same binding sites also yielded potentiators and, most notably, compounds with a dual corrector-potentiator activity (dual-acting). Compounds harboring both activity types may prove to be better leads for the development of CF therapeutics than either pure correctors or pure potentiators. To the best of our knowledge this is the first report of structure-based discovery of CFTR modulators.

The mitochondrial carrier Citrin plays a role in regulating cellular energy during carcinogenesis.

Citrin, encoded by SLC25A13 gene, is an inner mitochondrial transporter that is part of the malate-aspartate shuttle, which regulates the NAD+/NADH ratio between the cytosol and mitochondria. Citrullinemia type II (CTLN-II) is an inherited disorder caused by germline mutations in SLC25A13, manifesting clinically in growth failure that can be alleviated by dietary restriction of carbohydrates. The association of citrin with glycolysis and NAD+/NADH ratio led us to hypothesize that it may play a role in carcinogenesis. Indeed, we find that citrin is upregulated in multiple cancer types and is essential for supplementing NAD+ for glycolysis and NADH for oxidative phosphorylation. Consequently, citrin deficiency associates with autophagy, whereas its overexpression in cancer cells increases energy production and cancer invasion. Furthermore, based on the human deleterious mutations in citrin, we found a potential inhibitor of citrin that restricts cancerous phenotypes in cells. Collectively, our findings suggest that targeting citrin may be of benefit for cancer therapy.

Conserved interactions required for inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands' interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50 µM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.

Nanobodies Targeting Prostate-Specific Membrane Antigen for the Imaging and Therapy of Prostate Cancer.

The repertoire of methods for the detection and chemotherapeutic treatment of prostate cancer (PCa) is currently limited. Prostate-specific membrane antigen (PSMA) is overexpressed in PCa tumors and can be exploited for both imaging and drug delivery. We developed and characterized four nanobodies that present tight and specific binding and internalization into PSMA+ cells and that accumulate specifically in PSMA+ tumors. We then conjugated one of these nanobodies to the cytotoxic drug doxorubicin, and we show that the conjugate internalizes specifically into PSMA+ cells, where the drug is released and induces cytotoxic activity. In vivo studies show that the extent of tumor growth inhibition is similar when mice are treated with commercial doxorubicin and with a 42-fold lower amount of the nanobody-conjugated doxorubicin, attesting to the efficacy of the conjugated drug. These data highlight nanobodies as promising agents for the imaging of PCa tumors and for the targeted delivery of chemotherapeutic drugs.

Experimental and computational characterization of the dimerization of the PTS-regulation domains of BglG from Escherichia coli.

BglG and LicT are transcriptional antiterminators from Escherichia coli and Bacillus subtilis, respectively, that control the expression of genes and operons involved in transport and catabolism of carbohydrates. Both proteins contain a duplicate conserved domain, the PTS-regulation domain (PRD), and they are regulated by phosphorylation on specific, highly conserved histidine residues located in the PRDs. However, despite their similar function and the high sequence identity, experimental evidence implies different modes of regulation. Thus, BglG must be de-phosphorylated on PRD2 in order to form active dimers, whereas activation of LicT requires de-phosphorylation on PRD1 and phosphorylation on PRD2. Here we address two goals. First, we test in vivo and in silico the effect of point mutations in the PRDs of BglG on the PRD-PRD dimerization. Second, we explore computationally the effect of histidine phosphorylation on PRD dimerization in BglG and LicT. We find excellent correspondence between the experimental and computational measures of the influence of mutations on PRD dimerization in BglG. This establishes that the geometric-electrostatic complementarity scores computed with the program MolFit provide a good measure of the effects of mutations in this system. In addition, it indicates that the dimerization mode of the separately expressed PRDs of BglG is similar to the dimers formed by activated LicT. The computations also show that phosphorylation of the histidine residues in PRD1 of either BglG or LicT leads to a strong electrostatic repulsion. Conversely, the phosphorylation of one histidine residue in PRD2 of LicT leads to improved electrostatic complementarity at the PRD2-PRD2 interface, whereas the corresponding phosphorylation in BglG has negligible contribution. This different conduct may be attributed to a single replacement in the sequence of PRD2 in BglG compared to LicT, Ala262 versus Asp261, respectively.

A Novel Allosteric Mechanism of NF-κB Dimerization and DNA Binding Targeted by an Anti-Inflammatory Drug.

The NF-κB family plays key roles in immune and stress responses, and its deregulation contributes to several diseases. Therefore its modulation has become an important therapeutic target. Here, we used a high-throughput screen for small molecules that directly inhibit dimerization of the NF-κB protein p65. One of the identified inhibitors is withaferin A (WFA), a documented anticancer and anti-inflammatory compound. Computational modeling suggests that WFA contacts the dimerization interface on one subunit and surface residues E285 and Q287 on the other. Despite their locations far from the dimerization site, E285 and Q287 substitutions diminished both dimerization and the WFA effect. Further investigation revealed that their effects on dimerization are associated with their proximity to a conserved hydrophobic core domain (HCD) that is crucial for dimerization and DNA binding. Our findings established NF-κB dimerization as a drug target and uncovered an allosteric domain as a target of WFA action.

Weighted geometric docking: incorporating external information in the rotation-translation scan.

Weighted geometric docking is a prediction algorithm that matches weighted molecular surfaces. Each molecule is represented by a grid of complex numbers, storing information about the shape of the molecule in the real part and weight information in the imaginary part. The weights are based on experimental biochemical and biophysical data or on theoretical analyses of amino acid conservation or correlation patterns in multiple-sequence alignments of homologous proteins. Only a few surface residues on either one or both molecules are weighted. In contrast to methods that use postscan filtering based on biochemical information, our method incorporates the external data in the rotation-translation search, producing a different set of docking solutions biased toward solutions in which the up-weighted residues are at the interface. Similarly, interactions involving specified residues can be impeded. The weighted geometric algorithm was applied to five systems for which regular geometric docking of the unbound molecules gave poor results. We obtained much better ranking of the nearly correct prediction and higher statistical significance when weighted geometric docking was used. The method was successful even when the weighted portion of the surface corresponded only partially and approximately to the binding site.

Prediction of the unknown: inspiring experience with the CAPRI experiment.

We submitted predictions for all seven targets in the CAPRI experiment. For four targets, our submitted models included acceptable, medium accuracy predictions of the structures of the complexes, and for a fifth target we identified the location of the binding site of one of the molecules. We used a weighted-geometric docking algorithm in which contacts involving specified parts of the surfaces of either one or both molecules were up-weighted or down-weighted. The weights were based on available structural and biochemical data or on sequence analyses. The weighted-geometric docking proved very useful for five targets, improving the complementarity scores and the ranks of the nearly correct solutions, as well as their statistical significance. In addition, the weighted-geometric docking promoted formation of clusters of similar solutions, which include more accurate predictions.