Benchmark Study for the Cysteine–Histidine Proton Transfer Reaction in a Protein Environment: Gas Phase, COSMO, QM/MM Approaches

Proton transfer reactions are of crucial interest for the investigation of proteins. We have investigated the accuracy of commonly used quantum chemical methods for the description of proton transfer reactions in different environments (gas phase, COSMO, QM/MM) using the proton transfer between the catalytic dyad residues cysteine 145 and histidine 41 of SARS coronavirus main protease as a case study. The test includes thermodynamic, kinetic, and structural properties. The study comprises computationally demanding <i>ab initio</i> approaches (HF, CC2, MP2, SCS-CC2, SCS-MP2, CCSD­(T)), popular density functional theories (BLYP, B3LYP, M06-2X), and semiempirical methods (MNDO/d, AM1, RM1, PM3, PM6). The approximated coupled cluster approach LCCSD­(T) is taken as a reference method. We find that the robustness of the tested methods with respect to the environment correlates well with the level of theory. As an example HF, CC2, MP2, and their SCS variants show similar errors for gas phase, COSMO, or QM/MM computations. In contrast for semiempirical methods, the errors strongly diversify if one goes from gas phase to COSMO or QM/MM. Particular problems are observed for the recent semiempirical methods PM6 and RM1, which show the best performance for gas phase calculations but possess larger errors in conjunction with COSMO. Finally, a combination of SCS-MP2 and B3LYP or M06-2X allows reliable estimates about remaining errors

Evidence for Substrate Binding-Induced Zwitterion Formation in the Catalytic Cys-His Dyad of the SARS-CoV Main Protease

The coronavirus main protease (M^pro) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M^pro and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M^pro active site that lead to the Cys^–/His⁺ zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate–enzyme and inhibitor–enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M^pro employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured p<i>K</i>_a values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M^pro activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M^pro toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors

Drug combination studies of curcumin and genistein against rhodesain of <i>Trypanosoma brucei rhodesiense</i>

<p>Curcumin and genistein are two natural products obtained from <i>Curcuma longa</i> L. and soybeans, endowed with many biological properties. Within the last years they were shown to possess also a promising antitrypanosomal activity. In the present paper, we investigated the activity of both curcumin and genistein against rhodesain, the main cysteine protease of <i>Trypanosoma brucei rhodesiense</i>; drug combination studies, according to Chou and Talalay method, allowed us to demonstrate a potent synergistic effect for the combination curcumin-genistein. As a matter of fact, with our experiments we observed that the combination index of curcumin-genistein is < 1 for the reduction from 10 to 90% of rhodesain activity.</p

Cathepsin B in Antigen-Presenting Cells Controls Mediators of the Th1 Immune Response during <i>Leishmania major</i> Infection

<div><p>Resistance and susceptibility to <i>Leishmania major</i> infection in the murine model is determined by the capacity of the host to mount either a protective Th1 response or a Th2 response associated with disease progression. Previous reports involving the use of cysteine cathepsin inhibitors indicated that cathepsins B (Ctsb) and L (Ctsl) play important roles in Th1/Th2 polarization during <i>L. major</i> infection in both susceptible and resistant mouse strains. Although it was hypothesized that these effects are a consequence of differential patterns of antigen processing, the mechanisms underlying these differences were not further investigated. Given the pivotal roles that dendritic cells and macrophages play during <i>Leishmania</i> infection, we generated bone-marrow derived dendritic cells (BMDC) and macrophages (BMM) from <i>Ctsb</i>^−/− and <i>Ctsl</i>^−/− mice, and studied the effects of Ctsb and Ctsl deficiency on the survival of <i>L. major</i> in infected cells. Furthermore, the signals used by dendritic cells to instruct Th cell polarization were addressed: the expression of MHC class II and co-stimulatory molecules, and cytokine production. We found that <i>Ctsb</i>^−/− BMDC express higher levels of MHC class II molecules than wild-type (WT) and <i>Ctsl</i>^−/− BMDC, while there were no significant differences in the expression of co-stimulatory molecules between cathepsin-deficient and WT cells. Moreover, both BMDC and BMM from <i>Ctsb</i>^−/− mice significantly up-regulated the levels of interleukin 12 (IL-12) expression, a key Th1-inducing cytokine. These findings indicate that <i>Ctsb</i>^−/− BMDC display more pro-Th1 properties than their WT and <i>Ctsl</i>^−/− counterparts, and therefore suggest that Ctsb down-regulates the Th1 response to <i>L. major</i>. Moreover, they propose a novel role for Ctsb as a regulator of cytokine expression.</p></div

Comparison of <i>L. major</i> promastigote uptake and processing by BMDC from WT and cathepsin-deficient mice.

<p>(A) Representative histograms for WT BMDC (CD11c⁺-gated) infected for 2 hours with eGFP-tg <i>L. major</i> promastigotes, and further incubated for 4 and 24 hours in fresh medium. The percentage of eGFP⁺ cells was considered as percentage of remaining infected cells. (B) No significant differences between BMDC from WT and cathepsin-deficient mice were found in the uptake and processing of eGFP-tg promastigotes over the course of 24 hours. The results are expressed as mean ± SD of 3 independent experiments.</p

BMDC and BMM from cathepsin B-deficient mice express higher levels of IL-12 in response to <i>L. major</i> than cells derived from WT and cathepsin L-deficient mice.

<p>(A) IL-12p70 in supernatants from non-treated BMDC(NT), BMDC infected (Inf) with <i>L. major</i> promastigotes at 48 hours p.i., BMDC stimulated with parasite lysate (LmAg), or with heat-killed parasites (HK), for 48 hours. (B) IL-12p40 and (C) IL-10 concentration in supernatants of BMDC at 48 hours p.i., or stimulation with LmAg or HK parasites. (D) IL-12p70 production in supernatants from non-treated BMM (NT), BMM infected (Inf) with <i>L. major</i> promastigotes at 48 hours p.i., and BMM stimulated for 48 hours with LmAg or HK parasites. (E) IL-12p40 and (F) IL-10 concentration in supernatants of BMM at 48 hours p.i., or stimulation with either LmAg or HK parasites. The results are expressed as mean ± SD of 5 independent experiments. For each experimental group (NT, Inf, LmAg and HK), statistical significance was estimated between WT and Ctsb^−/− cells, and between WT and Ctsl^−/− cells, * p<0.05, **p<0.01, *** p<0.005.</p

BMDC from cathepsin B-deficient mice express higher levels of MHC class II molecules in comparison with BMDC from cathepsin L-deficient and WT mice, but no differences were observed in the expression of co-stimulatory molecules.

<p>The expression of MHC class II molecules, CD40, CD86 and CD80 was measured by flow cytometry in BMDC in response to infection with <i>L. major</i> promastigotes (Inf), LmAg, heat-killed parasites (HK), or LPS. (A) Average MFI of MHC class II molecules, normalized to non-treated (NT) WT BMDC. (B) Average MFI of CD40, normalized WT NT BMDC. (C) Average MFI of CD80 and CD86, normalized WT NT BMDC. MFI values are expressed as mean ± SD of 4 independent experiments. Statistical significance was assessed between WT BMDC and Ctsb^−/− BMDC, and between WT BMDC and Ctsl^−/− BMDC for every single treatment, * p<0.05, *** p<0.005.</p

Th1 polarization of OT-II CD4⁺ naïve T cells by BMDC from WT C57BL/6 and Ctsb^−/− mice.

<p>Isolated CD4⁺ CD25⁻ T cells from OT-II mice were co-cultured with BMDC generated from WT C57BL/6 and Ctsb^−/− mice in the presence of LPS as a stimulus and OVA peptide (327–339) or ovalbumin (OVA) as antigens. A) Zebra plots from one representative experiment. B) Average percentages of IFN-γ, IL-4, or IL-10⁺ CD4⁺ T cells from 3 independent experiments ± SD. * p<0.05.</p

IL-12 is up-regulated at the transcriptional level in BMM from cathepsin B-deficient mice in response to <i>L. major</i> infection and LPS stimulation.

<p>Relative expression levels of (A) IL-12p40 and (B) IL-12p35 transcripts in mRNA from BMM at 6 hours or 24 hours p.i. with <i>L. major</i> promastigotes or LPS stimulation. Non-treated (NT) BMM from each mouse line were used as negative controls. The expression levels were estimated using the 2^{− ΔΔC}_T method, using WT NT BMM at t = 6 hours as a reference. The results are shown as mean ± SD of 4 independent experiments, * p<0.05.</p

<i>L. major</i> promastigotes survive comparably in BMM from cathepsin B- and cathepsin L-deficient mice, and these BMM show similar levels of NO production in response to <i>L. major</i> and LPS.

<p>WT, Ctsb^−/− and Ctsl^−/− BMM were infected with eGFP-tg <i>L. major</i> promastigotes, and the percentage of infected cells 24 hours post infection (p.i.) and 48 hours p.i. was determined by fluorescence microscopy (A). No statistically significant differences were found in the amounts of infected cells between WT and cathepsin-deficient BMM. (B) As in (A) the number of parasites per infected cells was determined by fluorescence microscopy, and used as an indicator for parasite proliferation. Although no significant differences were found between WT and cathepsin-deficient BMM, each line showed significant differences in the counts of parasites per infected cell between 24 hours and 48 hours p.i. (C) Parasite proliferation determined by luminescence of Luc-tg <i>L. major</i> promastigotes within BMM at 48 hours p.i. Data are shown as counts per second (CPS). (D) NO production in supernatants from BMM 48 hours after infection with <i>L. major</i> and stimulation with LPS. Results are expressed as mean ± SD from 3 independent experiments.</p