Identification and evaluation of potent

Middle East respiratory syndrome

coronavirus (MERS-CoV) 3CLPro
inhibitors Vathan Kumar
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 Antiviral Research 141 (2017) 101e106

Contents lists available at ScienceDirect

Antiviral Research
journal homepage: www.elsevier.com/locate/antiviral

Identification and evaluation of potent Middle East respiratory

syndrome coronavirus (MERS-CoV) 3CLPro inhibitors
Vathan Kumar a, 1, Jin Soo Shin b, 1, Jiun-Jie Shie c, Keun Bon Ku b, Chonsaeng Kim b,
Yun Young Go b, Kai-Fa Huang a, Meehyein Kim b, **, Po-Huang Liang a, d, *
a
Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan
b
Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea
c
Institute of Chemistry, Academia Sinica, Taipei, Taiwan
d
Institute of Biochemical Sciences, National Taiwan University, Taipei, Taiwan

a r t i c l e i n f o a b s t r a c t

Article history: Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe acute respiratory illness with
Received 13 September 2016 fever, cough and shortness of breath. Up to date, it has resulted in 1826 human infections, including 649
Received in revised form deaths. Analogous to picornavirus 3C protease (3Cpro), 3C-like protease (3CLpro) is critical for initiation of
10 February 2017
the MERS-CoV replication cycle and is thus regarded as a validated drug target. As presented here, our
Accepted 14 February 2017
peptidomimetic inhibitors of enterovirus 3Cpro (6b, 6c and 6d) inhibited 3CLpro of MERS-CoV and severe
Available online 17 February 2017
acute respiratory syndrome coronavirus (SARS-CoV) with IC50 values ranging from 1.7 to 4.7 mM and from
0.2 to 0.7 mM, respectively. In MERS-CoV-infected cells, the inhibitors showed antiviral activity with EC50
Keywords:
MERS-CoV
values ranging from 0.6 to 1.4 mM, by downregulating the viral protein production in cells as well as
SARS-CoV reducing secretion of infectious viral particles into culture supernatants. They also suppressed other a-
3C-like protease and b-CoVs from human and feline origin. These compounds exhibited good selectivity index (over 70
Peptidomimetic inhibitor against MERS-CoV) and could lead to the development of broad-spectrum antiviral drugs against
Coronavirus emerging CoVs and picornaviruses.
Picornavirus © 2017 Elsevier B.V. All rights reserved.

1. Introduction
Coronaviruses (CoVs) affecting upper respiratory tract were first
identified in humans in mid-1960 (Tyrrell and Bynoe, 1965). In late
2002, there was emergence of a life threatening CoV of atypical
pneumonia, named severe acute respiratory syndrome CoV (SARS-
CoV). SARS-CoV belongs to the family Coronaviridae, and is an
enveloped, positive-stranded RNA virus with ~30,000 nucleotides
(Rota et al., 2003). Its genome encodes two polyproteins, pp1a
(~490 kDa) and pp1ab (~790 kDa) which are processed by 3C-like
protease (3CLpro) and papain-like protease (PLpro) to generate non-
structural proteins essential for viral replication (Thiel et al., 2001,
* Corresponding author. Institute of Biological Chemistry, Academia Sinica, Taipei
115, Taiwan.
** Corresponding author. Center for Virus Research and Testing, Korea Research
Institute of Chemical Technology, 141 Gajeongro, Yuseong, Daejeon 34114, Republic
of Korea.
E-mail addresses: mkim@krict.re.kr (M. Kim), phliang@gate.sinica.edu.tw
(P.-H. Liang).
1
These authors contributed equally to this work.
2003). Due to its vital role in replication, 3CLpro has been regar-
ded as a validated drug target. Many inhibitors of SARS-CoV 3CLpro
were discovered by high throughput screening and structure-based
rational design as summarized in the review articles (Hilgenfeld
and Peiris, 2013; Kumar et al., 2013; Kuo and Liang, 2015;
Pillaiyar et al., 2016; Ramajayam et al., 2011; Tong, 2009; Zhao
et al., 2013). After SARS-CoV infection subsided, Middle East res-
piratory syndrome CoV (MERS-CoV), has emerged in Saudi Arabia
in 2012 and spread worldwide, killing 36% of the reported 1826
patients (http://www.who.int/mediacentre/factsheets/mers-cov/
en/). Due to the similar maturation pathway, MERS-CoV 3CLpro is
also regarded as a target for developing antiviral drugs (Tomar et al.,
2015). Though tremendous efforts have been made to develop in-
hibitors, therapeutic interventions for such continuous CoV out-
breaks are yet to reach market (Barnard and Kumaki, 2011; Kilianski
and Baker, 2014).
These CoVs' 3CLpro are functionally similar to the 3Cpro in pi-
cornaviruses and both adopt chymotrypsin fold (Anand et al.,
2003). However, 3CLpro is a dimer with Cys-His dyad, whereas
3Cpro is a monomer with Cys-His-Glu triad (Hsu et al., 2005; Lee

http://dx.doi.org/10.1016/j.antiviral.2017.02.007
0166-3542/© 2017 Elsevier B.V. All rights reserved.
102 V. Kumar et al. / Antiviral Research 141 (2017) 101e106
et al., 2009; Yang et al., 2003). Picornaviruses are small, non-
enveloped RNA virus with genome size of 7500e8000 nucleo-
tides. Based on their genetic organization, the family is composed
of 31 genera including Enterovirus (enterovirus and rhinovirus),
Aphthovirus (foot-and-mouth disease virus), Cardiovirus (encepha-
lomyocarditis virus), Hepatovirus (hepatitis A virus) and others
(http://www.picornaviridae.com/). As 3Cpro is produced in all
genera of Picornaviridae virus family, its inhibitors showed broad-
spectrum, potent antiviral activity against rhinovirus, coxsack-
ievirus and enterovirus (Jetsadawisut et al., 2016; Kim et al., 2015;
St John et al., 2015). Though 3Cpro and 3CLpro share similar struc-
tures at their active sites, subtle differences often discriminate in-
hibitors. AG7088, an established 3Cpro inhibitor, was inactive
against SARS-CoV 3CLpro prior to the modifications (Ghosh et al.,
2005; Shie et al., 2005; Thanigaimalai et al., 2013; Yang et al.,
2006). Unlike AG7088 which contains a, b-unsaturated ester for
forming covalent bond with the active-site Cys, our previously re-
ported potent peptidomimetic inhibitors of 3Cpro from enterovirus
71 (EV71) contains aldehyde as electrophilic warhead (Kuo et al.,
2008). In this work, we screened those EV71 3Cpro inhibitors
against MERS-CoV 3CLpro and further evaluated the hits by cell-
based assays using live MERS-CoV. Our best compounds 6b, 6c
and 6d inhibited MERS-CoV 3CLpro with IC50 values ranging from
1.7 to 4.7 mM and also suppressed viral replication with EC50 values
between 0.6 and 1.4 mM. These derivatives represent some of few
cell-based assay-confirmed anti-MERS-CoV agents and also
showed broad-spectrum activity against both a- and b-types of
CoVs as described herein.
2. Materials and methods
2.1. Synthesis of compounds
Compounds reported here were synthesized using previously
reported procedures with some modifications (Kuo et al., 2008).
Test compounds and gemcitabine hydrochloride (GEM; Sigma-
Aldrich, St. Louis, MO) were dissolved in dimethyl sulfoxide
(DMSO; Sigma-Aldrich) at 50 mM concentration.
2.2. Viruses and cells
Patient-derived isolate MERS-CoV (MERS-CoV/KOR/KNIH/
002_05_2015; GenBank accession No. KT029139) was provided by
the Korea Center for Disease Control and Prevention. Huh-7 and
Vero cells (Cat. No. CCL-81) were obtained from Prof. D.-E. Kim at
Konkuk University (Seoul, Republic of Korea) and American Type
Culture Collection (ATCC, Manassas, VA), respectively. The cells
were maintained in Dulbecco's Modified Eagle Medium (DMEM;
Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine
serum (FBS; Gibco BRL) at 37
C and 5% CO2. To minimize adaptive
mutation probability of MERS-CoV to another species during pas-
sage, MERS-CoV was amplified by infection of a human cell line,
Huh-7 cells. The infectious viral titers from culture supernatants at
day 2 post-infection (p.i.) were measured by a plaque assay using
Vero cells according to other reports (Chan et al., 2013; de Wilde
et al., 2013). MERS-CoV was maintained under biosafety level 3
conditions in Korea Research Institute of Chemical Technology
(KRICT).
Human CoV strains, 229E (Cat. No. VR-740) and OC43 (Cat. No.
VR-1558) were purchased from ATCC. They were amplified by
infecting human fetal lung fibroblast MRC-5 cells (ATCC, Cat. No.
CCL-171). Feline infectious peritonitis coronavirus (FIPV) strain
(Cat. No. VR-990) and its host cell line Crandall feline kidney (CRFK)
(Cat. No. 10094) were obtained from ATCC and Korean Cell Line
Bank (Seoul, Republic of Korea), respectively.
2.3. Expression and purification of SARS- and MERS-CoV 3CLpro
The expression and purification of SARS-CoV 3CLpro followed
our reported procedure (Kuo et al., 2004). For expression of MERS-
CoV 3CLpro (Kumar et al., 2016), the Factor Xa cleavage site (IEGR)
and the 3CLpro (accession KJ361502.1, Ser3248eGln3553) DNA
sequence was synthesized and cloned into the pET32 expression
vector by Mission Biotech. Company (Taiwan) and was transformed
into E. coli BL21 (DE3). A 10 ml overnight culture of a single
transformant was used to inoculate 1L of fresh LB medium con-
taining 100 mg/ml ampicillin. The cells were grown at 37
C to
A600 ¼ 0.8 and induced with 0.4 mM isopropyl-b-thiogalactopyr-
anoside (IPTG) for 22 h at 16
C. The cells were harvested by
centrifugation at 7000  g for 15 min and the pellet was suspended
in lysis buffer (12 mM Tris-HCl, 120 mM NaCl, 0.1 mM EDTA, and
5 mM DTT, pH 7.5). A French-press instrument (Constant Cell
Disruption System) was used to disrupt the cells at 20,000 psi and
centrifuged at 20,000  g for 1 h to discard the debris. The cell-free
extract was loaded onto Ni-NTA column which was equilibrated
with lysis buffer containing 5 mM imidazole. After exhaustive
washing with lysis buffer, the imidazole concentration of the
washing buffer was increased to 30 mM. The protein eluted by lysis
buffer containing 300 mM imidazole was dialyzed against lysis
buffer to remove imidazole and then Factor Xa was added to a final
concentration of 1% (w/w) and incubated at 16
C for 24 h to
remove the His-tag. Subsequently, the processed MERS-CoV 3CLpro
was passed through a Ni-NTA column for purification. The protein
concentration was determined by the protein assay kit (BioRad,
USA) and BSA was used as standard.
2.4. Measurement of IC50
A fluorometric assay by using the fluorogenic peptide, Dabcyl-
KTSAVLQSGFRKME-Edans as previously described (Kuo et al.,
2004) was used to determine the inhibition constants of com-
pounds. The enhanced fluorescence due to the cleavage of this
substrate catalyzed by the 3CLpro was monitored at 538 nm with
excitation at 355 nm. The IC50 value of individual sample was
measured in a reaction mixture containing 50 nM SARS-CoV 3CLpro
or 0.3 mM MERS-CoV 3CLpro and 10 mM of the fluorogenic substrate
in 20 mM Bis-Tris (pH 7.0).
2.5. Cytopathic effect inhibition assay
Huh-7 cells were seeded in 96-well plates (2  104 cells per
well). On the next day, cells were infected with MERS-CoV at a
multiplicity of infection (MOI) of 0.1 in DMEM without FBS for 1 h.
After washing with PBS, mock-infected or virus-infected cells were
treated with 3-fold serial dilutions of test compounds or GEM used
as a positive control. At day 2 p.i., cell lysate was harvested for
measuring cell viability using the CellTiter 96® AQueous One Solu-
tion Cell Proliferation Assay according to the manufacturer's in-
structions (Promega, Madison, WI). The 50% cytotoxic
concentration (CC50) and 50% effective concentration (EC50) values
were calculated using GraphPad Prism 6 software (GraphPad
Software, La Jolla, CA). Antiviral assay for other CoVs, including
229E, OC43 and FIPV strains, were performed as mentioned above
by using different cell lines. MRC-5 cells were used for culturing
human CoVs, 229E and OC43, while CRFK cells for feline CoV, FIPV.
2.6. Western blot analysis
Huh-7 cells seeded in 6-well plates (3  105 cells per well) were
infected with MERS-CoV at an MOI of 0.02 for 1 h. After washing
with PBS, cells were treated with 0.1, 1 and 10 mM of compounds 6b,
 V. Kumar et al. / Antiviral Research 141 (2017) 101e106 103

Table 1
Enzymatic and cell-based antiviral assays of selected compounds.

Compd Ar R IC50 (mM) CC50 (mM)a EC50 (mM) MERS-CoV S.I.b

pro pro
MERS-CoV 3CL SARS eCoV 3CL

4a 3,4-(OCH2O)C6H3 eCOOCH3 >25 >25 NDc ND ND

5a 3,4-(OCH2O)C6H3 eCH2OH >25 >25 ND ND ND
6a 3,4-(OCH2O)C6H3 eCHO >25 >25 >100 >100 ND
6b 3-BrC6H4 eCHO 2.4 ± 0.3 0.7 ± 0.2 >100 1.4 ± 0.0 >71.4
6c 4-Me2NC6H4 eCHO 4.7 ± 0.6 0.5 ± 0.1 >100 1.2 ± 0.6 >83.3
6d 4-Cl,2-FC6H3 eCHO 1.7 ± 0.3 0.2 ± 0.07 58.6 ± 1.2 0.6 ± 0.0 97.9
GEMd e e ND ND >100 8.3 ± 0.9 >12.1
a
50% cytotoxic concentration in MDCK cells.
b
Selectivity index.
c
Not determined.
d
Gemcitabine hydrochloride.

6c and 6d. In parallel, 0.02% DMSO was treated as a compound
vehicle. On day 1 p.i., 30 mg cell lysates suspended in sample loading
buffer (Biosesang, Gyeonggi-do, Republic of Korea) were subjected
to 10% SDS-PAGE and electro-transferred to a polyvinylidene fluo-
ride membrane (Millipore, Billerica, MA). MERS-CoV NP was
detected using a primary antibody specific for viral nucleocapsid
protein (NP) (Cat. 100211-RP02; Sino Biological Inc., Beijing, China),
followed by a horseradish peroxidase (HRP)-conjugated goat anti-
rabbit secondary antibody (Thermo Scientific, Waltham, MA). The
cellular b-actin protein, a loading control, was detected with an
anti-b-actin-specific primary antibody (Cat. No. A1987; Sigma-
Aldrich) and the HRP-conjugated goat anti-mouse secondary anti-
body (Thermo Scientific). After addition of a chemiluminescent
HRP substrate (SuperSignal West Pico Chemiluminescent Sub-
strate; Pierce, Rockford, IL), images were obtained using a LAS-4000
Luminescent Image Analyzer (Fujifilm, Tokyo, Japan).
2.7. Plaque inhibition assay
Huh-7 cells were inoculated in 6-well plates at a density of
1  106 cells per well for 1 day. Culture supernatants treated with
1 mM compounds were harvested at day 1 p.i. They were serially
diluted 10-fold in DMEM from 101 to 103 and 1 ml of each sample
was used to infect Vero cells for 1 h. After washing with PBS to
remove unabsorbed virus, DMEM containing 0.5% agarose (overlay
medium) was added. On day 3 p.i., plaques were visualized with
50 mg/ml neutral red (Sigma-Aldrich).
3. Results and discussion
3.1. Inhibition of MERS-CoV 3CLpro and viral infection by 6b, 6c and
6d
Peptidomimetic compounds were synthesized according to our
reported method (Kuo et al., 2008). Preliminary screening of these
peptidomimetic compounds against MERS 3CLpro were done at
50 mM. Compounds inhibiting more than half of the protease ac-
tivity under such condition were selected for further IC50 mea-
surements. Using enzymatic assay, the compounds 6b, 6c and 6d
showed IC50 of 2.4, 4.7 and 1.7 mM against purified 3CLpro of MERS-
CoV, respectively (Table 1). These compounds also inhibited SARS
3CLpro at lower IC50 values of 0.7, 0.5 and 0.2 mM, respectively.
To evaluate the ability of these compounds to block viral repli-
cation, we performed cytopathic inhibition assay using MERS-CoV-
infected Huh-7 cells. As shown in Fig. 1, compounds 6b, 6c and 6d
efficiently suppressed viral replication with EC50 of 1.4, 1.2 and
0.6 mM, respectively (Table 1). Though it is usual to see EC50 higher
than IC50, due to the presence of membrane barrier, we observed
EC50 to be smaller than IC50. This could be due to the higher con-
centration (300 nM) used for in-vitro enzymatic assay because it is a
weakly associated dimer (Tomar et al., 2015). In fact, they inhibited
50 nM SARS 3CLpro, a tight dimer, in the submicromolar range
(Table 1). These compounds had CC50 larger than 100 mM for 6b and
6c or 58.6 mM for 6d against uninfected cells, resulting in selectivity
index (S.I.) values larger than 71.4. As expected, compound 6a
which was inactive in the enzyme assay did not suppress viral
replication. GEM, used as a positive control according to a previous
report (Dyall et al., 2014), was less potent in inhibiting MERS-CoV
infection with an EC50 value of 8.3 mM. It also showed marginal
toxicity and thus decreased viability of mock cells by 20% or more at
the concentrations above 3.7 mM (Table 1 and Fig. 1).
3.2. Suppression of viral protein production and infectious MERS-
CoV generation
To confirm that the observed antiviral activity of compounds 6b,
6c and 6d reflects inhibition of MERS-CoV infection, both viral
protein and progeny production was measured after treatment of
virus-infected cells with these compounds. Western blot analysis
showed that viral NP was decreased by these inhibitors in a dose-
dependent manner (Fig. 2). It is noteworthy that no viral NP
detectable in the presence of 10 mM 3CLpro inhibitors.
We further compared the number of infectious MERS-CoV par-
ticles in the culture supernatants, both in the presence and absence
of 3CLpro inhibitors. The plaque assay showed that the viral titer in
the absence of compound was 4.4  105 plaque forming units (pfu)/
ml, but reduced to 1.7  104, 2.9  104, and 1.2  104 pfu/ml by 1 mM
of compounds 6b, 6c and 6d, respectively (Fig. 3). Taken together,
104 V. Kumar et al. / Antiviral Research 141 (2017) 101e106

Fig. 1. Antiviral activity of 6b, 6c and 6d against MERS-CoV in Huh-7 cells. Huh-7 cells in 96-well plates were mock-infected or infected with MERS-CoV at an MOI of 0.1 for 1 h at
37
C. After washing with PBS, cells were treated with 3-fold serial dilutions of test compounds (6a, 6b, 6c and 6d) or a control compound (GEM). On day 2 p.i., cell lysates were
harvested for measuring cell viability. The data represent the means ± standard deviations from three independent experiments.

generation. The data also indicate that the inhibitors can penetrate
virus-infected cell membrane to reach the active site of 3CLpro.

3.3. In silico molecular docking of 6d against MERS-CoV 3CLpro

To rationalize potent inhibition, we docked 6d into the active

Fig. 2. Inhibition of MERS-CoV NP production by 6b, 6c and 6d in a dose-
dependent manner. Huh-7 cells in 6-well plates were infected with MERS-CoV at
an MOI of 0.02 for 1 h at 37
C.The virus-infected cells were treated with increasing
concentrations (0.1, 1 and 10 mM) of each compound. Co-treatment of interferon-alpha
2A (IFN; 50 ng/ml) and ribavirin (RBV; 100 mM) was used as a positive control. On day 1
p.i., cells were harvested and loaded to 10% SDS-PAGE (30 mg per well). Immuno-
blotting was performed using rabbit anti-NP antibody and HRP-conjugated goat anti-
rabbit secondary antibody. b-Actin was used as a loading control.
the results suggest that the compounds originally selected as EV71
3Cpro blockers efficiently inhibited MERS-CoV 3CLpro activity and
suppressed viral protein production as well as viral progeny
site of MERS-CoV 3CLpro. The initial pose of the complex was
generated based on the X-ray structure (PDB code: 4RSP) of the
3CLpro in complex with a covalent inhibitor (Tomar et al., 2015) and
those of the human rhinovirus 3Cpro bound with irreversible in-
hibitors (Matthews et al., 1999; Webber et al., 1998). Then, the
simulation was done by Discovery Studio (Accelrys Inc., San Diego,
CA). A Cys at subsite S1 of 3CLpro acts as a nucleophile to cleave
substrates by attacking carbonyl carbon of the amide bond between
the conserved Gln at P1 and the small amino acids such as Ser, Ala
or Gly at P1' (Fan et al., 2004; Needle et al., 2015). Our modelling
shows that the g-sulfur of Cys148 forms a covalent bond with the
6d aldehyde carbon and the resulting oxyanion is stabilized by
His41 (Fig. 4). A cyclic lactam moiety with cis-amide geometry on
 V. Kumar et al. / Antiviral Research 141 (2017) 101e106
Fig. 3. Downregulation of MERS-CoV progeny generation by 3CLpro inhibitors, 6b,
6c and 6d. MERS-CoV-infected Huh-7 cells in 6-well plates were treated with 1 mM 6b,
6c and 6d for 1 day. Culture supernatants were harvested and serially diluted by 10-
fold in DMEM (101 to 103). Fresh Vero cells in 6-well plates were infected with
the diluted cell culture inoculum for 1 h. And the number of infectious viral particles
was counted by addition of the overlay medium for 3 days and by neutral red staining.
Fig. 4. Docking of inhibitor 6d with MERS-CoV 3CLpro. Cys148 of the protease makes
a covalent bond with the carbonyl carbon of the inhibitor aldehyde, forming a stable
tetrahedral species (the inset), and the resulting oxyanion being stabilized by His 41.
The protease is shown in a charge-potential surface. The putative substrate-binding
subsites S10, S1, S2, S3 and S4 are indicated. Moreover, the possible hydrogen bonds
of 6d to the protease are further drawn with dashed lines.
binding to 3Cpro was proposed to mimic the P1 Gln of peptide
substrates (Matthews et al., 1999). Based on our docking results, the
P1 lactam moiety of 6d binds to S1 subsite of 3CLpro by forming H-
bonds with His166 and Glu169. The P2 phenylalanine moiety pre-
fers to occupy S2 subsite. The cinnamoyl group of 6d occupies S3
and may be extended to S4. The amide group between phenylala-
nine and cinnamoyl group further forms H-bonds with Gln192 and
Glu169. We also docked 6d based on the apo-form MERS-CoV
3CLpro structure (PDB code: 5c3n) (Ho et al., 2015). Since the free-
105
Table 2
Antiviral activity of 6b, 6c, and 6d against 229E, OC43 and FIP.
Compd CC50a (mM) EC50 (mM)
(S.I)
229Eb OC43c FIPVd
6b >100 4.3 ± 0.1 13.5 ± 0.8 2.5 ± 1.1
(>25.0) (>7.3) (>40.0)
6c >100 4.2 ± 0.3 16.8 ± 0.3 1.9 ± 0.2
(>23.8) (>6.0) (>52.6)
6d >100 2.0 ± 0.2 17.7 ± 1.6 1.1 ± 0.3
(>50.0) (>5.7) (>90.9)
a
50% cytotoxic concentration in MRC-5 cells and in CRFK cells.
b
Human alpha coronavirus.
c
Human beta coronavirus.
d
Feline infectious peritonitis alpha coronavirus.
form and ligand-bound structures showed no significant differ-
ence in the active site, the binding modes of 6d in these two
structures are indeed very similar (data not shown).
From our experimental as well as modelling results, sub-
stituents on cinnamoyl groups of these peptidomimetic inhibitors
seem to be critical for the activity. Substituent p-chloro in 6d makes
halogen bonding with His194, leading to better potency. Compound
6b and 6c with slightly bulkier m-bromo and p-dimethylamino
moiety, respectively, displayed approximately 2-fold drop in IC50.
Compound 6a with the bulkiest 3,4-methylenedioxy substituent
failed to inhibit the 3CLpro even at 25 mM. Compounds 4a and 5a
that lacked aldehyde warhead failed to inhibit 3CLpro, emphasizing
the importance of reactive aldehyde electrophile.
3.4. Broad spectrum activity against human and feline CoVs
To investigate for broad spectrum activity, three compounds
were tested against human and feline CoVs. The result showed both
a-CoVs, human 229E strain and FIPV, and b-CoV (human OC43
strain) were sensitive to the compounds with EC50 of 1.1e17.7 mM
(Table 2), suggesting potent and broad-spectrum antiviral activities
of these 3CLpro inhibitors. The CC50 values measured using MRC-5
and CRFK cells, used to cultivate human and feline CoVs, respec-
tively, were found to be more than 100 mM. Therefore, the S.I. values
were ranged above 5.7 as shown in parentheses.
Peptide and peptidomimetic aldehyde inhibitors against 3CLpro
have been reported (Akaji et al., 2008, 2011; Zhu et al., 2011) but not
tested on live CoVs. However, a potent SARS-CoV 3CLpro peptido-
mimetic aldehyde inhibitor, TG-0205221, has been shown to block
SARS-CoV and human CoV 229E replications (Yang et al., 2006). As
shown in this study, we have identified potent and membrane-
permeable MERS-CoV inhibitors 6b, 6c and 6d using live MERS-
CoV virus with EC50 of 0.6e1.2 mM and S.I. over 71.4. These com-
pounds with IC50 < 0.5 mM against 3Cpro (Kuo et al., 2008) inhibited
3CLpro of MERS-CoV with IC50 of 1.7e4.7 mM and SARS-CoV with
IC50 of 0.2e0.7 mM. Moreover, we found these inhibitors were active
against other viruses, including a- and b-CoVs with EC50 of
1.1e17.7 mM, but were less potent (higher EC50) in killing human b-
CoV OC43. Although not as potent as inhibiting picornavirus EV71
with the EC50 of 18 and 7 nM for 6c and 6d, respectively (Kuo et al.,
2008), they are the most potent inhibitors of live MERS-CoV iden-
tified so far. Their inhibitory activities against picornaviruses and
CoVs make these compounds broad-spectrum antiviral agents.
With the escalating cost of drug discovery, development of an
antiviral agent with broad-spectrum activities might help in over-
coming financial hurdles. More compounds are being synthesized
for lead optimization. Animal study needs to be further conducted
for developing one of these potent inhibitors into an antiviral drug.
106 V. Kumar et al. / Antiviral Research 141 (2017) 101e106
Acknowledgements
We appreciate Dr. Sung Soon Kim at Korea Centers for Disease
Control and Prevention (KCDC) for providing MERS-CoV. This work
was supported by Academia Sinica, Taiwan and the grants from
KRICT (KK1603) and KCDC (2015-ER4808-00), Republic of Korea.
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adjournment, it is thereby removed from before the House, and does
not stand ipso facto before them at their next meeting, but must
come forward in the usual way. So, when it is interrupted by the
order of the day. Such other privileged questions also as dispose of
the main question, (e. g., the previous question, postponement, or
commitment,) remove it from before the House. But it is only
suspended by a motion to amend, to withdraw, to read papers, or by
a question of order or privilege, and stands again before the House
when these are decided. None but the class of privileged questions
can be brought forward while there is another question before the
House, the rule being that when a motion has been made and
seconded, no other can be received except it be a privileged one.

SEC. XXXVIII.—EQUIVALENT QUESTIONS.

If, on a question for rejection, a bill be retained, it passes, of

course, to its next reading. Hakew., 141; Scob., 42. And a question for
a second reading determined negatively, is a rejection without
further question. 4 Grey, 149. And see Elsynge’s Memor., 42, in what
cases questions are to be taken for rejection.
Where questions are perfectly equivalent, so that the negative of
the one amounts to the affirmative of the other, and leaves no other
alternative, the decision of the one concludes necessarily the other. 4
Grey, 157. Thus the negative of striking out amounts to the
affirmative of agreeing; and therefore to put a question on agreeing
after that on striking out, would be to put the same question in effect
twice over. Not so in questions of amendments between the two
Houses. A motion to recede being negatived, does not amount to a
positive vote to insist, because there is another alternative, to wit, to
adhere. A bill originating in one House is passed by the other with an
amendment. A motion in the originating House to agree to the
amendment is negatived. Does there result from this vote of
disagreement, or must the question on disagreement be expressly
voted? The question respecting amendments from another House
are—1st, to agree; 2d, disagree; 3d, recede; 4th, insist; 5th, adhere,
 Either of these concludes the other necessarily, for the positive of
either is exactly the equivalent of the negative of the other, and no
1st. To
other alternative remains. On either motion amendments to the
agree.
amendment may be proposed; e. g., if it be moved to disagree, those
2d. To
who are for the amendment have a right to propose amendments,
disagree.
and to make it as perfect as they can, before the question of
disagreeing is put.
You may then either insist or adhere.
3d. To You may then either recede or adhere.
recede. You may then either recede or insist.
4th. To Consequently the negative of these is not equivalent to a positive
insist. vote, the other way. It does not raise so necessary an implication as
5th. To may authorize the Secretary by inference to enter another vote; for
adhere. two alternatives still remain, either of which may be adopted by the
House.

SEC. XXXIX.—THE QUESTION.

The question is to be put first on the affirmative, and then on the

negative side.
After the Speaker has put the affirmative part of the question, any
member who has not spoken before to the question may rise and
speak before the negative be put; because it is no full question till the
negative part be put. Scob., 23; 2 Hats., 73.
But in small matters, and which are of course, such as receiving
petitions, reports, withdrawing motions, reading papers, &c., the
Speaker most commonly supposes the consent of the House where
no objection is expressed, and does not give them the trouble of
putting the question formally. Scob., 22; 2 Hats., 87; 5 Grey, 129; 9
Grey, 301.

SEC. XL.—BILLS, THIRD READING.

To prevent bills from being passed by surprise, the House, by a

standing order, directs that they shall not be put on their passage
before a fixed hour, naming one at which the House is commonly
full. Hakew., 153.
 [The usage of the Senate is, not to put bills on their passage till
noon.]
A bill reported and passed to the third reading, cannot on that day
be read the third time and passed; because this would be to pass on
two readings in the same day.
At the third reading the Clerk reads the bill and delivers it to the
Speaker, who states the title, that it is the third time of reading the
bill, and that the question will be whether it shall pass. Formerly the
Speaker, or those who prepared a bill, prepared also a breviate or
summary statement of its contents, which the Speaker read when he
declared the state of the bill, at the several readings. Sometimes,
however, he read the bill itself, especially on its passage. Hakew.,
136, 137, 153; Coke, 22, 115. Latterly, instead of this, he, at the third
reading, states the whole contents of the bill verbatim, only, instead
of reading the formal parts, “Be it enacted,” &c., he states that
“preamble recites so and so—the 1st section enacts that, &c.; the 2d
section enacts,” &c.
[But in the Senate of the United States, both of these formalities
are dispensed with; the breviate presenting but an imperfect view of
the bill, and being capable of being made to present a false one; and
the full statement being a useless waste of time, immediately after a
full reading by the Clerk, and especially as every member has a
printed copy in his hand.]
A bill on the third reading is not to be committed for the matter or
body thereof, but to receive some particular clause or proviso, it hath
been sometimes suffered, but as a thing very unusual. Hakew., 156.
Thus, 27 El., 1584, a bill was committed on the third reading, having
been formerly committed on the second, but is declared not usual.
D’Ewes, 337, col. 2; 414, col. 2.
When an essential provision has been omitted, rather than erase
the bill and render it suspicious, they add a clause on a separate
paper, engrossed and called a rider, which is read and put to the
question three times. Elsynge’s Memo., 59; 6 Grey, 335; 1 Blackst.,
183. For examples of riders, see 3 Hats., 121, 122, 124, 156. Every one
is at liberty to bring in a rider without asking leave. 10 Grey, 52.
It is laid down as a general rule, that amendments proposed at the
second reading shall be twice read, and those proposed at the third
reading thrice read; as also all amendments from the other House.
Town., col. 19, 23, 24, 25, 26, 27, 28.
It is with great and almost invincible reluctance that amendments
are admitted at this reading, which occasion erasures or
interlineations. Sometimes a proviso has been cut off from a bill;
sometimes erased. 9 Grey, 513.
This is the proper stage for filling up blanks; for if filled up before,
and now altered by erasure, it would be peculiarly unsafe.
At this reading the bill is debated afresh, and for the most part is
more spoken to at this time than on any of the former readings.
Hakew., 153.
The debate on the question whether it should be read a third time,
has discovered to its friends and opponents the arguments on which
each side relies, and which of these appear to have influence with the
House; they have had time to meet them with new arguments, and to
put their old ones into new shapes. The former vote has tried the
strength of the first opinion, and furnished grounds to estimate the
issue; and the question now offered for its passage is the last
occasion which is ever to be offered for carrying or rejecting it.
When the debate is ended, the Speaker, holding the bill in his
hand, puts the question for its passage, by saying, “Gentlemen, all
you who are of opinion that this bill shall pass, say aye;” and after the
answer of the ayes, “All those of the contrary opinion, say no.”
Hakew., 154.
After the bill is passed, there can be no further alteration of it in
any point. Hakew., 159.

SEC. XLI.—DIVISION OF THE HOUSE.

The affirmative and negative of the question having been both put
and answered, the Speaker declares whether the yeas or nays have it
by the sound, if he be himself satisfied, and it stands as the judgment
of the House. But if he be not himself satisfied which voice is the
greater, or if before any other member comes into the House, or
before any new motion made, (for it is too late after that,) any
member shall rise and declare himself dissatisfied with the Speaker’s
decision, then the Speaker is to divide the House. Scob., 24; 2 Hats.,
140.
When the House of Commons is divided, the one party goes forth,
and the other remains in the House. This has made it important
which go forth and which remain; because the latter gain all the
indolent, the indifferent, and inattentive. Their general rule,
therefore, is, that those who give their vote for the preservation of the
orders of the House shall stay in; and those who are for introducing
any new matter or alteration, or proceeding contrary to the
established course, are to go out. But this rule is subject to many
exceptions and modifications. 2 Hats., 134; 1 Rush., p. 3, fol. 92;
Scob., 43, 52; Co., 12, 116; D’Ewes, 505, col. 1; Mem. in Hakew., 25,
29; as will appear by the following statement of who go forth:
Petition that it be received
Ayes.
Read

Lie on the table

Noes.
Rejected after refusal to lie on table

Referred to a committee, for further proceeding Ayes.

Bill, that it be brought in

Read first or second time
Engrossed or read third time Ayes.
Proceeding on every other stage
Committed

To Committee of the whole Noes.

To a select committee Ayes.

Report of bill to lie on table Noes.

Be now read Ayes.

Be taken into consideration three months hence 30, P.J. 251.

Amendments to be read a second time Noes.

Clause offered on report of bill to be read second time

For receiving a clause Ayes. 334.
With amendments be engrossed 395.

That a bill be now read a third time Noes. 398.

Receive a rider 260.

Pass Ayes.
259.
Be printed

Committees. That A take the chair Noes. 291.

To agree to the whole or any part of report
That the House do now resolve into committee
Speaker. That he now leave the chair, after order to go into
committee
That he issue warrant for a new writ
Member. That none be absent without leave

Witness. That he be further examined Ayes. 344.

Previous question Noes.

Blanks. That they be filled with the largest sum.

Ayes.
Amendments. That words stand part of

Lords. That their amendment be read a second time Noes.

Messenger be received
Ayes.
Orders of day to be now read, if before 2 o’clock

If after 2 o’clock Noes.

Adjournment. Till the next sitting day, if before 4 o’clock Ayes.

If after 4 o’clock Noes.

Over a sitting day, (unless a previous resolution.) Ayes.

Over the 30th of January Noes.

For sitting on Sunday, or any other day not being a sitting day Ayes.
The one party being gone forth, the Speaker names two tellers
from the affirmative and two from the negative side, who first count
those sitting in the House and report the number to the Speaker.
Then they place themselves within the door, two on each side, and
count those who went forth as they come in, and report the number
to the Speaker. Mem. in Hakew., 26.
A mistake in the report of the tellers may be rectified after the
report made. 2 Hats., 145, note.
[But in both Houses of Congress all these intricacies are avoided.
The ayes first rise, and are counted standing in their places by the
President or Speaker. Then they sit, and the noes rise and are
counted in like manner.]
[In Senate, if they be equally divided, the Vice-President
announces his opinion, which decides.]
 [The Constitution, however, has directed that “the yeas and nays of
the members of either House on any question, shall at the desire of
one-fifth of those present, be entered on the journal.” And again: that
in all cases of reconsidering a bill disapproved by the President, and
returned with is objections, “the votes of both Houses shall be
determined by yeas and nays, and the names of persons voting for
and against the bill shall be entered on the journals of each House
respectively.”]
[By the 16th and 17th rules of the Senate, when the yeas and nays
shall be called for by one-fifth of the members present, each member
called upon shall, unless for special reasons he be excused by the
Senate, declare openly, and without debate, his assent or dissent to
the question. In taking the yeas and nays, and upon the call of the
House, the names of the members shall be taken alphabetically.]
[When the yeas and nays shall be taken upon any question in
pursuance of the above rule, no member shall be permitted, under
any circumstances whatever, to vote after the decision is announced
from the Chair.]
[When it is proposed to take the vote by yeas and nays, the
President or Speaker states that “the question is whether, e. g., the
bill shall pass—that it is proposed that the yeas and nays shall be
entered on the journal. Those, therefore, who desire it, will rise.” If
he finds and declares that one-fifth have risen, he then states that
“those who are of opinion that the bill shall pass are to answer in the
affirmative; those of the contrary opinion in the negative.” The Clerk
then calls over the names, alphabetically, note the yea or nay of each,
and gives the list to the President or Speaker, who declares the result.
In the Senate, if there be an equal division, the Secretary calls on the
Vice-President and notes his affirmative or negative, which becomes
the decision of the House.]
In the House of Commons, every member must give his vote the
one way or the other, Scob., 24, as it is not permitted to any one to
withdraw who is in the House when the question is put, nor is any
one to be told in the division who was not in when the question was
put. 2 Hats., 140.
This last position is always true when the vote is by yeas and nays;
where the negative as well as affirmative of the question is stated by
the President at the same time, and the vote of both sides begins and
proceeds pari passu. It is true also when the question is put in the
usual way, if the negative has also been put; but if it has not, the
member entering, or any other member, may speak, and even
propose amendments, by which the debate may be opened again,
and the question be greatly deferred. And as some who have
answered ay may have been changed by the new arguments, the
affirmative must be put over again. If, then, the member entering
may, by speaking a few words, occasion a repetition of a question, it
would be useless to deny it on his simple call for it.
While the House is telling, no member may speak or move out of
his place; for if any mistake be suspected, it must be told again.
Mem. in Hakew., 26; 2 Hats., 143.
If any difficulty arises in point of order during the division, the
speaker is to decide peremptorily, subject to the future censure of the
House if irregular. He sometimes permits old experienced members
to assist him with their advice, which they do sitting in their seats,
covered, to avoid the appearance of debate; but this can only be with
the Speaker’s leave, else the division might last several hours. 2
Hats., 143.
The voice of the majority decides; for the lex majoris partis is the
law of all councils, elections, &c., where not otherwise expressly
provided. Hakew., 93. But if the House be equally divided, semper
presumatur pro negante; that is, the former law is not to be changed
but by a majority. Towns., col. 134.
[But in the Senate of the United States, the Vice-President decides
when the House is divided. Const. U. S., I, 3.]
When from counting the House on a division it appears that there
is not a quorum, the matter continues exactly in the state in which it
was before the division, and must be resumed at that point on any
future day. 2 Hats., 126.
1606, May 1, on a question whether a member having said yea may
afterwards sit and change his opinion, a precedent was remembered
by the Speaker, of Mr. Morris, attorney of the wards, in 39 Eliz., who
in like case changed his opinion. Mem. in Hakew., 27.

SEC. XLII.—TITLES.
 After the bill has passed, and not before, the title may be amended,
and is to be fixed by a question; and the bill is then sent to the other
House.

SEC. XLIII.—RECONSIDERATION.

[When a question has been once made and carried in the

affirmative or negative, it shall be in order for any member of the
majority to move for the reconsideration thereof; but no motion for
the reconsideration of any vote shall be in order after a bill,
resolution, message, report, amendment, or motion upon which the
vote was taken shall have gone out of the possession of the Senate
announcing their decision; nor shall any motion for reconsideration
be in order unless made on the same day on which the vote was
taken, or within the two next days of actual session of the Senate
thereafter. Rule 20.]
[1798, Jan. A bill on its second reading being amended, and on the
question whether it shall be read a third time negatived, was restored
by a decision to reconsider that question. Here the votes of negative
and reconsideration, like positive and negative quantities in
equation, destroy one another, and are as if they were expunged from
the journals. Consequently the bill is open for amendment, just so far
as it was the moment preceding the question for the third reading;
that is to say, all parts of the bill are open for amendment except
those on which votes have been already taken in its present stage. So,
also, it may be recommitted.]
[[102]The rule permitting a reconsideration of a question affixing to
it no limitation of time or circumstance, it may be asked whether
there is no limitation? If, after the vote, the paper on which it is
passed has been parted with, there can be no reconsideration; as if a
vote has been for the passage of a bill, and the bill has been sent to
the other House. But where the paper remains, as on a bill rejected;
when, or under what circumstances, does it cease to be susceptible of
reconsideration? This remains to be settled; unless a sense that the
right of reconsideration is a right to waste the time of the House in
repeated agitations of the same question, so that it shall never know
when a question is done with, should induce them to reform this
anomalous proceeding.]
In Parliament a question once carried cannot be questioned again
at the same session, but must stand as the judgment of the House.
Towns., col. 67; Mem. in Hakew., 33. And a bill once rejected,
another of the same substance cannot be brought in again the same
session. Hakew., 158; 6 Grey, 392. But this does not extend to
prevent putting the same question in different stages of a bill;
because every stage of a bill submits the whole and every part of it to
the opinion of the House, as open for amendment, either by insertion
or omission, though the same amendment has been accepted or
rejected in a former stage. So in reports of committees, e. g., report
of an address, the same question is before the House, and open for
free discussion. Towns., col. 26; 2 Hats., 98, 100, 101. So orders of
the House, or instructions to committees, may be discharged. So a
bill, begun in one House, and sent to the other, and there rejected,
may be renewed again in that other, passed and sent back Ib., 92: 3
Hats., 161. Or if, instead of being rejected, they read it once and lay it
aside or amend it, and put it off a month, they may order in another
to the same effect, with the same or a different title. Hakew., 97, 98.
Divers expedients are used to correct the effects of this rule; as, by
passing an explanatory act, if anything has been omitted or ill
expressed, 3 Hats., 278, or an act to enforce, and make more
effectual an act, &c., or to rectify mistakes in an act, &c., or a
committee on one bill may be instructed to receive a clause to rectify
the mistakes of another. Thus, June 24, 1685, a clause was inserted
in a bill for rectifying a mistake committed by a clerk in engrossing a
bill of supply. 2 Hats., 194, 6. Or the session may be closed for one,
two, three or more days, and a new one commenced. But then all
matters depending must be finished, or they fall, and are to begin de
novo. 2 Hats., 94, 98. Or a part of the subject may be taken up by
another bill, or taken up in a different way. 6 Grey, 304, 316.
And in cases of the last magnitude, this rule has not been so
strictly and verbally observed as to stop indispensable proceedings
altogether. 2 Hats., 92, 98. Thus when the address on the
preliminaries of peace in 1782 had been lost by a majority of one, on
account of the importance of the question, and smallness of the
majority, the same question in substance, though with some words
not in the first, and which might change the opinion of some
members, was brought on again and carried, as the motives for it
were thought to outweigh the objection of form. 2 Hats., 99, 100.
A second bill may be passed to continue an act of the same session,
or to enlarge the time limited for its execution. 2 Hats., 95, 98. This
is not in contradiction to the first act.

SEC. XLIV.—BILLS SENT TO THE OTHER HOUSE.

[All bills passed in the Senate shall, before they are sent to the
House of Representatives, be examined by a committee, consisting of
three members, whose duty it shall be to examine all bills,
amendments, resolutions, or motions, before they go out of the
possession of the Senate, and to make report that they are correctly
engrossed; which report shall be entered on the journal. Rule 34.]
A bill from the other House is sometimes ordered to lie on the
table. 2 Hats., 97.
When bills, passed in one House and sent to the other, are
grounded on special facts requiring proof, it is usual, either by
message or at a conference, to ask the grounds and evidence; and
this evidence, whether arising out of papers, or from the examination
of witnesses, is immediately communicated. 3 Hats., 48.

SEC. XLV.—AMENDMENTS BETWEEN THE HOUSES.

When either House, e. g., the House of Commons, send a bill to the
other, the other may pass it with amendments. The regular
progression in this case is, that the Commons disagree to the
amendment; the Lords insist on it; the Commons insist on their
disagreement; the Lords adhere to their amendment; the Commons
adhere to their disagreement. The term of insisting may be repeated
as often as they choose to keep the question open. But the first
adherence by either renders it necessary for the other to recede or
adhere also; when the matter is usually suffered to fall. 10 Grey, 148.
Latterly, however, there are instances of their having gone to a
second adherence. There must be an absolute conclusion of the
subject somewhere, or otherwise transactions between the Houses
would become endless. 3 Hats., 268, 270. The term of insisting, we
are told by Sir John Trevor, was then (1679) newly introduced into
parliamentary usage, by the Lords. 7 Grey, 94. It was certainly a
happy innovation, as it multiplies the opportunities of trying
modifications which may bring the Houses to a concurrence. Either
House, however, is free to pass over the term of insisting, and to
adhere in the first instance; 10 Grey, 146; but it is not respectful to
the other. In the ordinary parliamentary course, there are two free
conferences, at least, before an adherence. 10 Grey, 147.
Either House may recede from its amendment and agree to the
bill; or recede from their disagreement to the amendment, and agree
to the same absolutely, or with an amendment; for here the
disagreement and receding destroy one another, and the subject
stands as before the agreement. Elsynge, 23, 27; 9 Grey, 476.
But the House cannot recede from or insist on its own
amendment, with an amendment; for the same reason that it cannot
send to the other House an amendment to its own act after it has
passed the act. They may modify an amendment from the other
House by ingrafting an amendment on it, because they have never
assented to it; but they cannot amend their own amendment,
because they have, on the question, passed it in that form. 9 Grey,
363; 10 Grey, 240. In the Senate, March 29, 1798. Nor where one
House has adhered to their amendment, and the other agrees with
an amendment, can the first House depart from the form which they
have fixed by an adherence.
In the case of a money bill, the Lords’ proposed amendments,
become, by delay, confessedly necessary. The Commons, however,
refused them, as infringing on their privilege as to money bills; but
they offered themselves to add to the bill a proviso to the same effect,
which had no coherence with the Lords’ amendments; and urged
that it was an expedient warranted by precedent, and not
unparliamentary in a case become impracticable, and irremediable
in any other way. 3 Hats., 256, 266, 270, 271. But the Lords refused,
and the bill was lost. 1 Chand., 288. A like case, 1 Chand., 311. So the
Commons resolved that it is unparliamentary to strike out, at a
conference, anything in a bill which hath been agreed and passed by
both Houses. 6 Grey, 274; 1 Chand., 312.
 A motion to amend an amendment from the other House takes
precedence of a motion to agree or disagree.
A bill originating in one House is passed by the other with an
amendment.
The originating House agrees to their amendment with an
amendment. The other may agree to their amendment with an
amendment, that being only in the 2d and not the 3d degree; for, as
to the amending House, the first amendment with which they passed
the bill is a part of its text; it is the only text they have agreed to. The
amendment to that text by the originating House, therefore, is only
in the 1st degree, and the amendment to that again by the amending
House is only in the 2d, to wit, an amendment to an amendment, and
so admissible. Just so, when, on a bill from the originating House,
the other, at its second reading, makes an amendment; on the third
reading this amendment is become the text of the bill, and if an
amendment to it be moved, an amendment to that amendment may
also be moved, as being only in the 2d degree.

SEC. XLVI.—CONFERENCES.

It is on the occasion of amendments between the Houses that

conferences are usually asked; but they may be asked in all cases of
difference of opinion between the two Houses on matters depending
between them. The request of a conference, however, must always be
by the House which is possessed of the papers. 3 Hats., 31; 1 Grey,
425.
Conferences may be either simple or free. At a conference simply,
written reasons are prepared by the House asking it, and they are
read and delivered, without debate, to the managers of the other
House at the conference; but are not then to be answered. 4 Grey,
144. The other House then, if satisfied, vote the reasons satisfactory,
or say nothing; if not satisfied, they resolve them not satisfactory and
ask a conference on the subject of the last conference, where they
read and deliver, in like manner, written answers to those reasons. 3
Grey, 183. They are meant chiefly to record the justification of each
House to the nation at large, and to posterity, and in proof that the
miscarriage of a necessary measure is not imputable to them. 3 Grey,
255. At free conferences, the managers discuss, viva voce and freely,
and interchange propositions for such modifications as may be made
in a parliamentary way, and may bring the sense of the two Houses
together. And each party reports in writing to their respective
Houses the substance of what is said on both sides, and it is entered
on their journals. 9 Grey, 220; 3 Hats., 280. This report cannot be
amended or altered, as that of a committee may be. Journal Senate,
May 24, 1796.
A conference may be asked, before the House asking it has come to
a resolution of disagreement, insisting or adhering. 3 Hats., 269, 341.
In which case the papers are not left with the other conferees, but are
brought back to be the foundation of the vote to be given. And this is
the most reasonable and respectful proceeding; for, as was urged by
the Lords on a particular occasion, “it is held vain, and below the
wisdom of Parliament, to reason or argue against fixed resolutions,
and upon terms of impossibility to persuade.” 3 Hats., 226. So the
Commons say, “an adherence is never delivered at a free conference,
which implies debate.” 10 Grey, 137. And on another occasion the
Lords made it an objection that the Commons had asked a free
conference after they had made resolutions of adhering. It was then
affirmed, however, on the part of the Commons, that nothing was
more parliamentary than to proceed with free conferences after
adhering, 3 Hats., 269, and we do in fact see instances of conference,
or of free conference, asked after the resolution of disagreeing, 3
Hats., 251, 253, 260, 286, 291, 316, 349; of insisting, ib., 280, 296,
299, 319, 322, 355; of adhering, 269, 270, 283, 300; and even of a
second or final adherence. 3 Hats., 270. And in all cases of
conference asked after a vote of disagreement, &c., the conferees of
the House asking it are to leave the papers with the conferees of the
other; and in one case where they refused to receive them, they were
left on the table in the conference chamber. ib., 271, 317, 323, 354; 10
Grey, 146.
After a free conference, the usage is to proceed with free
conferences, and not to return again to a conference. 3 Hats., 270; 9
Grey, 229.
After a conference denied, a free conference may be asked. 1 Grey,
45.
 When a conference is asked, the subject of it must be expressed, or
the conference not agreed to. Ord. H Com., 89; Grey, 425; 7 Grey,
31. They are sometimes asked to inquire concerning an offense or
default of a member of the other House. 6 Grey, 181; 1 Chand., 304.
Or the failure of the other House to present to the King a bill passed
by both Houses, 8 Grey, 302. Or on information received, and
relating to the safety of the nation. 10 Grey, 171. Or when the
methods of Parliament are thought by the one House to have been
departed from by the other, a conference is asked to come to a right
understanding thereon. 10 Grey, 148. So when an unparliamentary
message has been sent, instead of answering it, they ask a
conference. 3 Grey, 155. Formerly an address or articles of
impeachment, or a bill with amendments, or a vote of the House, or
concurrence in a vote, or a message from the King, were sometimes
communicated by way of conference. 6 Grey, 128, 300, 387; 7 Grey,
80; 8 Grey, 210, 255; 1 Torbuck’s Deb., 278; 10 Grey, 293; 1
Chandler, 49, 287. But this is not the modern practice. 8 Grey, 255.
A conference has been asked after the first reading of a bill. 1 Grey,
194. This is a singular instance.

SEC. XLVII.—MESSAGES.

Messages between the Houses are to be sent only while both

Houses are sitting. 3 Hats., 15. They are received during a debate
without adjourning the debate. 3 Hats., 22.
[In Senate the messengers are introduced in any state of business,
except, 1. While a question is being put. 2. While the yeas and nays
are being called. 3. While the ballots are being counted. Rule 51. The
first case is short; the second and third are cases where any
interruption might occasion errors difficult to be corrected. So
arranged June 15, 1798.]
In the House of Representatives, as in Parliament, if the House be
in committee when a messenger attends, the Speaker takes the chair
to receive the message, and then quits it to return into committee,
without any question or interruption. 4 Grey, 226.
Messengers are not saluted by the members, but by the Speaker
for the House. 2 Grey, 253, 274.
 If messengers commit an error in delivering their message, they
may be admitted or called in to correct their message. 4 Grey, 41.
Accordingly, March 13, 1800, the Senate having made two
amendments to a bill from the House of Representatives, their
Secretary, by mistake, delivered one only; which being inadmissible
by itself, that House disagreed, and notified the Senate of their
disagreement. This produced a discovery of the mistake. The
Secretary was sent to the other House to correct his mistake, the
correction was received, and the two amendments acted on de novo.
As soon as the messenger, who has brought bills from the other
House, has retired, the Speaker holds the bills in his hand, and
acquaints the House “that the other House have by their messenger
sent certain bills,” and then reads their titles, and delivers them to
the Clerk, to be safely kept till they shall be called for to be read.
Hakew., 178.
It is not the usage for one House to inform the other by what
numbers a bill is passed. 10 Grey, 150. Yet they have sometimes
recommended a bill, as of great importance, to the consideration of
the House to which it is sent. 3 Hats., 25. Nor when they have
rejected a bill from the other House, do they give notice of it; but it
passes sub silentio, to prevent unbecoming altercations. 1 Blackst.,
183.
[But in Congress the rejection is notified by message to the House
in which the bill originated.]
A question is never asked by the one House of the other by way of
message, but only at a conference; for this is an interrogatory, not a
message. 3 Grey, 151, 181.
When a bill is sent by one House to the other, and is neglected,
they may send a message to remind them of it. 3 Hats., 25; 5 Grey,
154. But if it be mere inattention, it is better to have it done
informally by communications between the Speakers or members of
the two Houses.
Where the subject of a message is of a nature that it can properly
be communicated to both Houses of Parliament, it is expected that
this communication should be made to both on the same day. But
where a message was accompanied with an original declaration,
signed by the party to which the message referred, its being sent to
one House was not noticed by the other, because the declaration,
being original, could not possibly be sent to both Houses at the same
time. 2 Hats., 260, 261, 262.
The King having sent original letters to the Commons, afterward
desires they may be returned, that he may communicate them to the
Lords. 1 Chandler, 303.

SEC. XLVIII.—ASSENT.

The House which has received a bill and passed it may present it
for the King’s assent, and ought to do it, though they have not by
message notified to the other their passage of it. Yet the notifying by
message is a form which ought to be observed between the two
Houses from motives of respect and good understanding. 2 Hats.,
242. Were the bill to be withheld from being presented to the King, it
would be an infringement of the rules of Parliament. Ib.
[When a bill has passed both Houses of Congress, the House last
acting on it notifies its passage to the other, and delivers the bill to
the Joint Committee of Enrolment, who see that it is truly enrolled in
parchment]. When the bill is enrolled, it is not to be written in
paragraphs, but solidly, and all of a piece, that the blanks between
the paragraphs may not give room for forgery. 9 Grey, 143. [It is then
put into the hands of the Clerk of the House of Representatives to
have it signed by the Speaker. The Clerk then brings it by way of
message to the Senate to be signed by their President. The Secretary
of the Senate returns it to the Committee of Enrolment, who present
it to the President of the United States. If he approve, he signs, and
deposits it among the rolls in the office of the Secretary of State, and
notifies by message the House in which it originated that he has
approved and signed it; of which that House informs the other by
message. If the President disapproves, he is to return it, with his
objections, to that House in which it shall have originated; who are to
enter the objections at large on their journal, and proceed to
reconsider it. If, after such reconsideration, two-thirds of that House
shall agree to pass the bill, it shall be sent, together with the
President’s objections, to the other House, by which it shall likewise
be reconsidered; and if approved by two-thirds of that House, it shall
become a law. If any bill shall not be returned by the President
within ten days (Sundays excepted) after it shall have been presented
to him, the same shall be a law, in like manner after he had signed it,
unless the Congress, by their adjournment, prevent its return; in
which case it shall not be a law. Const., I, 7.]
[Every order, resolution, or vote, to which the concurrence of the
Senate and House of Representatives may be necessary, (except on a
question of adjournment), shall be presented to the President of the
United States, and, before the same shall take effect, shall be
approved by him; or, being disapproved by him, shall be repassed by
two-thirds of the Senate and House of Representatives, according to
the rules and limitations prescribed in the case of a bill. Const., I, 7.]

SEC. XLIX.—JOURNALS.

[Each House shall keep a journal of its proceedings, and from time
to time publish the same, excepting such parts as may, in their
judgment, require secrecy. Const., I, 5.]
[The proceedings of the Senate, when not acting as in a Committee
of the Whole, shall be entered on the journals as concisely as
possible, care being taken to detail a true account of the proceedings.
Every vote of the Senate shall be entered on the journals, and a brief
statement of the contents of each petition, memorial, or paper
presented to the Senate, be also inserted on the journal. Rule 5.]
[The titles of bills, and such parts thereof, only, as shall be affected
by proposed amendments, shall be inserted on the journals. Rule 5.]
If a question is interrupted by a vote to adjourn, or to proceed to
the orders of the day, the original question is never printed in the
journal, it never having been a vote, nor introductory to any vote; but
when suppressed by the previous question, the first question must be
stated, in order to introduce and make intelligible the second. 2
Hats., 83.
So also when a question is postponed, adjourned, or laid on the
table, the original question, though not yet a vote, must be expressed
in the journals; because it makes part of the vote of postponement,
adjourning, or laying it on the table.
 Where amendments are made to a question, those amendments
are not printed in the journals, separated from the question; but only
the question as finally agreed to by the House. The rule of entering in
the journals only what the House has agreed to, is founded in great
prudence and good sense; as there may be many questions proposed,
which it may be improper to publish to the world in the form in
which they are made. 2 Hats., 85.
[In both Houses of Congress, all questions whereon the yeas and
nays are desired by one-fifth of the members present, whether
decided affirmatively or negatively, must be entered in the journals.
Const., I, 5.]
The first order for printing the votes of the House of Commons
was October 30, 1685. 1 Chandler, 387.
Some judges have been of opinion that the journals of the House of
Commons are no records, but only remembrances. But this is not
law. Hob., 110, 111; Lex Parl., 114, 115; Jour. H. C., Mar. 17, 1592;
Hale, Parl., 105. For the Lords in their House have power of
judicature, the Commons in their House have power of judicature,
and both Houses together have power of judicature; and the book of
the Clerk of the House of Commons is a record, as is affirmed by act
of Parl., 6 H. 8, c. 16; 4 Inst., 23, 24; and every member of the House
of Commons hath a judicial place. 4 Inst., 15. As records they are
open to every person, and a printed vote of either House is sufficient
ground for the other to notice it. Either may appoint a committee to
inspect the journals of the other, and report what has been done by
the other in any particular case. 2 Hats., 261; 3 Hats., 27–30. Every
member has a right to see the journals and to take and publish votes
from them. Being a record, every one may see and publish them. 6
Grey, 118, 119.
On information of a mis-entry or omission of an entry in the
journal, a committee may be appointed to examine and rectify it, and
report it to the House. 2 Hats., 194, 1195.

SEC. L.—ADJOURNMENT.

The two Houses of Parliament have the sole, separate, and

independent power of adjourning each their respective Houses. The